Cell Signalling Biology Michael J.

Berridge r Module 2 r Cell Signalling Pathways 2 r1

Module 2

Cell Signalling Pathways

Synopsis

Cells use a large number of clearly defined signalling pathways to regulate their activity. In this module,
attention is focused on the ON mechanisms responsible for transmitting information into the cell. These
signalling pathways fall into two main groups depending on how they are activated. Most of them are
activated by external stimuli and function to transfer information from the cell surface to internal effector
systems. However, some of the signalling systems respond to information generated from within the
cell, usually in the form of metabolic messengers. For all of these signalling pathways, information is
conveyed either through protein–protein interactions or it is transmitted by diffusible elements usually
referred to as second messengers. Cells often employ a number of these signalling pathways, and
cross-talk between them is an important feature. In this section, attention is focused on the
properties of the major intracellular signalling pathways operating in cells to regulate their cellular
activity.
During the processes of development, specific cell types select out those signalling systems that are
suitable to control their particular functions. One of the aims of this website is to understand how these
unique cell-specific signalsomes function to regulate different mammalian cell types.

Intracellular signalling pathways AMP signalling pathway, which led to the second mes-
There are a large number of intracellular signalling path- senger concept that applies to many other signalling
ways responsible for transmitting information within the systems. The idea is that the external stimulus arriving
cell. They fall into two main categories. The majority re- at the cell surface is the first messenger, which is then
spond to external stimuli arriving at the cell surface, usu- transformed at the cell surface by adenylyl cyclase
ally in the form of a chemical signal (neurotransmitter, (AC) into a second messenger, cyclic AMP, which is
hormone or growth factor), which is received by recept- part of the signalling cascade that then activates down-
ors at the cell periphery that function as molecular an- stream effectors.
tennae embedded in the plasma membrane. These recept- 2. Cyclic ADP-ribose (cADPR) signalling and nicotinic
ors then transfer information across the membrane using acid–adenine dinucleotide phosphate (NAADP) sig-
a variety of transducers and amplifiers that engage a di- nalling systems function in Ca2+ signalling. An ADP–
verse repertoire of intracellular signalling pathways (Path- ribosyl cyclase (ADP-RC) is responsible for generat-
ways 1–16 in Module 2: Figure cell signalling pathways). ing these two second messengers.
The phosphoinositide signalling and Ca2+ signalling sys- 3. Voltage-operated channels (VOCs) contribute to
tems (Pathways 2–6) have been grouped together because Ca2+ signals by controlling the entry of external Ca2+
they contain a number of related signalling pathways that in excitable cells.
often interact with each other. The other categories are 4. Receptor-operated channels (ROCs) contribute to
the pathways that are activated by signals generated from Ca2+ signals by controlling Ca2+ entry in both ex-
within the cell (Pathways 17 and 18). There are a number citable and non-excitable cells.
of metabolic messengers that act from within the cell to 5. Stimuli that activate phospholipase C (PLC) to hy-
initiate a variety of signalling pathways. drolyse PtdIns4,5P2 (also known as PIP2 ) generate a
All of these signalling pathways generate an internal number of signalling pathways:
messenger that is responsible for relaying information to • Inositol 1,4,5-trisphosphate (InsP3 )/Ca2+ signalling
the sensors that then engage the effectors that activate cel- cassette
lular responses. The main features of the signalling path- • Diacylglycerol (DAG)/protein kinase C (PKC) sig-
ways summarized in Module 2: Figure cell signalling path- nalling cassette
ways are outlined below: • PtdIns4,5P2 signalling cassette
1. Cyclic AMP signalling pathway. One of the first sig- • Multipurpose inositol polyphosphate signalling
nalling systems to be characterized was the cyclic pathway
6. PtdIns 3-kinase signalling is activated by stimuli that
stimulate PtdIns 3-kinase to phosphorylate PIP2 to
Green text indicates links to content within this module; blue text indicates links form the lipid second messenger PtdIns3,4,5P3 (PIP3 ).
to content in other modules.

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r2

Module 2: Figure cell signalling pathways

1 AC cAMP

2 ADP-RC NAADP
cADPR
3 VOC
Ca 2+
4 ROC
IP DAG
3
5 PLC
PIP2

RECEPTORS & TRANSDUCERS 6 PI 3-K PIP3

CELLULAR RESPONSES
cGMP
7 NOS NO
Nitrosylation

EFFECTORS
8 NOX O2 + H2O2

SENSORS
STIMULI

JNK
9 Ras Erk1/2
p38
10 IκBs
NF-κB
11 PLD PA

12 SMase ceramide/ S1P

13 JAK STAT

14 TGF-β Smad

15 Wnt β-catenin

16 Hh GLI

17 Notch NICD

ATF6, PERK
ER 18
SCAP, bHLH
Metabolism 19 AMP AMPK

Summary of the major signalling pathways used by cells to regulate cellular processes.
Cells have a number of signalling systems that are capable of responding either to external stimuli or to internal stimuli. In the case of the former, external
stimuli acting on cell-surface receptors are coupled to transducers to relay information into the cell using a number of different signalling pathways
(Pathways 1–17). Internal stimuli derived from the endoplasmic reticulum (ER) or from metabolism activate signalling pathways independently of
external signals (Pathways 18 and 19). All of these pathways generate an internal messenger that then acts through an internal sensor to stimulate
the effectors that bring about different cellular responses. As described in the text, the names of these signalling pathways usually reflect a major
component(s) of the pathway.

7. Nitric oxide (NO)/cyclic GMP signalling pathway. many cellular processes and particularly those related
Nitric oxide (NO) synthase (NOS) generates the gas to cell proliferation, cell stress and apoptosis.
NO that acts both through cyclic GMP and nitrosyla- 10. Nuclear factor κB (NF-κB) signalling pathway. This
tion reactions. NO has a particularly important role signalling system has a multitude of functions. It is
in modulating the activity of other pathways such as particularly important in initiating inflammatory re-
Ca2+ signalling. sponses in macrophages and neutrophils as part of an
8. Redox signalling. Many receptors act through innate immune response to invading pathogens.
NADPH oxidase (NOX) to form reactive oxygen 11. Phospholipase D (PLD) signalling pathway. This is a
species, such as the superoxide radical (O2 −• ) and hy- lipid-based signalling system that depends upon the
drogen peroxide (H2 O2 ), which act to regulate the hydrolysis of phosphatidylcholine by phospholipase
activity of specific signalling proteins such as tyrosine D (PLD) to give phosphatidic acid (PA), which func-
phosphatases, transcription factors and ion channels. tions as a second messenger to control a variety of
The O2 −• participates in the nitrosylation reaction in cellular processes.
Pathway 7. 12. Sphingomyelin signalling pathway. Certain growth
9. Mitogen-activated protein kinase (MAPK) signalling. factors and cytokines hydrolyse sphingomyelin to
This is a classical example of a protein phosphorylation generate two second messengers that appear to have
cascade that often begins with Ras and consists of a opposing effects in the cell. Ceramide seems to pro-
number of parallel pathways that function to control mote apoptosis, whereas sphingosine 1-phosphate

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r3

(S1P) stimulates cell proliferation. S1P may also re- ation usually depends upon the activation of G protein-
lease Ca2+ from internal stores. The action of S1P is coupled receptors (GPCRs) that use heterotrimeric G pro-
complicated in that it is released from the cell, where teins to activate the amplifier adenylyl cyclase (AC), which
it can act as a hormone to stimulate external receptors. is a large family of isoforms that differ considerably in both
13. Janus kinase (JAK)/signal transducer and activator of their cellular distribution and the way they are activated.
transcription (STAT) signalling pathway. This is a fast- There are a number of cyclic AMP signalling effectors
track signal transduction pathway for transferring in- such as protein kinase A (PKA), the exchange proteins ac-
formation from cell-surface receptors into the nucleus. tivated by cyclic AMP (EPACs) that activate the small
The Janus kinases (JAKs) are tyrosine kinases that GTP-binding protein Rap1 and the cyclic nucleotide-
phosphorylate the signal transducers and activators gated channels (CNGCs). These various effectors are then
of transcription (STATs), which carry the information responsible for carrying out the cyclic AMP signalling
into the nucleus. functions that include control of metabolism, gene tran-
14. Smad signalling pathway. This pathway mediates the scription and ion channel activity. In many cases, these
action of the transforming growth factor β (TGF-β) functions are modulatory in that cyclic AMP often acts
superfamily, which controls transcription through the to adjust the activity of other signalling pathways and
Smad transcription factors. thus has a central role to play in the cross-talk between
15. Wnt signalling pathways. These pathways play an im- signalling pathways. This modulatory function is particu-
portant role in both development and cell prolifera- larly evident in the case of Ca2+ signalling in both neur-
tion. onal and muscle cells. Many of the functions of cyclic
16. Hedgehog signalling pathway. This pathway re- AMP depend upon the precise location of PKA relative
sembles the Wnt signalling pathway in that it also to both its upstream and downstream effectors. A family
functions to regulate development and cell prolifer- of A-kinase-anchoring proteins (AKAPs) determines this
ation. The ligand Hedgehog (Hh) acts through the cellular localization of PKA as well as a number of other
transcription factor GLI. signalling components. The OFF reactions responsible for
17. Notch signalling pathway. This is a highly conserved removing cyclic AMP are carried out by either cyclic AMP
signalling system that functions in developmental pro- hydrolysis or by cyclic AMP efflux from the cell.
cesses related to cell-fate determination particularly in
stem cells. The notch receptor generates the transcrip-
tion factor NICD (Notch intracellular domain). Cyclic AMP formation
18. Endoplasmic reticulum (ER) stress signalling. The en- The formation of cyclic AMP can be activated by a very
doplasmic reticulum (ER) stress signalling pathway large number of cell stimuli, mainly neurotransmitters
concerns the mechanisms used by the ER to transmit and hormones (Module 1: Figure stimuli for cyclic AMP
information to the nucleus about the state of protein signalling). All these stimuli are detected by G protein-
processing within the lumen of the ER. coupled receptors (GPCRs) that use heterotrimeric G pro-
19. AMP signalling pathway. This pathway is regulated by teins, which are the transducers that are responsible for
adenosine monophosphate (AMP), which functions as either activating or inhibiting the enzyme adenylyl cy-
a metabolic messenger to activate an important path- clase (AC) (Module 2: Figure heterotrimeric G protein
way for the control of cell proliferation. signalling). In the case of AC stimulation, the external
stimulus binds to the GPCR that functions as a guanine
Not included in Module 2: Figure cell signalling path- nucleotide exchange factor (GEF) to replace GDP with
ways are some additional signalling pathways that have GTP, which dissociates the heterotrimeric complex into
specific functions in regulating various aspects of cell meta- their Gβγ and Gα subunits (Module 2: Figure cyclic AMP
bolism, such as sterol sensing and cholesterol biosynthesis, signalling). The GαS ·GTP complex activates AC, whereas
that control the level of cholesterol in cell membranes. An- Gαi ·GTP inhibits AC. The Gα subunits have GTPase
other example is found in the NAD signalling pathways, activity that hydrolyses GTP to GDP, thus terminating
where NAD+ functions to regulate a number of cellular their effects on AC. The endogenous GTPase of GαS ·GTP
processes, including energy metabolism, gene transcrip- is inhibited by cholera toxin and this causes the persistent
tion, DNA repair and perhaps ageing as well. activation of the intestinal fluid secretion that results in the
These cassettes then engage a variety of effectors that symptoms of cholera.
are responsible for activating cellular responses. All of
these mechanisms (Module 1: Figure signal transmission
mechanisms) depend upon information transfer mechan- Adenylyl cyclase (AC)
isms whereby information is transferred along an orderly The adenylyl cyclase (AC) family is composed of ten iso-
sequence of events to activate the internal effectors re- forms: nine of them are membrane-bound (AC1–AC9),
sponsible for inducing a great variety of cellular responses. while one of them is soluble (AC10) (Module 2: Table
adenylyl cyclases). The domain structure of AC1–AC9 is
characterized by having two regions where there are six
Cyclic AMP signalling pathway transmembrane regions (Module 2: Figure adenylyl cyc-
Cyclic AMP is a ubiquitous second messenger that regu- lase structure). The large cytoplasmic domains C1 and C2,
lates a multitude of cellular responses. Cyclic AMP form- which contain the catalytic region, form a heterodimer

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r4

Module 2: Table adenylyl cyclases

Regulatory properties and distribution of adenylyl cyclase.
Modulation by Ca2+ , calmodulin
(CaM), Ca2+ /CaM kinase II (CaMKII),
Adenylyl cyclase protein kinase C (PKC), protein
(AC) isoform Gα Gβγ Gαi or Gαio kinase A (PKA) Tissue distribution
AC1 ↑ ↓ ↓ Gαo ↑ CaM and PKC Brain, adrenal medulla
↓ CaMKII
AC2 ↑ ↑ – ↑ PKC Brain, skeletal and cardiac muscle, lung
AC3 ↑ ↑ CaM and PKC Brain, olfactory epithelium
↓ CaMKII
AC4 ↑ ↑ ↑ PKC Brain, heart, kidney, liver
AC5 ↑ ↓ ↓ ↓ Ca2+ and PKCα Brain, heart, kidney, liver, lung, adrenal
↓ PKA
AC6 ↑ ↓ ↓ ↑ PKC Ubiquitous
↓ Ca2+ and PKA
AC7 ↑ ↑ ↑ PKC Ubiquitous, high in brain
AC8 ↑ ↑ CaM Brain, lung, heart, adrenal
AC9 ↑ Brain, skeletal muscle
AC10 – – – Activated by HCO3 − Testis
The membrane-bound adenylyl cyclases (AC1–AC9) are widely distributed. They are particularly rich in brain, but are also expressed in
many other cell types. The soluble AC10 is restricted to the testis. The primary regulation of the AC1–AC9 isoforms is exerted through
components of the heterotrimeric G proteins, which are dissociated upon activation of neurotransmitter and hormonal receptors into their
α and βγ subunits. They are all activated by Gαs , but only some of the isoforms are inhibited by Gαi . The Gβγ subunit is able to activate
some isoforms, but inhibits others. The isoforms also differ in the way they are modulated by components of other signalling pathways
such as Ca2+ and PKC. Some of the others are inhibited by PKA, which thus sets up a negative-feedback loop whereby cyclic AMP can
inhibit its own production. Modified from Table I in Whorton and Sunahara 2003. Reproduced from Handbook of Cell Signaling, Volume 2
(edited by R.A. Bradshaw and E.A. Dennis), Whorton, M.R. and Sunahara, R.K., Adenylyl cyclases, pp. 419–426. Copyright (2003), with
permission from Elsevier.

Module 2: Figure adenylyl cyclase structure

TM 1-6 TM 7-12
C1 C2 AC 1 - 9

C1 C2 AC 10

Plasma membrane
1 2 3 4 5 6 7 8 9 10 11 12

C1 C2
O O
O P O P O
O O Pyrophosphate
NH 2 NH 2

N N
N N
H H O O O H H
N C O P O P O P O N C
N O N O O
O O O
O P
ATP OH OH Cyclic AMP OH O O

Domain structure of adenylyl cyclase (AC).

The nine membrane-bound adenylyl cyclases (AC1–AC9) have a similar domain structure. The single polypeptide has a tandem repeat of six
transmembrane domains (TM) with TM1–TM6 in one repeat and TM7–TM12 in the other. Each TM cassette is followed by large cytoplasmic domains
(C1 and C2), which contain the catalytic regions that convert ATP into cyclic AMP. As shown in the lower panel, the C1 and C2 domains come together
to form a heterodimer. The ATP-binding site is located at the interface between these two domains. The soluble AC10 isoform lacks the transmembrane
regions, but it retains the C1 and C2 domains that are responsible for catalysis.

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r5

Module 2: Figure cyclic AMP signalling

Stimulatory agonists Inhibitory agonists

Adenylyl
cyclase

γ β αS GDP γ β αi
Cholera GDP γ β
α
toxin
- GTP
S + - αi
GTP
ABCC4

HCO + Cyclic
3
Soluble adenylyl cyclase AMP PDE 5’AMP

EPAC + + CNGC
Rap1 PKA
PLCε
+ Ca2+
+ P
InsP 3
DAG
+ + CFTR

+ + + +
P
Cyclic
GMP P + + Ca2+
AMPAR
P P
cGMP PDE
P P P RYR
CREB P
5’GMP P CaV1.1
F-2,6-P2 P
phosphatase Lipase Phosphorylase PLN Ca V1.2
kinase ER/SR
SERCA

Organization and function of the cyclic AMP signalling pathway.

Cyclic AMP is formed both by membrane-bound adenylyl cyclase and by the bicarbonate-sensitive soluble adenylyl cyclase. The former is regulated
by both stimulatory agonists that act through the αS subunit or through inhibitory agonists that act through either the αi or the βγ subunits. The
increase in cyclic AMP then acts through three different effector systems. It acts through the exchange protein activated by cyclic AMP (EPAC), which
functions to activate Rap1. It can open cyclic nucleotide-gated channels (CNGCs). The main action of cyclic AMP is to activate protein kinase A (PKA)
to phosphorylate a large number of downstream targets. Some of these drive specific processes such as gene transcription through phosphorylation
of cyclic AMP response element-binding protein (CREB), and activation of ion channels [e.g. AMPA receptors and cystic fibrosis transmembrane
conductance regulator (CFTR)] and various enzymes that control metabolism [e.g. fructose 2,6-bisphosphate (F-2,6-P2 ) 2-phosphatase, lipase and
phosphorylase kinase]. Other downstream targets are components of other signalling pathways such as the cyclic GMP phosphodiesterase (cGMP
PDE), the phospholamban (PLN) that controls the sarco/endo-plasmic reticulum Ca2+ -ATPase (SERCA), the ryanodine receptor (RYR) and the Ca2+
channels CaV 1.1 and CaV 1.2.

and co-operate with each other to convert ATP into cyclic and has a high affinity for cyclic AMP, whereas protein
AMP. kinase A (PKA) II has a much more precise location by be-
ing coupled to the A-kinase-anchoring proteins (AKAPs).
Cyclic AMP signalling effectors The AKAPs are examples of the scaffolding proteins that
Cyclic AMP is a highly versatile intracellular messen- function in the spatial organization of signalling pathways
ger capable of activating a number of different effectors by bringing PKA into contact with its many substrates.
(Module 2: Figure cyclic AMP signalling). An example of The scaffolding function of the AKAPs is carried out by
such effectors is the exchange proteins activated by cyc- various domains such as the conserved PKA-anchoring
lic AMP (EPACs), which act to stimulate Rap. Another domain [yellow region in Module 2: Figure protein kinase
group of effectors are the cyclic nucleotide-gated channels A (PKA)], which is a hydrophobic surface that binds to
(CNGCs) (Module 3: Figure Ca2+ entry mechanisms) that an extended hydrophobic surface on the N-terminal di-
play a particularly important role in the sensory systems merization region of the R subunits. At the other end of
responsible for smell and taste. Most of the actions of cyclic the molecule, there are unique targeting domains (blue)
AMP are carried out by protein kinase A (PKA). that determine the way AKAPs identify and bind specific
cellular targets in discrete regions of the cell.
Protein kinase A (PKA)
Many of the actions of cyclic AMP are carried out by pro- Protein kinase A (PKA) I
tein kinase A (PKA), which phosphorylates specific sites Type I protein kinase A (PKA) associates with the RI iso-
on downstream effector processes (Module 2: Figure cyc- forms. As for all isoforms, the R subunits form dimers
lic AMP signalling). PKA is composed of two regulatory through their N-terminal dimerization/docking domains
(R) subunits and two catalytic (C) subunits. The way in [yellow bar in Module 2: Figure protein kinase A (PKA)].
which cyclic AMP activates PKA is to bind to the R sub- In addition to holding two R subunits together, this N-
units, which then enables the C subunits to phosphorylate terminal region is also responsible for docking to the A-
a wide range of different substrates [Module 2: Figure pro- kinase-anchoring proteins (AKAPs), as occurs for PKA
tein kinase A (PKA)]. Of the two types of PKA, protein II. However, the RI isoforms have a very low affinity for
kinase A (PKA) I is found mainly free in the cytoplasm the AKAPs and are thus mainly soluble. Cyclic AMP acts

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r6

Module 2: Figure protein kinase A (PKA)

PKA I PKA II
RI RI RII RII
C Tandem C
C C

AKAP
cyclic AMP-binding
domains

Substrate Substrate

Cellular target

Cyclic AMP

RI RI
Cyclic AMP

RII RII

AKAP
C
P
C P P
Substrate Substrate
Substrate Substrate

Cellular target

The functional organization of protein kinase A (PKA).

There are two types of PKA, type I PKA (PKA I) and type II PKA (PKA II), which differ primarily in the type of R subunits that associate with the C
subunits. There are four R subunit isoforms (RIα, RIβ, RIIα and RIIβ), which have somewhat different properties with regard to their affinity for cyclic
AMP and their ability to associate with the A-kinase-anchoring proteins (AKAPs). It is these different R subunits that define the properties of the two
types of PKA.

by binding to the tandem cyclic AMP-binding domains to • In insulin-secreting β-cells, the salt-inducible kinase 2
release active C subunits that then phosphorylate specific (SIK2) that phosphorylates transducer of regulated cyc-
substrates. Since the RI subunits have a higher cyclic AMP- lic AMP response element-binding protein (TORC)
binding affinity, PKA I will be able to respond to the lower is inhibited by a cyclic AMP/PKA-dependent phos-
cyclic AMP concentrations found globally within the bulk phorylation (Module 7: Figure β-cell signalling).
cytoplasm. • PKA phosphorylates the hormone-sensitive lipase
(HLS) that initiates the hydrolysis of triacylglycerol
Protein kinase A (PKA) II
to free fatty acids and glycerol in both white fat cells
A characteristic feature of Type II protein kinase A (PKA)
(Module 7: Figure lipolysis and lipogenesis) and in
is that the regulatory dimer is made up of RII subunits.
brown fat cells (Module 7: Figure brown fat cell).
Since this RII subunit has a much higher affinity for the
• PKA phosphorylates the phosphorylase kinase that
A-kinase-anchoring proteins (AKAPs), PKA II is usually
converts inactive phosphorylase b into active phos-
docked to this scaffolding protein and thus has a much
phorylase a in skeletal muscle (Module 7: Figure skeletal
more precise localization to specific cellular targets.
muscle E-C coupling) and in liver cells (Module 7: Fig-
The substrates phosphorylated by cyclic AMP (Mod-
ure glycogenolysis and gluconeogenesis).
ule 2: Figure cyclic AMP signalling) fall into two main
• PKA activates the transcription factor cyclic AMP re-
groups: the cyclic AMP substrates that regulate specific
sponse element-binding protein (CREB) (Module 4:
cellular processes and the cyclic AMP substrates that are
Figure CREB activation). This activation is a critical
components of other signalling systems.
event in the induction of gluconeogenesis in liver cells
Cyclic AMP substrates that regulate specific cellular
processes: (Module 7: Figure liver cell signalling).
• PKA inhibits the salt-inducible kinase 2 (SIK2) that nor-
• In neurons, cyclic AMP acts through PKA to phos- mally acts to phosphorylate TORC2, thereby prevent-
phorylate Ser-845 on the AMPA receptor (Module 3: ing it from entering the nucleus to facilitate the activity
Figure AMPA receptor phosphorylation). of CREB (Module 7: Figure liver cell signalling).
• Cyclic AMP acting through PKA stimulates the • PKA phosphorylates inhibitor 1 (I1), which assists the
fructose-2,6-bisphosphatase component to lower the protein phosphorylation process by inactivating protein
level of fructose 2,6-bisphosphate, which reduces glyco- phosphatase 1 (PP1).
lysis and promotes gluconeogenesis (Module 2: Figure • PKA contributes to the translocation and fusion of ves-
AMPK control of metabolism). icles with the apical membrane during the onset of acid

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r7

secretion by parietal cells (Module 7: Figure HCl secre- in insulin-secreting β-cells (Module 7: Figure β-cell sig-
tion). nalling).
• PKA phosphorylates the regulatory (R) domain on the
cystic fibrosis transmembrane conductance regulator Exchange proteins activated by cyclic AMP
(CFTR) to enable it to function as an anion channel (EPACs)
(Module 3: Figure CFTR channel). Other targets for the second messenger cyclic AMP are the
• In the small intestine, PKA phosphorylates the cystic exchange proteins activated by cAMP (EPACs). One of the
fibrosis transmembrane conductance regulator (CFTR) functions of EPACs is to activate Rap1, which has many
channel (Module 3: Figure CFTR channel) that is re- functions in cells, many of which are related to controlling
sponsible for activating fluid secretion (Module 7: Fig- actin dynamics. In addition, the EPAC/Rap pathway can
ure intestinal secretion). Uncontrolled activation of cyc- activate phospholipase Cε (PLCε).
lic AMP formation by cholera toxin results in cholera.
• In kidney collecting ducts, cyclic AMP acts through
Cyclic AMP signalling functions
PKA to phosphorylate Ser-256 on the C-terminal cyto-
The cyclic AMP signalling pathway functions in the con-
plasmic tail of aquaporin 2 (AQP2), enabling this water
trol of a wide range of cellular processes:
channel to fuse with the apical membrane to allow wa-
ter to enter the cell (Module 7: Figure collecting duct • Cyclic AMP suppresses spontaneous Ca2+ oscillations
function). during oocyte maturation.
• In blood platelets, cyclic AMP activates the • Cyclic AMP has a potent anti-inflammatory action by
phosphorylation of vasodilator-stimulated phos- inhibiting the activity of macrophages (Module 11: Fig-
phoprotein (VASP), which is a member of the ure macrophage signalling) and mast cells (Module 11:
Ena/vasodilator-stimulated phosphoprotein (VASP) Figure mast cell inhibitory signalling).
family resulting in a decrease in the actin-dependent • Melanocortin 4 receptors (MC4Rs) on second-order
processes associated with clotting (Module 11: Figure neurons use the cyclic AMP signalling pathway
platelet activation). to induce the hypothalamic transcription factor
Single-minded 1 (Sim1) to decrease food intake and
Cyclic AMP substrates that are components of other weight loss (Module 7: Figure control of food intake).
signalling systems: • Cyclic AMP mediates the action of lipolytic hormones
in white fat cells by stimulating a hormone-sensitive
• Phosphorylation of the cyclic GMP phosphodiesterase lipase (Module 7: Figure lipolysis and lipogenesis).
PDE1A by PKA results in a decrease in its sensitivity • Heat production by brown fat cells is controlled by
to Ca2+ activation. noradrenaline acting through cyclic AMP (Module 7:
• Entry of Ca2+ through the L-type CaV 1.1 channel Figure brown fat cells).
(Module 3: Figure CaV 1.1 L-type channel) and the L- • The phosphorylation of DARPP-32 co-ordinates the
type CaV 1.2 channel (Module 3: Figure CaV 1.2 L-type activity of the dopamine and glutamate signalling path-
channel) is enhanced through PKA-dependent phos- ways in medium spiny neurons (Module 10: Figure me-
phorylation. dium spiny neuron signalling).
• PKA-dependent phosphorylation of dopamine- and • Adrenaline-induced glycogenolysis in skeletal muscle
cyclic AMP-regulated phosphoprotein of apparent mo- cells depends upon a cyclic AMP-dependent phos-
lecular mass 32 kDa (DARPP-32) functions as a molecu- phorylation of phosphorylase kinase (Module 7: Figure
lar switch to regulate the activity of protein phosphatase skeletal muscle E-C coupling).
1 (PP1). • Activation of the cyclic AMP-dependent transcription
• The ryanodine receptor 2 (RYR2) is modulated by phos- factor cyclic AMP response element-binding protein
phorylation through PKA, which is associated with the (CREB) contributes to the regulation of glucagon bio-
cytoplasmic head through an AKAP (Module 3: Figure synthesis in glucagon-secreting α-cells (Module 7: Fig-
ryanodine receptor structure). ure α-cell signalling).
• Sarco/endo-plasmic reticulum Ca2+ -ATPase 2a
(SERCA2a) increases its Ca2+ -pumping activity when
the inhibitory effect of phospholamban (PLN) is Cyclic AMP hydrolysis
removed following its phosphorylation by PKA on There are two OFF reactions of the cyclic AMP sig-
Ser-16 (Module 5: Figure phospholamban mode of nalling pathway, cyclic AMP efflux from the cell and cyclic
action). AMP hydrolysis. The latter is carried out by a family of
phosphodiesterase enzymes that hydrolyse cyclic AMP to
Salt-inducible kinase 2 (SIK2) AMP (Module 2: Figure cyclic AMP signalling).
The salt-inducible kinase 2 (SIK2) functions to phos-
phorylate the transducer of regulated CREB (TORC2), Cyclic AMP efflux
thereby preventing it from entering the nucleus to facilitate There are two OFF reactions for the cyclic AMP sig-
the activity of the transcriptional factor CREB (Module 4: nalling pathway, cyclic AMP hydrolysis and cyclic AMP
Figure CREB activation). It functions to regulate TORC2 efflux from the cell (Module 2: Figure cyclic AMP sig-
in liver cells (Module 7: Figure liver cell signalling) and nalling). The latter is carried out by ABCC4, which is one

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r8

Module 2: Figure G protein binary switching

Input from cell surface receptors

Guanine nucleotide
exchange factors
GEFs
Signalling responses

Cyclic AMP signalling

InsP 3 /DAG signalling
GTP GDP MAP kinase signalling
G G
protein protein PtdIns 3-kinase signalling

GDP GTP PLD signalling

Redox signalling
Cytoskeletal remodelling
GAPs Ion channel modulation
GTPase-activating
Phosphate factors

Signal transduction through G protein binary switching.

All GTP-binding proteins (G proteins) function through a binary switching mechanism that is driven by the binding of GTP. The G protein is inactive
when bound to GDP. When this GDP is exchanged for GTP, the G protein is activated and can stimulate a number of different signalling responses.
The rapid switching to the active state is facilitated by the guanine nucleotide exchange factors (GEFs) that receive the information coming in from
the receptors on the cell surface. By contrast, the GTPase-activating proteins (GAPs) accelerate the OFF reaction by enhancing the hydrolysis of GTP
to GDP.

of the ATP-binding cassette (ABC) transporters (Mod- GPCRs and to relay it to various amplifiers. The Gα sub-
ule 3: Table ABC transporters). units are either palmitoylated or myristoylated near the
N-terminus, whereas the Gβγ subunits are prenylated.
The heterotrimeric G proteins are extremely versatile sig-
GTP-binding proteins nalling elements, and both the Gα subunit and the Gβγ
There are a large number of GTP-binding proteins, which subunit are able to relay information to downstream com-
usually are referred to as G proteins, which play a cent- ponents (Module 2: Figure heterotrimeric G protein sig-
ral role in cell signalling as molecular switches (Module 2: nalling).
Figure G protein binary switching). These G proteins are These heterotrimeric G proteins exist in two states.
also GTPases, and the switching is driven by both the When the Gα subunit is bound to GDP, it forms a com-
binding of GTP and its hydrolysis to GDP. The G protein plex with Gβγ subunits to form the inactive heterotri-
is inactive when bound to GDP, but when the GDP is ex- meric complex. When the GPCRs, which are sensitive to a
changed for GTP, the G protein/GTP complex is active and wide range of stimuli (Module 1: Figure stimuli for cyclic
transfers information down the signalling pathway until AMP signalling), are activated, they function as a guan-
the endogenous GTPase activity hydrolyses GTP to GDP. ine nucleotide exchange factor (GEF) to replace the GDP
The G proteins belong to two groups, the heterotrimeric with GTP. The binding of GTP not only activates the
G proteins and the monomeric G proteins, which have Gα subunit, but also liberates the Gβγ subunit, both of
separate, but overlapping, functions. which can activate a range of signalling systems. This signal
transduction process is switched off when the endogen-
Heterotrimeric G proteins ous GTPase activity of the Gα subunit hydrolyses GTP
The heterotrimeric G proteins function as transducers for to GDP and the Gα/GDP complex recombines with the
the G protein-coupled receptors (GPCRs) that activate a Gβγ subunit to reform the inactive heterotrimeric com-
number of cell signalling pathways (Module 1: Table G plex. The normal rate of GTPase activity is very low (four
protein coupled receptors). These G proteins are made up to eight conversions per s), which means that the two sub-
from 16 Gα, five Gβ and 11 Gγ genes (Module 2: Table units have a long time to find their targets. However, there
heterotrimeric G proteins). These different subunits are are two mechanisms for speeding up the GTPase activity.
characterized by having various lipid modifications that Firstly, some of the targets can act to accelerate the GT-
serve to insert them into the plasma membrane, where they Pase activity. Secondly, a family of regulators of G protein
are positioned to detect information coming in from the signalling (RGS) proteins function as GTPase-activating

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r9

Module 2: Table heterotrimeric G proteins

The heterotrimeric proteins are assembled from subunits taken from the G protein α (Gα), G protein β (Gβ) and G protein γ (Gγ) families.
Heterotrimeric G protein Function
G protein α (Gα) subunits
Gαs Stimulate adenylyl cyclase
Gαolf Stimulate adenylyl cyclase
Gαi1 Inhibit adenylyl cyclase
Gαi2 Inhibit adenylyl cyclase
Gαi3 Inhibit adenylyl cyclase
Gαo1 Inhibit adenylyl cyclase
Gαo2 Inhibit adenylyl cyclase
Gαt1 Stimulate cyclic GMP phosphodiesterase in rod photoreceptors
Gαt2 Stimulate cyclic GMP phosphodiesterase in rod photoreceptors
Gz Close K+ channels. Inhibits exocytosis (see Module 10: Figure lactotroph regulation)
Gαgust Stimulate phospholipase Cβ (PLCβ) (see Module 7: Figure L cell)
Gαq Stimulate phospholipase Cβ (PLCβ)
Gα11 Stimulate phospholipase Cβ (PLCβ)
Gα14 Stimulate phospholipase Cβ (PLCβ)
Gα15 Stimulate phospholipase Cβ (PLCβ)
Gα16 Stimulate phospholipase Cβ (PLCβ)
Gα12 Stimulate RhoGEFs to activate Rho (Module 2: Figure Rho signalling)
Gα13 Stimulate RhoGEFs to activate Rho (Module 2: Figure Rho signalling)
G protein β (Gβ) subunits; β1–β5 These β subunits combine with γ subunits to form βγ dimers that have a number of control functions
(for details see Module 2: Figure heterotrimeric G protein signalling)
G protein γ (Gγ) subunits; γ1–γ11 These γ subunits combine with β subunits to form βγ dimers that have a number of control functions
(for details see Module 2: Figure heterotrimeric G protein signalling)

proteins (GAPs) that accelerate the Gα subunit GTPase ible for binding to Gα/GTP. However, many of the RGS
activity more than 1000-fold. proteins contain a number of other protein–protein in-
There are G protein receptor kinases (GRKs) such as teraction domains, such asPDZ, phosphotyrosine-binding
β-adrenergic receptor kinase 1 (βARK1), which phos- (PTB), pleckstrin homology (PH) and phox homology
phorylate active receptors to provide binding sites for (PX) domains, indicating that, in addition to their GAP
arrestin that prevent the heterotrimeric proteins from activity, they may have other functions. One of these might
binding the receptor and this leads to receptor desensit- be the regulation of the G protein-activated inwardly rec-
ization (Module 1: Figure homologous desensitization). tifying K+ (GIRK) channel.
The active Gα/GTP and Gβγ subunits are able to re- The function of RGS proteins has been clearly defined
lay information to a large number of signalling pathways, in phototransduction, where RGS9 acts to accelerate GTP
which are described in more detail for the different sig- hydrolysis by Gαt (step 6 in Module 10: Figure photo-
nalling pathways: transduction).
• The cyclic AMP signalling pathway (Module 2: Figure
cyclic AMP signalling). Monomeric G proteins
• The activation of phospholipase Cβ (PLCβ) in the The monomeric GTP-binding proteins (G proteins) be-
inositol 1,4,5-trisphosphate (InsP3 )/Ca2+ signalling cas- long to a large family of approximately 150 members. Ras
sette (Module 2: Figure PLC structure and function). was the founding member, and the family is often referred
• Modulation of the CaV 2 family of N-type and P/Q-type to as the Ras family of small G proteins. Within this family,
channels (Module 3: Figure CaV 2 channel family). it is possible to recognize five subfamilies: Ras, Rho, Rab,
• Activation of the PtdIns 3-kinase signalling pathway Ran and ADP-ribosylation factor (Arf) (Module 2: Table
(Module 2: Figure PtdIns 3-kinase signalling). monomeric G protein toolkit). The Ran family plays a role
• Activation of phosphodiesterase 6 (PDE6) during pho- in nuclear transport, whereas the large Rab family func-
totransduction in photoreceptors (Module 10: Figure tions in membrane trafficking. The Ras and Rho family are
phototransduction). primarily involved in cell signalling, where they function as
• G-αolf functions in sperm motility and chemotaxis. binary switches to control a number of cell signalling sys-
• G-αgust , which is also known as gustudin, functions in tems. This binary switch is driven by the binding of GTP,
taste cells and in the L cells that detect food components which represents the ON reaction, and is followed by the
in the lumen of the intestine (Module 7: Figure L cells). hydrolysis of the GTP by the endogenous GTPase activ-
Regulators of G protein signalling (RGS) ity. Most attention has focused on a small number of these
The regulators of G protein signalling (RGS) are a large signal transducers, and the following will be described in
family of approximately 30 proteins that function as detail to illustrate their role in cell signalling:
GTPase-activating proteins (GAPs) for the heterotrimeric
G proteins (Module 2: Figure heterotrimeric G protein sig- • Ras signalling mechanisms
nalling). The Gα subunit has a low intrinsic GTPase activ- • Rho signalling mechanisms
ity and this is greatly increased by the RGS proteins. RGS • Rac signalling mechanisms
structure is defined by an RGS-box region that is respons- • Cdc42 signalling mechanisms

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r10

Module 2: Figure heterotrimeric G protein signalling

Ca 2+
InsP3
Gα responses
PLC
Stimulus DAG
αq
AC Cyclic AMP
αs
AC Cyclic AMP
GPCR αi
αt PDE6 Cyclic GMP

α 12/13
P
GRK
α Rho A Cytoskeleton
Arrestins
GTP

α βγ
GTP GDP + Rac Cytoskeleton

GDP βγ Cytoskeleton
Cdc42
G protein
PI 3-K PIP3
RGS
GIRK ∆V
proteins
CaV1.2 Ca 2+

AC Cyclic AMP

InsP3 Ca 2+
PLC
DAG Gβγ responses

Heterotrimeric G proteins function as transducers to activate many signalling pathways.

External stimuli that bind to G protein-coupled receptors (GPCRs) act as guanine nucleotide exchange factors (GEFs) for the heterotrimeric G proteins.
When the GDP on the Gα subunit is replaced with GTP, the complex dissociates into Gα/GTP and Gβγ subunits that are then capable of activating
or inhibiting a wide range of signalling systems. Most of the actions are stimulatory, but some are inhibitory, as illustrated by the yellow arrows. The
regulators of G protein signalling (RGS) proteins function as GTPase-activating proteins (GAPs) to facilitate the GTPase activity of the Gα subunit,
which is the OFF reaction that terminates signalling. G protein receptor kinase (GRK) phosphorylates active receptors and provides binding sites for
arrestin that result in receptor desensitization by preventing the heterotrimeric G proteins from binding the receptor.

Ras signalling mechanisms ing up the hydrolysis of GTP to GDP by Ras. Examples
Ras is a small (21 kDa) GTPase that plays a central role of such RasGAPs include p120 RasGAP, neurofibromin,
as a signal transducer for a number of signalling sys- SynGAP, Ca2+ -promoted Ras inactivator (CAPRI) and
tems. It was first identified as a transducer for the tyr- Ras GTPase-activating-like (RASAL). The function of
osine kinase-linked receptors (Module 1: Figure stimuli some of these RasGAPs such as CAPRI and RASAL are
for enzyme-linked receptors), where it functions to re- attracting considerable attention because they are Ca2+ -
lay information to the mitogen-activated protein kinase sensitive proteins that rapidly translocate to the mem-
(MAPK) signalling pathway. It is now known that ac- brane through their C2 domains whenever there is an in-
tivated Ras is able to relay information to a number of crease in the level of Ca2+ . SynGAP, which is expressed
other signalling pathways. Like other G proteins (Mod- mainly in brain, is also activated by Ca2+ following its
ule 2: Figure G protein binary switching), Ras functions as phosphorylation by Ca2+ /calmodulin-dependent protein
a binary switch that is activated by binding GTP. There kinase II (CaMKII). The role of Ca2+ in modulating the
are thus two critical aspects to Ras action: how is the activity of both the RasGEFs and RasGAPs indicates an
switch controlled and how is information relayed out to important feedback interaction between the Ca2+ and Ras
different signalling pathways (Module 2: Figure Ras sig- signalling pathways.
nalling)? With regard to the first question, the ON reac- The Ras signalling mechanism relays information to a
tion of the binary switch is controlled by a number of Ras number of signalling targets:
guanine nucleotide exchange factors (RasGEFs) such as
Son-of-sevenless (SoS), Ras guanine nucleotide release-in- • It activates Raf to initiate the MAPK signalling pathway
ducing factors (RasGRFs) and Ras guanine nucleotide (Module 2: Figure ERK signalling).
releasing proteins (RasGRPs) (Module 2: Table mono- • It activates phospholipase Cε (PLCε) (Module 2: Figure
meric G protein toolkit). This conversion of Ras·GDP into PLC structure and function).
Ras·GTP is inhibited by the tumour suppressor protein • It activates the PtdIns 3-kinase signalling pathway.
merlin. Neurofibromatosis type 2 is caused by mutations • Ras can activate a family of RalGEFs such as Ral-
in merlin. GDP dissociation stimulator that then activates the Ral
There are a number of Ras GTPase-activating proteins proteins (RalA and RalB) (Module 2: Figure Ras sig-
(RasGAPs) that accelerate the OFF reaction by speed- nalling). One of the functions of RalA is to stimulate

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Module 2: Figure Ras signalling

Glutamate Growth
factors
Ca2+
PTKR
Ras responses
NMDAR
2+ DAG
Ca
+ +
TK TK
RasGRP
RasGRF SOS Raf ERK1/2
2+ CaM
Ca
PI 3-K PIP3
+
Ras GTP GDP Ras InsP3 Ca 2+
PLCε
GDP GTP
DAG

NEUROFIBROMIN Ral-GDS Ral PLD1

RASAL
CAPRI p120
SynGAP Ras GAP
CaMKII
+
+ GAPs
+
Ca2+

Function of the monomeric Ras G protein in cell signal transduction.

Ras plays a role as a signal transducer to relay information from various external stimuli to a range of different Ras-dependent responses. When bound
to GDP, Ras is inactive. Cell stimuli act through different guanine nucleotide exchange factors (GEFs) such as Son-of-sevenless (SoS), RasGRF and
RasGRP to facilitate an exchange of GDP for GTP to create the activated Ras/GTP complex that can relay information through a number of signalling
pathways. The action of Ras is terminated by a variety of GTPase-activating proteins (GAPs) that accelerate the ability of Ras to hydrolyse GTP back
to GDP.

the phospholipase D signalling pathway (Module 2: Fig- which then acts through calmodulin (CaM) to stimulate
ure PLD signalling). RasGRF. This is a highly localized signalling event, be-
cause the RasGRF is associated with the NMDA receptor
Son-of-sevenless (SoS) and thus responds to the microdomain of Ca2+ near the
Son-of-sevenless (SoS) is a classical Ras guanine nucle- mouth of the channel. This signalling mechanism func-
otide exchange factor (RasGEF). Its mode of action is tions in neuronal gene transcription (Module 10: Figure
evident in the way the platelet-derived growth factor re- neuronal gene transcription).
ceptor (PDGFR) is linked to the mitogen-activated pro-
tein kinase (MAPK) signalling pathway (Module 1: Fig- Ras guanine nucleotide releasing proteins (RasGRPs)
ure PDGFR activation). SoS binds to the Src homology 3 The Ras guanine nucleotide releasing proteins (RasGRPs)
(SH3)-containing adaptor growth factor receptor-bound are mainly expressed in haematopoietic cells (Module 2:
protein 2 (Grb2), which is attached to the phosphotyr- Table monomeric G protein toolkit). These GRPs are also
osine residues of the activated receptor. Once it is associ- known as CalDAG-GEFs because they are sensitive to di-
ated with the receptor, SoS comes into contact with Ras acylglycerol (DAG) and Ca2+ . They contain a C1 domain
and can begin to facilitate the exchange of GDP for GTP, that binds DAG and they also have a pair of Ca2+ -binding
thus creating the active Ras/GTP complex that begins to EF-hands. The sensitivity to both DAG and Ca2+ sug-
stimulate the MAPK signalling pathway (for further de- gests that these RasGRPs may couple phosphoinositide
tails see Module 2: Figure ERK signalling). signalling to the activation of the various pathways that
Ras guanine nucleotide release-inducing factors are linked to Ras (Module 2: Figure Ras signalling).
(RasGRFs)
The Ras guanine nucleotide release-inducing factors Rac signalling mechanisms
(RasGRFs) and Ras guanine nucleotide releasing proteins The Rac signalling pathway has an important role in ac-
(RasGRPs) are also important GEFs that can relay inform- tivating a number of signalling pathways (Module 2: Fig-
ation to Ras. The RasGRFs, which are strongly expressed ure Rac signalling). Like other G proteins, Rac functions
in brain, are particularly important for the activation of as a binary switch. It is inactive when bound to GDP,
the MAPK signalling pathway in neurons, where they re- but when this GDP is exchanged for GTP, the Rac/GTP
spond to activation of N-methyl-d-aspartate (NMDA) re- complex becomes active. External stimuli can activate this
ceptors. One of the functions of the latter is to gate Ca2+ , switch using different guanine nucleotide exchange factors

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r12

Module 2: Figure Rac signalling

Stimulus Stimulus

PTKR GPCR Rac responses

PIP
3
βγ NOX ROS signalling
Ca2+

TK
CaMKII TK Vav1 Sos
Kalirin Cofilin
P-Rex GEFs
TIAM P

Rac PAK1 Cofilin

Rac GTP GDP LIM-K1
GDP GTP

3BP-1 GAPs
Actin
IRS p53 WAVE Arp2/3 assembly
Actin
polymerization

Actin-Profilin Profilin

Function of the Rac monomeric G protein in signal transduction.

Rac is inactive when bound to GDP, but switches into an active form when this GDP is exchanged for GTP. This GTP for GDP exchange is facilitated by
a number of guanine nucleotide exchange factors (GEFs) such as Tiam, Kalirin, Vav, SoS and P-Rex, which are sensitive to various messengers such
as Ca2+ , PtdIns3,4,5P3 (PIP3 ) or Gβγ subunits of heterotrimeric G proteins. The activated Rac/GTP then relays information out to different signalling
pathways as described in the text. Further details concerning the role of Wiskott–Aldrich syndrome protein (WASP) verprolin homologous (WAVE) are
shown in Module 4: Figure actin remodelling.

(GEFs) such as T cell lymphoma invasion and metastasis changes that occur during long-term potentiation (LTP)
(Tiam), Kalirin, Vav, SoS and P-Rex. Kalirin functions in (Module 10: Figure Ca2+ -induced synaptic plasticity).
the Ephrin (Eph) receptor signalling pathway, where it
links the EphB receptor to Rac and actin assembly (Mod-
ule 1: Figure Eph receptor signalling). The lipid second Rho signalling mechanisms
messenger PtdIns3,4,5P3 , which is produced by the PtdIns The Rho signalling pathway has an important role in reg-
3-kinase signalling pathway, is particularly effective in ulating a number of systems, in particular the operation
stimulating many of these GEFs. of the cytoskeleton. It regulates the function of actin in
The activated Rac/GTP complex has a number of two main ways. Firstly, it controls some of the processes
important actions: that function in actin assembly and it also operates one of
the control mechanisms that regulate the myosin II–actin
interaction responsible for contraction both in smooth
• Rac is responsible for mediating the action of the PtdIns muscle cells and non-muscle cells (Module 2: Figure Rho
3-kinase signalling pathway in stimulating the NADPH signalling). Like other G proteins, Rho functions as a bin-
oxidase to initiate the reactive oxygen species (ROS) ary switch. It is inactive when bound to GDP, but when
signalling pathway (Module 2: Figure plasma membrane this GDP is exchanged for GTP, the Rho/GTP complex
ROS formation). becomes active. External stimuli can activate this switch
• It stimulates the JNK (c-Jun N-terminal kinase) sig- using different Rho guanine nucleotide exchange factors
nalling pathway (Module 2: Figure JNK signalling). (RhoGEFs). There are a large number of such RhoGEFs
• It promotes actin stability by stimulating p21-activated that are characterized by having a Dbl homology (DH) do-
kinase (PAK) to phosphorylate LIM kinase, which main. The term Dbl comes from diffuse B-cell lymphoma,
phosphorylates cofilin to inhibit its ability to cut actin. which is the cell line where the first RhoGEF was identi-
• It promotes actin remodelling by acting on in- fied. The family of Dbl-containing GEFs has now expan-
sulin receptor substrate p53 (IRSp53), which activates ded to 69 members, some of which are shown in Mod-
Wiskott-Aldrich syndrome protein (WASP) verprolin ule 2: Table monomeric G protein toolkit. These GEFs are
homologous (WAVE) to co-ordinate the activity of the not necessarily specific for Rho: some are rather promis-
actin-related protein 2/3 complex (Arp2/3 complex) that cuous and will also function as Rac and Cdc42 GEFs.
initiates actin polymerization (Module 4: Figure actin In the case of Rho, ephexin appears to be specific for
remodelling). Such a role is evident in neurons during coupling ephrin receptors to Rho (Module 2: Figure Rho
spine morphogenesis (Module 10: Figure postsynaptic signalling). Also leukaemia-associated RhoGEF (LARG),
density) and during the Ca2+ and synaptic plasticity p115-RhoGEF and PDZ-RhoGEF are specific for

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r13

Module 2: Figure Rho signalling

Rho responses
CaM
MLCK
s
Stimulu Stimulus Myosin II
MLC P
Contraction
P
PPI
GPCR MYPT1
PTKR
PIP
3 α12/1 βγ P
PPI
3
MYPT1
TK
TK

ROK
Net1 LARG p115-RhoGEF
Ephexin
PDZ-RhoGEF
LIM-K1 Cofilin
PtdIns4P Actin
GTP GDP assembly
P
Rho Rho PtdIns4P Cofilin
GDP GTP 5-kinase
Actin
PtdIns4,5P2 polymerization
DLC1 p190Rho
GAPs GAP
mDia
Profilin
Actin-Profilin

Function of the Rho monomeric G protein in cell signal transduction.

Rho is a typical G protein that is activated when GDP is exchanged for GTP. This exchange is facilitated by a number of Rho guanine nucleotide exchange
factors (RhoGEFs). For example, the ephexins mediate the action of protein tyrosine-linked receptors (PTKRs), such as the ephrin receptors, whereas
the G protein-coupled receptors (GPCRs) use the α12/13 subunit of heterotrimeric G proteins to activate leukaemia-associated RhoGEF (LARG),
p115-RhoGEF or PDZ-RhoGEF. The activated Rho/GTP complex then activates a number of signalling systems as outlined in the text.

coupling G protein-coupled receptors (GPCRs) to Rho • Activation of uropod contraction during neutrophil
activation. Net-1, which appears to be sensitive to chemotaxis (Module 11: Figure neutrophil chemotactic
PtdIns3,4,5P3 (PIP3 ), is normally found in the nucleus, signalling).
but translocates to the cytoplasm, where it functions to • Physical interactions between endothelial cells and
activate RhoA. Another important GEF is Ect2, which is the extracellular matrix (ECM) activate p190RhoGAP
also found in the nucleus during interphase, but then as- that is responsible for the mechanosensitive control of
sociates with microtubules during mitosis, where it stim- VEGF receptor expression.
ulates the activation of Rho cytokinesis (Module 9: Figure
cytokinesis). Cdc42 signalling mechanisms
The activated Rho/GTP complex is able to stimulate a The Cdc42 signalling mechanism has an important role
number of signalling processes, many of which are dir- in controlling the actin cytoskeleton. It regulates some of
ected towards remodelling of actin and its contraction. the processes that function in actin assembly (Module 2:
Many of the actions of Rho are carried out through a Rho Figure Cdc42 signalling). Like other G proteins, Cdc42
kinase (ROK). Diaphanous-related forming 1 (Dia1) is one functions as a binary switch. It is inactive when bound
of the downstream effectors that responds to Rho/GTP to GDP, but when this GDP is exchanged for GTP, the
by interacting with profilin to increase actin polymeriz- Cdc42/GTP complex becomes active. External stimuli ac-
ation. tivate this switch using different guanine nucleotide ex-
The following are some examples of the function of Rho change factors (GEFs), such as intersectin-long (ITSN-
signalling: L) and PAK-interacting exchange factor α (α-Pix). Just
how these Cdc42 RhoGEFs are activated is still not prop-
• Activation of smooth muscle contraction (Module 7: erly understood. In the case of the Ephrin (Eph) receptor
Figure smooth muscle cell E-C coupling). signalling pathway, intersectin is activated by binding to
• Assembly of the actomyosin contractile ring and the EphA receptor (Module 1: Figure Eph receptor sig-
activation of contraction during cytokinesis (Module nalling). The activated Cdc42/GTP complex then stimu-
9: Figure cytokinesis). lates various elements that regulate actin polymerization.
• Endothelial cell contraction to open the permeability It promotes actin stability by stimulating p21-activated
barrier (Module 7: Figure endothelial cell contraction). kinase (PAK) to phosphorylate LIM kinase, which phos-
• Assembly of the actin ring in osteoclast podosomes phorylates cofilin to inhibit its ability to cut actin. One
(Module 7: Figure osteoclast podosome). of its main actions is to stimulate the Wiskott-Aldrich
• Eph receptor-induced growth cone collapse in develop- syndrome protein (WASP) that acts on the actin-related
ing neurons (Module 1: Figure Eph receptor signalling). protein 2/3 complex (Arp2/3 complex) to initiate actin

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Module 2: Table monomeric G protein toolkit

Summary of the monomeric G proteins with their guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs).
Monomeric G protein, GEF or GAP Comments
Monomeric G proteins (approximately 150 members)
Ras family (36 members) Contribute to the activation of multiple signalling pathways (Module 2: Figure Ras
signalling)
H-Ras
K-Ras
N-Ras
R-Ras
RalA
RalB
Rap1
Rap2
Rho family (22 members)
Rho Primary function is cytoskeletal remodelling (Module 2: Figure Rho signalling)
RhoA
RhoD
RhoE
RhoF
RhoH
RhoV
Mito Rho
Rho BTB
Rac Cytoskeletal remodelling and ROS signalling (Module 2: Figure Rac signalling)
Rac1
Rac2
Rac3
Cdc42 Primary function is cytoskeletal remodelling (Module 2: Figure Cdc42 signalling)
Rab family A large family of GTPases that function in membrane trafficking
Rab1–Rab35
Arf family
Arf1–Arf6 ADP-ribosylation factors function in membrane trafficking and can activate
phospholipase D (Module 2: Figure PLD signalling)
Ran A small GTPase that functions in nucleocytoplasmic transport
Guanine nucleotide exchange factors (GEFs)
RasGEFs
SoS1 Son-of-sevenless-1
SoS2 Son-of-sevenless-2
RasGRF1 Expressed in brain, particularly hippocampus
RasGRF2
RasGRF3 Expressed in brain
RasGRP1 Expressed in T cells
RasGRP2 Expressed in neutrophils and platelets
RasGRP3 Expressed in B cells
RasGRP4 Expressed in mast cells
Rho family GEFs
The Dbl family The 69 family members have a Dbl homology domain (DH)
Dbl Diffuse B-cell lymphoma
Dbs Dbl’s big sister
Ect2 Epithelial-cell transforming gene-2 that functions in cytokinesis(Module 9: Figure
cytokinesis)
Ephexin Functions in growth cone collapse mediated by EphA receptors (Module 1: Eph
receptor signalling)
Fgd1 Facial genital dysplasia
ITSN-L Intersectin-long controls Cdc42 activity during EphB receptor signalling (Module 1:
Eph receptor signalling)
Kalirin Functions to activate Rac during the action of Eph receptors on dendritic spine
morphogenesis (Module 1: Figure Eph receptor signalling)
LARG Leukaemia-associated RhoGEF
NET1 Neuroepithelioma transforming gene 1
p115-RhoGEF Links G protein-coupled receptors to Rho
PDZ-RhoGEF Links G protein-coupled receptors to Rho
P-Rex1 PtdIns3,4,5P3 -dependent Rac exchanger 1
α-Pix PAK-interacting exchange factor α
β-Pix PAK-interacting exchange factor β
Tiam1 T cell lymphoma invasion and metastasis-1
Tiam2 T cell lymphoma invasion and metastasis-2
Trio
Vav1
Vav2
Vav3
Arf GEFs
Cytohesin 1
Cytohesin 4
ARNO/Geα2

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Module 2: Table continued

Monomeric G protein, GEF or GAP Comments
GTPase-activating proteins (GAPs)
Ras GAPs
p120RasGAP
Neurofibromin This tumour suppressor is lost in the inherited disorder neurofibromatosis type 1
SynGAP
GAP1m
GAP1IP4 BP
Ins1,3,4,5P4 binds to this protein, causing it to dissociate from the plasma membrane
CAPRI Ca2+ -promoted Ras inactivator
RASAL Ras GTPase-activating-like
DAB2IP
Miro Mitochondrial Rho-GTPase functions in mitochondrial motility (Module 5: Figure
mitochondrial motility)
Rac GAPs
3BP-1 3BP-1 inactivates Rac.GTP by enhancing GTP hydrolysis (Module 2: Figure Rac
signalling)
Rho GAPS
DLC1 (p122RhoGAP in mice) Deleted in liver cancer 1 is altered in many tumours. It regulates actin formation in
adhesion complexes (Module 6: Figure integrin signalling)
p190-RhoGAP Inhibits Rho activity during neutrophil chemotaxis (Module 11: Figure neutrophil
chemotactic signalling)
Since some of the families are very large, only representative members have been included.

Module 2: Figure Cdc42 signalling

ulus Stimulus
Stim

PTKR GPCR Cdc42 responses

PIP
3
βγ Cofilin
?
TK
TK

P
ITSN-L Dbs
α-Pix GEFs
PAK4 LIM-K1 Cofilin

Cdc42 GTP GDP Cdc42

GDP GTP
WASP Actin
assembly
Arp2/3
GAPs Actin
IRS p53
polymerization

Actin-Profilin
Profilin

Function of the Cdc42 monomeric G protein in signal transduction.

When bound to GDP, Cdc42 is inactive, but it is activated when the GDP is exchanged for GTP. This exchange is accelerated by guanine nucleotide
exchange factors (GEFs), but how these are activated is still somewhat of a mystery. The primary action of the Cdc42/GTP complex is to stimulate
actin assembly by inhibiting the action of cofilin, by promoting actin polymerization by acting on actin/profilin and Wiskott–Aldrich syndrome protein
(WASP). Further details on the role of WASP are shown in Module 4: Figure actin remodelling.

polymerization (Module 4: Figure actin remodelling). This cycle control, transcription and apoptosis. There are six
action is facilitated by Cdc42 acting on profilin through in- isoforms (PAK1–PAK6) with slightly different functions.
sulin receptor substrate p53 (IRSp53) and Mena. Rac acts through LIM kinase 1 (LIMK1) to phosphorylate
cofilin (Module 2: Figure Rac signalling). Rho acts through
p21-activated kinase (PAK) PAK4 to phosphorylate the same LIMK1 (Module 2: Fig-
The p21-activated kinases (PAKs) are some of the major ure Cdc42 signalling). Another function of PAK1 is to
downstream targets of Rac and Rho (Module 2: Figure phosphorylate myosin light chain kinase (MLCK), res-
Rho-regulated kinases). They have been implicated in a ulting in a decrease in the activity of the actin–myosin
large number of processes such as actin remodelling, cell contractile system.

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Module 2: Figure Rho-regulated kinases

Rac Cdc42 LIMK1/2

GTP GTP MLCK

PBD Kinase
P21-activated kinase (PAK)
Kinase PBD

Cdc42
MYPT1 GTP
CRD

Myotonic dystrophy Kinase CC1 CC2 3 PH CH PBD

kinase-related Cdc42-binding
kinase (MRCK) Kinase CC1 CC2 3 PH CH PBD

LIMK1/2 Rho
FilGAP GTP
CRD

Kinase CC PBD PH
Rho kinase (ROK)
Kinase CC PBD PH

Structure of Rho GTPase-regulated kinases.

The Rho family exert many of their actions by stimulating protein kinases such as PAK, MRCK and ROK. These kinases exist as homodimers with
PAK being arranged in a head-to-tail manner, whereas MRCK and ROK have a parallel alignment. The GTP-bound form of these p21 monomeric
G proteins bind to a p21-binding domain (PBD). CC, coiled-coil domain: CH, citron homology domain; CRD, cysteine-rich domain; FilGap, filamin
GTPase-activating protein; MLCK, myosin light chain kinase; PH, pleckstrin homology domain.

The fragile X mental retardation protein 1 RMRP1), in non-muscle cells. The activity of myosin II is regulated
which is mutated in fragile X syndrome (FXS), may by the phosphorylation status of the myosin light chain
act to inhibit the function of PAK particularly during (MLC) that is associated with the myosin head. When
actin remodelling in neuronal spines (Module 10: Figure MLC is dephosphorylated, myosin is inactive, but when it
Ca2+ -dependent synaptic plasticity). is phosphorylated by the Ca2+ -sensitive myosin light chain
kinase (MLCK), myosin can begin to interact with actin to
Myotonic dystrophy kinase-related Cdc42-binding induce contraction. ROK can influence the phosphoryla-
kinase (MRCK) tion of MLC by inhibiting the protein phosphatase 1 (PP1)
Myotonic dystrophy kinase-related Cdc42-binding kinase by phosphorylating the scaffolding protein myosin phos-
(MRCK) has a number of functional domains and be- phatase targeting subunit 1 (MYPT1) (Module 5: Table
longs to the same family as Rho kinase (ROK) (Module 2: PP1 regulatory, targeting and inhibitory subunits).
Figure Rho-regulated kinases). MRCK, which is activated
by Cdc42·GTP, may play a role in actin formation dur- Diaphanous-related formin 1 (Dia1)
ing the growth of neurites. One of the downstream tar- There are two diaphanous-related formin proteins (Dia1
gets of MRCK is the MYPT1 subunit (Module 5: Table and Dia2) that belong to a formin-related family. They con-
PP1 regulatory and inhibitory subunits and proteins) that tain three formin homology domains that are used to bind
binds protein phosphatase 1δ (PP1δ) and which dephos- to various effectors. Dia1 is activated by the Rho signalling
phorylates the myosin light chain (MLC). mechanism and functions to control actin polymarization
by binding to profilin (Module 2: Figure Rho signalling).
Rho kinase (ROK) Dia1 has also been implicated in the control of poly-
Rho kinase (ROK) is a serine/threonine kinase that phos- cystin 2.
phorylates key regulators of actin and myosin function
(Module 2: Figure Rho-regulated kinases). ROK acts to
stabilize actin by phosphorylating LIM, which then phos- Ca2+ signalling
phorylates cofilin to prevent it from severing actin. Rho Ca2+ signalling is one of the major signalling systems in
also acts on diaphanous-related formin protein (Dia), cells (Module 2: Figure cell signalling pathways). It func-
which belongs to a formin-related protein family that in- tions to regulate many different cellular processes through-
teracts with profilin to promote actin polymerization. out their life history. It triggers new life at the time of
The other major function of ROK is to control contrac- fertilization. It controls many processes during develop-
tion by activating myosin II in smooth muscle cells and ment, and once cells have differentiated, it governs the

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Module 2: Figure basic Ca2+ signalling mechanism

STIMULUS Time
domain
Exocytosis µs

ON REACTIONS
Contraction ms
2+ Metabolism sec
Ca
2+ Ca Gene
100 nM 500nM
OFF REACTIONS transcription min
Fertilization
Proliferation hr
Hypertrophy

The basic mechanism of Ca2+ signalling.

The concentration of Ca2+ in cells at rest is approximately 100 nM, but this increases to 500 nM or more following a stimulus that activates the Ca2+
ON reactions. When the stimulus is removed, the Ca2+ OFF reactions return the concentration of Ca2+ to its resting level. Ca2+ is a universal signal
capable of activating many different cellular processes operating over a very wide time domain.

activity of most cellular processes, effectively determining effectors that are responsible for translating Ca2+ signals
how we metabolize, secrete, move and think. There also is into a change in cellular activity.
a darker side to its action, because larger than normal elev-
ations can cause cell death, either in the controlled manner Basic mechanism of Ca2+ signalling
of programmed cell death (apoptosis) or in the more cata- Cells at rest maintain a low intracellular concentration
strophic necrotic changes that occur during processes such of Ca2+ (approximately 100 nM), but this increases rap-
as stroke or cardiac ischaemia. idly into the micromolar range when cells are stimulated
The basic mechanism of Ca2+ signalling is relatively (Module 2: Figure basic Ca2+ signalling mechanism). This
simple in that it depends upon an increase in the intracel- increase in intracellular Ca2+ can operate over a very wide
lular concentration of this ion. The Ca2+ concentration is time domain (e.g. microseconds to hours) to regulate many
low when cells are at rest, but when an appropriate stimu- different cellular processes. This very wide temporal range
lus arrives, there is a sudden elevation, which is responsible of Ca2+ signalling is an intrinsic property of the Ca2+ sig-
for a change in cellular activity. However, there are multiple nalling modules.
variations of this relatively simple theme. The versatility An important feature of Ca2+ signalling is its dynamic
of Ca2+ signalling is achieved by having an extensive Ca2+ nature, as exemplified by the fact that Ca2+ signals in-
toolkit from which a large number of Ca2+ signalling sig- variably appear as a brief transient. The rising phase of
nalsomes are assembled. This large toolkit contains many each transient is produced by the ON reactions, whereas
different components that can be mixed and matched to the falling phase depends on the OFF reactions (Module
create many different Ca2+ signalling modules. There are 2: Figure Ca2+ signalling dynamics). At any moment, the
Ca2+ entry channels, which control the entry of Ca2+ from level of Ca2+ is determined by the balance between the
the outside. There are Ca2+ release channels, which control Ca2+ ON reactions that increase Ca2+ and the Ca2+ OFF
the release of Ca2+ from internal stores. The Ca2+ buffers reactions that remove it from the cytosol. An import-
ensure that the concentration of Ca2+ remains within its ant aspect of the ON and OFF reactions is their spa-
operation range and does not rise to levels that can induce tial location. An example of this spatial organization is
cell death. Once Ca2+ has carried out its signalling func- the ER/mitochondrial Ca2+ shuttle (Module 5: Figure
tion, there are Ca2+ pumps and exchangers that remove ER/mitochondrial shuttle), where events at the ER are
it from the cytoplasm by either extruding it from the cell closely linked to those in the mitochondria. Another im-
or returning it to the internal stores. Ca2+ signalling func- portant spatial aspect is that the ON reactions are often
tions are carried out by various Ca2+ sensors and Ca2+ closely associated with the effector systems that respond

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Module 2: Figure Ca2+ signalling dynamics

STIMULUS 2+
[ Ca ]
PMCA

2nd [Ca 2+ ]
messengers Resting
+ +
SERCA

ER

Na + Na +
Mitochondria

2+
[Ca 2+ ] Ca
Activated
Buffers

ON REACTIONS Effectors OFF REACTIONS

The dynamics of Ca2+ signalling.

The dynamics of Ca2+ signalling are governed by an interplay between the ON and OFF reactions that control the fluxes of Ca2+ across both the
plasma membrane and the internal organelles such as the endoplasmic reticulum (ER) and mitochondria. External stimuli activate the ON reactions,
which introduce Ca2+ into the cytoplasm either through channels in the plasma membrane or from internal stores such as the ER. Most cells make use
of both sources, but there are examples of cells using either external or internal sources to control specific processes. Most of the Ca2+ that enters
the cytoplasm is adsorbed on to buffers, while a much smaller proportion activates the effectors to stimulate cellular processes. The OFF reactions
remove Ca2+ from the cytoplasm using a combination of mitochondria and different pumping mechanisms. When cells are at rest, these OFF reactions
keep the concentration low, but these are temporarily overwhelmed when external stimuli activate the ON reactions. Sequential activation of the ON
and OFF reactions gives rise to the Ca2+ transients (Module 2: Figure Ca2+ transient mechanisms), which are such a characteristic feature of Ca2+
signalling systems. PMCA, plasma membrane Ca2+ -ATPase; SERCA, sarco/endo-plasmic reticulum Ca2+ -ATPase.

to Ca2+ . For example, voltage-operated channels (VOCs) it will require to control its particular functions (Mod-
in presynaptic endings are associated with the synaptic ule 8: Figure signalsome expression). There are an enorm-
vesicles, thus producing a highly localized puff of Ca2+ to ous number of cell-specific Ca2+ signalsomes (Module
trigger exocytosis (Module 4: Figure Ca2+ -induced mem- 2: Figure cell-specific Ca2+ signalsomes). The important
brane fusion). Similarly, the type 2 ryanodine receptors point is that each signalsome generates a cell-specific Ca2+
(RYR2s) of cardiac cells are lined up close to the contract- signal with characteristic spatial and temporal properties.
ile filaments to ensure that Ca2+ will rapidly stimulate A signalsome is defined here as the collection of sig-
contraction. nalling components that make up each cell-specific sig-
In cases where cells need to be stimulated over a long nalling system. The Ca2+ signalsomes of different cell types
time, these transients are repeated at set intervals to set up often display recurring themes in the form of Ca2+ sig-
Ca2+ oscillations. These oscillations are part of the spati- nalling modules (Module 2: Figure Ca2+ modules).
otemporal aspects of Ca2+ signalling.
Ca2+ signalling modules
Ca2+ signalling signalsome The Ca2+ signalling system in specific cell types is often
Cells have access to a large Ca2+ signalling toolkit (Mod- not a single entity, but is made up of distinct modules,
ule 2: Table Ca2+ signalling toolkit). Many of the toolkit which are mixed and matched to produce the cell-specific
components have similar functions (Module 2: Figure Ca2+ systems found in different cell types. Some of the main
signalling toolkit), which represents a generic Ca2+ sig- modules used by cells are summarized in Module 2: Figure
nalling system. In reality, however, each cell type has a Ca2+ modules:
clearly defined subset of toolkit components that will be
referred to as a Ca2+ signalling signalsome. These cell- 1. Agonists such as the neurotransmitters glutamate
specific signalsomes are put in place during development and ATP act directly on receptor-operated channels
when a process of signalsome expression enables each dif- (ROCs) in the plasma membrane to allow external Ca2+
ferentiating cell to select out those signalling components to enter the cell.

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Module 2: Table Ca2+ signalling toolkit

The Ca2+ signalling toolkit.
Component Comments
RECEPTORS AND TRANSDUCERS
G protein-coupled receptors (GPCRs) See Module 1: Table G protein-coupled receptors for a list of those
GPCRs that act by stimulating phospholipase Cβ
Tyrosine-kinase-linked receptors See Module 1: Figure tyrosine kinase-linked receptors. (The receptors
shown opposite can activate phospholipase Cγ)
Platelet-derived growth factor receptor (PDGFR) See Module 1: Figure PDGFR activation
PDGFRα
PDGFRβ
Epidermal growth factor receptor (EGFR)
ERBB1–ERBB4
Vascular endothelial growth factor receptor (VEGFR) See Module 9: Figure VEGF-induced proliferation
VEGFR1–VEGFR3
G Proteins
Gαq , Gα11 , Gα14 , Gα15 , Gα16 , Gβγ These G protein subunits activate phospholipase C (Module 2: Figure
heterotrimeric G protein signalling)
Guanine nucleotide exchange factors
RasGRF1 A Ca2+ -activated GEF expressed in brain (Module 2: Figure Ras
signalling)
Regulators of G protein signalling (RGS)
RGS1, RGS2, RGS4, RGS16
Phospholipase C (PLC)
PLCβ1–4, PLCγ1–2, PLCδ1–4, PLCε, PLCζ PLC hydrolyses PtdIns4,5P2 to form InsP3 and DAG (Module 2: Figure
PLC structure and function)
ADP-ribosyl cyclase Functions to generate cADPR and NAADP (Module 2: Figure
cADPR/NAADP function)
CD38
CHANNELS
Plasma membrane channels
Voltage-operated channels (VOCs) See Module 3: Table VOC classification for further details
CaV 1.1 (L-type)
CaV 1.2 (L-type)
CaV 1.3 (L-type)
CaV 1.4 (L-type)
CaV 2.1 (P/Q-type)
CaV 2.2 (N-type)
CaV 2.3 (R-type)
CaV 3.1 (T-type)
CaV 3.2 (T-type)
CaV 3.3 (T-type)
Ca2+ -sensitive ion channels
Ca2+ -activated K+ channels See Module 3: Table properties of Ca2+ -sensitive K+ channels for
further information
SK (small conductance Ca2+ -sensitive channel)
IK (intermediate conductance Ca2+ -sensitive channel)
BK (large conductance Ca2+ -sensitive channel)
Ca2+ -activated chloride channel
HCLCA1 (human chloride channel, Ca2+ -activated)
Receptor-operated channels (ROCs) See Module 3: Table receptor-operated channel toolkit for further
details)
Nicotinic acetylcholine receptors
5-HT3 5-Hydroxytryptamine receptor
AMPA receptors α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor
NMDA receptors N-methyl-D-aspartate receptor
P2X receptors
Second messenger-operated channels (SMOCs) Module 3: Figure Ca2+ entry mechanisms
Arachidonate-regulated Ca2+ channel (IARC )
Cyclic nucleotide-gated channels (CNGs) Module 3: Figure cyclic nucleotide-gated channels
CNGA1–CNGA4, CNGB1, CNGB3
Transient receptor potential (TRP) ion channel family Module 3: Figure TRP channel phylogeny
TRPC1–TRPC7 Canonical
TRPV1–TRPV6 Vanilloid
TRPM1–TRPM8 Melastatin
Polycystins Module 3: Figure polycystin domain structure
PC1
PC2
Endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) Ca2+
release channels
InsP3 receptors (InsP3 Rs) Module 3: Figure InsP3 R structure
InsP3 R1
InsP3 R2
InsP3 R3
Ryanodine receptors (RYRs) Module 3: Figure ryanodine receptor structure
RYR1
RYR2
RYR3

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Module 2: Table continued

Component Comments
Channel regulators
Triadin
Junctin
Sorcin EF-hand protein that can bind to annexin 7
FKBP12 FK506-binding protein 12 kDa
FKBP12.6 FK506-binding protein 12.6 kDa
Phospholamban Module 5: Figure phospholamban mode of action
Ca2+ BUFFERS
Cytosolic buffers
Calbindin D-28k
Calretinin
Parvalbumin
Endoplasmic reticulum (ER)/sarcoplasmic
reticulum (SR) buffers and chaperones
Calnexin
Calreticulin
Calsequestrin
GRP78 Glucose-regulatory protein of 78 kDa. Also known as BiP
GRP94 Glucose-regulatory protein of 94 kDa. Also known as endoplasmin
Ca2+ SENSORS
EF-hand Ca2+ -binding proteins
Calmodulin (CaM) A ubiquitous Ca2+ sensor
Calcineurin B (CaNB) A sensor that resembles CaM and controls the function of calcineurin
(Module 4: Figure calcineurin)
Troponin C (TnC) The Ca2+ sensor in striated muscle
Miro Mitochondrial Rho-GTPase functions in mitochondrial motility (Module
5: Figure mitochondrial motility)
DAG kinase α A Ca2+ -sensitive enzyme that phosphorylates diacylglycerol (DAG)
(Module 2: Figure InsP3 /DAG recycling)
Stromal interaction molecule (STIM) Senses Ca2+ in the ER lumen (Module 3: Figure STIM-induced Ca2+
entry)
S100 proteins
S100 protein clustered on chromosome 1 S100A1-S100A14 are all clustered on 1q21 (Module 4: Figure S100
phylogenetic tree)
S100A1
S100A2
S100A3
S100A4
S100A5
S100A6
S100A7
S100A8
S100A9
S100A10
S100A11
S100A12
S100A13
S100A14
S100 protein clustered on other chromosomes
S100B
S100C
S100P
Neuronal Ca2+ sensor proteins (NCS) A family of 14 EF-hand Ca2+ -binding proteins
NCS-1 Brain and retina
Hippocalcin Restricted to hippocampal neurons
Neurocalcin δ Brain and retina
Recoverin Retina
Visinin-like proteins (VILIPs)
VILIP-1 Brain and retina
VILIP-2 Retina
VILIP-3 Retina and cerebellar Purkinje cells
Guanylyl cyclase-activating proteins (GCAPs) Module 10: Figure phototransduction
GCAP1 Retina
GCAP2 Retina
GCAP3 Retina
Kv-channel-interacting proteins (KChIPs)
KChIP1 Brain
KChIP2 Brain
KChIP3/downstream regulatory element modulator This protein has three names which are currently in use
(DREAM)/calsenilin
KChIP4 Brain

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Module 2: Table continued

Component Comments
Ca2+ -binding proteins (CaBPs)
Caldendrin Brain and retina
L-CaBP1 A long splice variant of caldendrin
S-CaBP1 A short splice variant of caldendrin
CaBP2 Retina
CaBP3 Retina
CaBP4 Brain and retina
CaBP5 Retina
C2 domain Ca2+ -binding proteins
Synaptotagmins The Ca2+ sensors that control exocytosis (Module 4: Figure
Ca2+ -induced membrane fusion)
Synaptotagmin I
Synaptotagmin II
Synaptotagmin III
Annexins Ca2+ -dependent phospholipid-binding proteins (Module 4: Figure
annexin structure)
Annexin A1
Annexin A2
Annexin A3
Annexin A4
Annexin A5
Annexin A6
Annexin A7
Annexin A8
Annexin A9
Annexin A10
Annexin A11
Annexin A13
Ca2+ -SENSITIVE ENZYMES AND PROCESSES
Ca2+ -regulated enzymes
Ca2+ -dependent protein kinases (CaMKs)
CaMKI
CaMKII
CaMKIII
CaMKIV
Myosin light chain kinase (MLCK)
Phosphorylase kinase
Ins1,4,5P3 3-kinase
Proline-rich tyrosine kinase 2 (Pyk2) A Ca2+ -sensitive tyrosine kinase that functions in osteoclast podosomes
(Module 7: Figure osteoclast podosome)
Lipid kinase
hVps34 See Module 9: Figure target of rapamycin signalling
PKC-α
PKC-βI
PKC-βII
PKC-γ
Phosphodiesterases
Cyclic GMP phosphodiesterase (PDE) Some of the phosphodiesterases are sensitive to Ca2+ see Module 5:
Table PDE family properties
PDE1A
PDE1B
PDE1C
Adenylyl cyclases (ACs) See Module 2: Table adenylyl cyclases for details of these
Ca2+ -sensitive enzymes
AC-1
AC-III
AC-VIII
AC-V
AC-VI
Dual oxidases (DUOX1–2) See Module 2: Table redox signalling components
Nitric oxide synthase (NOS)
Module 2: Figure NO and cyclic GMP signalling
Endothelial NOS (eNOS)
Neural NOS (nNOS)
Ca2+ -activated proteases
Calpain I
Calpain II
Transcription factors
Nuclear factor of activated T cells (NFAT) Module 4: Figure NFAT activation
NFATc1
NFATc2
NFATc3
NFATc4
Cyclic AMP response element-binding protein (CREB) Module 4: Figure CREB activation
Downstream regulatory element modulator (DREAM)
CREB-binding protein (CBP)

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Module 2: Table continued

Component Comments
Ca2+ PUMPS AND EXCHANGERS
Na+ /Ca2+ exchanger (NCX) See Module 5: Table Ca2+ pumping toolkit for further details
NCX1
NCX2
NCX3
Na+ /Ca2+ /-K+ exchanger (NCKX)
NCKX1
NCKX2
NCKX3
NCKX4
Mitochondrial channels and exchangers Module 5: Figure mitochondrial Ca2+ signalling
Permeability transition pore (PMT)
Na+ /Ca2+ exchanger
Ca2+ uniporter
Plasma membrane Ca2+ -ATPases (PMCAs) See Module 5: Table Ca2+ pumping toolkit for further details
PMCA1
PMCA2
PMCA3
PMCA4
Sarco/endo-plasmic reticulum Ca2+ -ATPases (SERCAs) See Module 5: Table Ca2+ pumping toolkit for further details
SERCA1
SERCA2
SERCA3
Secretory-pathway Ca2+ -ATPase (SPCA) pumps
SPCA1 Ubiquitous; located in the Golgi
SPCA2
The Ca2+ signalling system has a very large toolkit of signalling components. The ways in which the major components are organized are
summarized in Module 2: Figure Ca2+ signalling toolkit. Components from this toolkit can be mixed and matched to create a diverse
array of cell-specific Ca2+ signalsomes (Module 2: Figure cell-specific Ca2+ signalsomes) that are capable of delivering Ca2+ signals
with very different spatial and temporal properties.

Module 2: Figure cell-specific Ca2+ signalsomes

SKELETAL CARDIAC Ca1

MUSCLE CELL ATRIAL CELL NEURON T CELL

Receptors ET-1R/α1R mGluR1 TCR

AngIIR M1
PLC PLCβ PLCβ PLCγ1
Entry CaV 1.1 Ca V 1.2 Ca V1.2/ Ca V2.1
channels Orai1
Ca V 2.2/ NMDAR

Release RYR1 RYR2 RYR2

channels InsP3 R2 InsP3 R2 InsP3 R1

PMCAs PMCA1a, PMCA1c, PMCA1a, 2a PMCA4b

1c,1d 1d,2a 3a
SERCAs SERCA1a, 1b SERCA2a SECA2b, 3 SERCA2b, 3
Na+ /Ca 2+ NCX NCX1 NCX1, 3 –
exchanger
Parvalbumin
Buffers Parvalbumin
Calbindin 28K
Troponin c Troponin c Calmodulin Calmodulin
Sensors
Calmodulin Calmodulin

Some examples of cell-specific Ca2+ signalling signalsomes.

The four cell types represented here generate Ca2+ signals with very different spatial and temporal properties. For example, the skeletal muscle
signalsome selects out those components specialized to deliver rapid pulses of Ca2+ to activate contraction, whereas the T cell signalsome has
different components that generate the much slower repetitive pulses of Ca2+ necessary to stimulate cell proliferation.

2. Second messengers such as diacylglycerol (DAG), 3. Membrane depolarization (
V) activates voltage-
cyclic AMP, cyclic GMP and arachidonic acid act- operated channels (VOCs) in the plasma membrane to
ing from the cytoplasmic side open second mes- allow a rapid influx of external Ca2+ .
senger-operated channels (SMOCs) in the plasma 4. Membrane depolarization (
V) activates a specific
membrane. VOC isoform, the CaV 1.1 L-type channel, that

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Module 2 : Figure Ca2+ signalling toolkit

Stimulus 2+ 2+ 2+ 2+
Ca Ca Ca Ca
VOC ROC SOC SMOC
Receptors
PLC

G proteins
InsP3

Cellular responses
ER/SR InsP3R
PMCA SERCA

Ca
2+
Ca
2+
Ca2+
2+ RYR Sensors
Ca CaBP
Effectors
NCX

Uniporter
2+
Ca
Mitochondrion

Summary of the major components that contribute to the Ca2+ signalling signalsome.
A generic Ca2+ signalling signalsome is made up from components of the extensive Ca2+ signalling toolkit. The duplication of the individual
components represents the fact that there are numerous isoforms that further enhance the diversity of the Ca2+ signalling systems. The yellow arrows
illustrate the ON reactions that introduce Ca2+ into the cell, and the blue arrows depict the OFF reactions that pump Ca2+ either out of the cell or back
into the endoplasmic reticulum (ER). During its passage through the cytoplasm, Ca2+ resides temporarily on the buffers or within the mitochondria.
To carry out its signalling function, Ca2+ binds to sensors that then employ a range of effectors to stimulate cellular processes. These different
components are mixed and matched to construct Ca2+ modules (Module 2: Figure Ca2+ modules) that are then assembled to produce cell-specific
signalsomes. CaBP, Ca2+ -binding protein; InsP3 R, inositol 1,4,5-trisphosphate receptor; NCX, Na+ /Ca2+ exchanger; PLC, phospholipase C; PMCA,
plasma membrane Ca2+ -ATPase; ROC, receptor-operated channel; RYR, ryanodine receptor; SERCA, sarco/endo-plasmic reticulum Ca2+ -ATPase;
SMOC, second messenger-operated channel; SOC, store-operated channel; VOC, voltage-operated channel.

activates the ryanodine receptor 1 (RYR1) in skeletal release channels to create Ca2+ signals with markedly dif-
muscle through a direct conformational-coupling ferent spatial and temporal properties.
mechanism. The entry of Ca2+ across the plasma membrane is carried
5. Membrane depolarization (
V) activates voltage- out by many different channels whose names indicate how
operated channels (VOCs) in the plasma membrane to they are opened:
allow a rapid influx of external Ca2+ (see Module 3)
• Voltage-operated channels (VOCs)
to provide a Ca2+ trigger that then activates the
• Agonist-operated channels (AOCs)
ryanodine receptor 2 (RYR2) to release Ca2+ stored
• Receptor-operated channels (ROCs)
in the sarcoplasmic reticulum (SR) through a process of
• Second messenger-operated channels (SMOCs)
Ca2+ -induced Ca2+ release (CICR). This mechanism is
• Store-operated channels (SOCs)
found in cardiac muscle and neurons.
6. Agonists acting on cell-surface receptors generate inos- Release of Ca2+ from internal stores is carried out by
itol 1,4,5-trisphosphate (InsP3 ) (Module 2: Figure InsP3 different types of channels and control mechanisms:
and DAG formation), which then diffuses into the cell
to activate the InsP3 receptor (InsP3 R) to release Ca2+ • Ryanodine receptors (RYRs)
from the ER. • InsP3 receptors (InsP3 Rs)
• NAADP control of Ca2+ release
Ca2+ ON reactions
One of the major problems in Ca2+ signalling has been
In response to external stimuli, channels in the plasma
to determine how stimuli arriving at the cell surface gain
membrane or ER are opened, and Ca2+ flows into the
access to these internal stores. Two main mechanisms have
cytoplasm to bring about the elevation of cytosolic Ca2+
been identified:
responsible for cell activation (Module 2: Figure Ca2+ sig-
nalling dynamics). During these ON reactions, the cell 1. Conformational coupling through a protein–protein
employs a variety of both Ca2+ entry channels and Ca2+ interaction. This is a very fast mechanism that depends

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Module 2: Figure Ca2+ modules

AGONIST
2+
1 Ca
ROC

Second messengers
2 SMOC
2+
Ca

VOC
2+
Ca
3
∆V

RYR1

SR
4
∆V Ca
2+

2+ RYR2
Ca
SR
5
∆V Ca
2+

InsP InsP R
3 3
AGONIST
ER

6 2+
Ca

Ca2+ signalling modules.

Some of the main Ca2+ signalling modules that cells employ to generate Ca2+ signals are depicted. Ca2+ is either derived from the external medium
or released from internal stores such as the sarcoplasmic reticulum (SR) in muscle or the endoplasmic reticulum (ER) in other cell types. The ion
channels responsible for gating Ca2+ in these different modules are explained in the text.

upon a sensor in the plasma membrane interacting dir- ant Ca2+ -mobilizing messengers is Ca2+ itself, which
ectly with an internal release channel. The receptor on is a potent activator of the two main internal re-
the cell surface is the CaV 1.1 L-type channel (a voltage lease channels, the ryanodine receptors (RYRs) and the
sensor), which is coupled to the type 1 ryanodine inositol 1,4,5-trisphosphate receptors (InsP3 Rs). This
receptor (RYR1)(Ca2+ module 4 in Module 2: Fig- Ca2+ -induced Ca2+ release (CICR) mechanism has the
ure Ca2+ modules). Information is transferred through unique property of being autocatalytic and plays a
a process of conformational coupling. This mechan- central role in generating those Ca2+ signals that ap-
ism is restricted to skeletal muscle (Module 7: Figure pear as regenerative Ca2+ waves (Module 2: Figure
skeletal muscle E-C coupling) and perhaps also to some Ca2+ -induced Ca2+ release).
neurons.
2. Generation of diffusible second messengers. Activa- Another classical process for releasing internal Ca2+
tion of receptors or channels on the cell surface gen- is the inositol 1,4,5-trisphosphate (InsP3 )/Ca2+ signalling
erate second messengers that then diffuse into the cell cassette (Ca2+ module 6 in Module 2: Figure Ca2+
to activate release channels. One of the most signific- modules). Other Ca2+ -mobilizing messengers have been

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r25

Module 2: Figure Ca2+ -induced Ca2+ release

Stimulus

VOC ROC

Ca2+ InsP3
Intracellular CICR
calcium wave

+
+
+

+
CICR CICR
+

+
+
Spark Puff
R R R I I I
Blink

+
+
+
+

Ca2+ ER

The role of Ca2+ -induced Ca2+ release (CICR) in mobilizing Ca2+ from internal stores.
The Ca2+ -sensitive channels are the ryanodine receptors (R) and the InsP3 receptors (I) located on the endoplasmic reticulum (ER). CICR has two
important functions. Firstly, it provides a mechanism for transferring information from the plasma membrane to these internal release channels. The
primary mechanism is based on the voltage-operated channels (VOCs) that open in response to membrane depolarization to allow a small amount
of Ca2+ to enter, which then diffuses into the cell to activate either R or I. The other important function of CICR is to link together these intracellular
channels so that the Ca2+ being released from one channel diffuses across to neighbouring channels that are excited to release further Ca2+ , thereby
setting up regenerative waves (yellow arrows).

described such as sphingosine 1-phosphate (S1P), cyclic in one region of the cell (the initiation site) propagates
ADP ribose (cADPR) and nicotinic acid–adenine dinuc- throughout the rest of the cytoplasm as a regenerative Ca2+
leotide phosphate (NAADP). wave. Waves can progress by recruiting either RYRs or in-
The channels responsible for these Ca2+ ON reactions ositol 1,4,5-trisphosphate receptors (InsP3 Rs) (Module 2:
usually have powerful inactivation mechanisms that rap- Figure Ca2+ -induced Ca2+ release). These Ca2+ waves are
idly curtail the entry or release processes to prevent the cell made up of elementary Ca2+ events such as the sparks and
being swamped with Ca2+ , which can result in cell stress puffs produced by the RYRs and InsP3 Rs respectively. It
and apoptosis. Once the ON reactions have been curtailed, is these unitary events that are used to generate the regen-
the Ca 2+ OFF reactions rapidly take over to return the erative waves that make up global Ca2+ signals.
activated level of Ca2+ back to its resting level.

Ca2+ -induced Ca2+ release (CICR) Ca2+ OFF reactions

A process of Ca2+ -induced Ca2+ release (CICR) plays a Cells use a variety of mechanisms to remove Ca2+ from
central role in the way Ca2+ signals are generated. This the cytoplasm (Module 2: Figure Ca2+ signalling dynam-
positive-feedback mechanism whereby Ca2+ triggers its ics). The introduction of Ca2+ into the cell during the
own release has two important functions in cells. It en- Ca2+ ON reactions usually occurs for a relatively brief
ables Ca2+ entering across the plasma membrane to func- period during which there is a rapid increase in the in-
tion as a messenger to release Ca2+ from the internal store tracellular concentration of Ca2+ . In fact, the increase in
(Module 2: Figure Ca2+ -induced Ca2+ release). This func- free Ca2+ that can be measured in the cytoplasm using
tion of CICR was first described in cardiac cells, where the aequorin or fluorescent indicators is a very small propor-
CaV 1.2 L-type channel provides an influx of trigger Ca2+ tion of the total amount of Ca2+ that enters during the
that then diffuses into the cell to activate the ryanodine re- ON reactions. Much of this Ca2+ is rapidly bound by the
ceptor 2 (RYR2) (Ca2+ module 5 in Module 2: Figure Ca2+ cytosolic buffers or is taken up by the mitochondria. As
modules). A similar interaction is particularly evident for the Ca2+ concentration returns to its resting level, Ca2+
neuronal Ca2+ entry and release channels. leaves the buffers and the mitochondria and is returned to
The other main function of CICR is to set up the ER or is pumped out of the cell resulting in a brief Ca2+
intracellular Ca2+ waves where an elevated level of Ca2+ transient.

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Module 2: Figure Ca2+ transient mechanisms

Ca2+ entry
Ca2+ release
Ca 2+ buffers
Mitochondria
Na+ /Ca 2+ exchanger
Ca 2+ pumps
(SERCA/PMCA)

ON OFF
Reactions Reactions

CALCIUM TRANSIENT

The sequence of ON and OFF reactions during the generation of a typical Ca2+ transient.
The rising phase of the Ca2+ spike results from the activation of Ca2+ entry and release mechanisms (yellow bars), which are then terminated by
inactivation processes. Once the ON reactions have been inactivated, a series of OFF reactions operate in a sequential manner to restore Ca2+ to
its resting level (blue bars). During the rising phase of the Ca2+ transient, large amounts of Ca2+ are rapidly bound to the Ca2+ buffers (calbindin
D-28k and parvalbumin) and are taken up by the mitochondria. The mitochondria and cytosolic buffers help to shape the Ca2+ signal by reducing the
impact of the ON reactions. In effect, they enable the cell to generate very fast transients without running the risk of being overwhelmed by Ca2+ .

The recovery process thus depends upon a complex in- Ca2+ within a fairly narrow range. This calciostat is a dy-
terplay between cytosolic Ca2+ buffers, mitochondria and namic system in that the OFF reactions are operating con-
Ca2+ pumps and exchangers on the internal stores and on tinuously to reverse the constant basal rate of Ca2+ entry
the plasma membrane (Module 2: Figure Ca2+ signalling through various ‘Ca2+ leak pathways’ that remain poorly
dynamics). These Ca2+ OFF reactions operate at different defined. There is increasing evidence that presenilins may
stages during the recovery phase of a typical Ca2+ spike. function as such a leak channel and this has been incorpor-
The buffers and mitochondria operate early, and the Ca2+ ated into the calcium hypothesis of Alzheimer’s disease (see
pumps and exchangers are responsible for restoring the step 10 in Module 12: Figure amyloid cascade hypothesis).
status quo by pumping Ca2+ out of the cell or back into the The latter can result in an elevation of cytosolic Ca2+ if the
ER. These pumps and exchangers operate at different times OFF reactions are inhibited. For example, agents such as
during the recovery process. The Na+ /Ca2+ exchangers thapsigargin, which inhibit the SERCA pumps on the ER,
have low affinities for Ca2+ , but have very high capacities will result in an increase in Ca2+ concentration. Indeed,
and this enables them to function at the beginning of the re- the rate at which the Ca2+ concentration rises following
covery process to rapidly remove large quantities of Ca2+ . pump inhibition provides a measure of the activity of these
On the other hand, the plasma membrane Ca2+ -ATPase leak pathways.
(PMCA) and sarco/endo-plasmic reticulum Ca2+ -ATPase In pathological situations, the lack of oxygen reduces
(SERCA) pumps have lower capacities, but their higher af- the supply of energy, thus compromising the function of
finities mean that they can complete the recovery process the OFF reactions, resulting in the rise of Ca2+ that is
and can continue to pump at lower Ca2+ levels, thus en- so damaging during stroke or cardiac ischaemia. Under
abling them to maintain the internal stores and the resting normal circumstances, however, the fully energized OFF
level (Module 5: Figure Ca2+ uptake and extrusion). reactions can rapidly reduce the pulse of Ca2+ introduced
Some of these OFF reactions interact with each other by the ON mechanisms, thus generating a brief Ca2+ tran-
during the recovery period, and this is particularly evident sient (Module 2: Figure Ca2+ transient mechanisms). Such
in the case of the ER/mitochondrial Ca2+ shuttle (Module brief pulses of Ca2+ are a characteristic feature of the Ca2+
5: Figure ER/mitochondrial shuttle). When Ca2+ is re- signalling pathway and contribute to the spatiotemporal
leased from the ER, Ca2+ is rapidly taken up by the mito- aspects of Ca 2+ signalling.
chondria and this is then released slowly back to the ER
once Ca2+ has returned to the resting level. Ca2+ buffers
In addition to its role of returning the level of Ca2+ to its Cells express a large number of Ca2+ -binding proteins,
resting level following a stimulus, the Ca2+ OFF reactions which fall into two main groups: Ca2+ sensors and
are in constant operation to maintain the resting level of Ca2+ buffers. The Ca2+ sensors respond to changes in

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r27

intracellular Ca2+ by activating some downstream effector the size of the elementary Ca2+ events that form around
process. In a sense, all proteins capable of binding Ca2+ will Ca2+ channels. CB is thus of central importance for the
act as a buffer, and this applies to the sensors. However, the ability of neurons to create the highly localized Ca2+ events
concentration of these sensors is usually rather low, so they that occur in spines. The existence of these buffers has en-
have little buffering capacity. The role of Ca2+ buffering abled neurons to increase the numbers of their synaptic
is carried out by the other major group of Ca2+ -binding connections, and this neuronal miniaturization greatly en-
proteins. Cells have a number of buffers (Module 2: Table hances the signal processing capacity of the brain. Some
Ca2+ signalling toolkit) that are capable of binding to Ca2+ support for such a notion emerged from the finding of a re-
both in the cytoplasm and within the lumen of the endo- markable compensatory mechanism whereby the volume
plasmic reticulum (ER). The sole function of these Ca2+ of the spines increases markedly in neurons when these
buffers is to bind Ca2+ , and this has an important func- buffers are knocked out. In effect, the lack of these buffers
tion in shaping both the spatial and temporal properties of reduces the capacity of neurons to miniaturize the Ca2+
Ca2+ signals. component of their signalling systems.
The major cytosolic buffers in cells are parvalbumin The ability of CB to buffer internal Ca2+ may play an
(PV) and calbindin D-28k (CB). The major buffers that op- important role in facilitating Ca2+ reabsorption by the kid-
erate within the lumen of the ER are calsequestrin (CSQ) in ney tubule (Module 7: Figure kidney Ca2+ reabsorption).
the sarcoplasmic reticulum of muscle cells and calreticulin In the case of Ca2+ reabsorption by the intestine, the flux
(CRT) in the ER of non-muscle cells. The latter is unusual of Ca2+ is facilitated by calbindin D-9k (Module 7: Figure
in that it functions both as a cytosolic and a luminal buffer. intestinal Ca2+ reabsorption).
The role of calsequestrin is discussed elsewhere and here An important aspect of the calcium hypothesis of
we concentrate on the function of the cytosolic buffers. Alzheimer’s disease is that there is a decrease in the ex-
The latter have subtly different Ca2+ -binding properties pression of CB, which increases the sensitivity of neurons
and are expressed in cells in differing combinations and to the enhanced Ca2+ signals that arise during the onset of
concentrations to create Ca2+ signals that are tailored to Alzheimer’s disease (Module 12: Figure amyloid cascade
carry out different functions. For example, neurons such hypothesis).
as Purkinje cells express large amounts of PV and CB.
As a consequence, Purkinje cells have a large endogenous Calreticulin (CRT)
Ca2+ buffering capacity, e.g. their buffers bind approxim- Calreticulin (CRT) is a low-affinity Ca2+ -binding protein
ately 2000 Ca2+ ions for each free ion. Lower capacities that is located within the lumen of the endoplasmic retic-
of 50–100:1 are found in other cells. Motor neurons have ulum (ER). It has also been detected in the nucleus and
a very low buffering capacity and consequently have large cytoplasm and it may also be secreted into the extracellu-
Ca2+ signals in both the soma and dendrites during normal lar environment during periods of cell stress. Its primary
physiological responses, and this makes them particularly location, however, is within the lumen of the ER, where
susceptible to neurodegeneration. Buffer concentration is it functions both as a chaperone protein and as the major
one of the important parameters in determining buffer ca- ER Ca2+ buffer. CRT has three main domains; there is
pacity. The other key parameters include affinity for Ca2+ a globular N-domain of unknown function followed by
and other metal ions, the kinetics of Ca2+ binding, release a proline-rich P-domain, which has sequences unique to
and mobility. CRT and its homologous proteins calnexin and calmegin.
Alterations in Ca2+ buffers have been linked to Finally, there is a highly acidic C-terminal domain, which
schizophrenia. has a large number of low-affinity Ca2+ -binding sites cap-
able of binding 20–30 mol of Ca2+ /mol of protein. This
Parvalbumin (PV) C-terminal region terminates in the KDEL sequence re-
Parvalbumin (PV) is a slow-onset buffer. It has relatively sponsible for retaining CRT in the lumen of the ER.
low on and off rates, which means that it cannot respond The chaperone function of CRT is carried out in
to the rapid onset of most Ca2+ signals. However, PV conjunction with a related chaperone calnexin. A cal-
can soak up Ca2+ once the signal has appeared and thus nexin/calreticulin cycle ensures the correct folding and
influences the rate at which Ca2+ recovers. It is strongly subunit assembly of glycoproteins and is thus essential
expressed in skeletal muscle, where it plays an important for protein trafficking and secretion. The proper folding
role in facilitating the rate of relaxation. In PV−/ − mice, and assembly of proteins is very dependent on a constant
there is a slowing in the recovery of the Ca2+ transient. level of Ca2+ within the ER lumen and the chaperones.
Some of the effects of removing PV are compensated for The ability of CRT to bind large amounts of Ca2+ en-
by an increase in the volume of mitochondria that has ables it to function as an ER Ca2+ buffer to help main-
a similar ability to PV of removing free Ca2+ from the tain this constancy of ER Ca2+ . In addition to functioning
cytoplasm during the recovery phase. as a passive buffer, CRT may play a more direct role in
maintaining homoeostatic control of their working envir-
Calbindin D-28k (CB) onment by modulating the activity of the Ca2+ channels
Calbindin D-28k (CB) is one of the major cytosolic buf- and pumps. When the Ca2+ level within the lumen gets too
fers, particularly in neurons. It is a fast buffer that can have high, CRT and calnexin inhibit the sarco/endo-plasmic re-
a major effect on both the spatial and temporal properties ticulum Ca2+ -ATPase (SERCA) pump and, when Ca2+ is
of Ca2+ transients. CB thus plays a major role in restricting released, this inhibition is relieved and the SERCA pumps

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Module 2: Figure temporal aspects

CALCIUM TRANSIENT

ON OFF
Reaction Reaction

Periodic stimulation Continuous stimulation

Skeletal and cardiac cell Fertilization, liver metabolism, fluid secretion,

contraction, synaptic smooth muscle contraction, c ell proliferation,
transmission

Temporal aspects of Ca2+ signalling.

In almost every example where Ca2+ is used as a signal, it is presented as a brief transient, which is the digital signal used to set up complex temporal
patterns of Ca2+ signalling. In some cells, these pulses are produced on demand in that they are generated in response to periodic stimulation (blue
arrows) as occurs in muscle contraction where a brief burst of Ca2+ activates the contractile machinery, which then recovers when the Ca2+ signal is
removed. Likewise, the release of neurotransmitters from nerve terminals is triggered by a brief localized pulse of Ca2+ . In many other tissues, which
receive a continuous stimulation over a prolonged period (blue bar), the Ca2+ signal is again presented as brief spikes that are produced rhythmically
to give highly regular Ca2+ oscillations whose frequencies vary with the level of cell stimulation.

can restore Ca2+ to its normal level. In addition to its role to cells if its level remains high for a prolonged period.
as an ER buffer, CRT may play an active role in ensuring Such toxicity is avoided by presenting Ca2+ signals in a
that the Ca2+ concentration within the ER lumen is main- pulsatile manner (Module 2: Figure temporal aspects). In
tained at the optimal level for protein folding to occur. addition to this temporal aspect, the Ca2+ signal is also
highly organized in space. The elementary and global as-
Ca2+ signalling function pects of Ca2+ signalling greatly increase the versatility of
The function of Ca2+ as an intracellular second messen- the Ca2+ signalling system in that it can act either locally
ger is carried out by a combination of Ca2+ sensors and or globally. For example, muscle contraction is activated
Ca2+ effectors. The major sensors are the EF-hand pro- by a global elevation in Ca2+ , whereas the release of neur-
teins troponin C (TnC), calmodulin (CaM), neuronal Ca2+ otransmitters results from a minute punctate pulse of Ca2+
sensor proteins (NCS) and the S100 proteins. However, delivered directly to the docked vesicle by a Ca2+ sensor
there are a number of other sensors (Module 2: Table Ca2+ tightly associated with exocytotic machinery (Module 4:
signalling toolkit). Figure Ca2+ -induced membrane fusion). In between these
These sensors are then responsible for relaying inform- two extremes, there are many variations in the way the
ation through a range of effectors: Ca2+ signal is presented to cells. Perhaps the most dra-
matic are the Ca2+ waves that initiate at fixed localities
• Ca2+ -sensitive K+ channels and then process through the cytoplasm in a regenerative
• Ca2+ -sensitive Cl− channels (CLCAs) manner through the process of CICR (Module 2: Figure
• Ca2+ /calmodulin-dependent protein kinases (CaMKs) Ca2+ -induced Ca2+ release).
• Calcineurin These spatiotemporal aspects greatly enhance the ver-
• Phosphorylase kinase satility of Ca2+ signalling, thus providing the flexibility to
• Myosin light chain kinase (MLCK) regulate so many cellular processes.
• Ca2+ -promoted Ras inactivator (CAPRI) (Module 2:
Figure Ras signalling)
Cyclic ADP-ribose (cADPR) signalling
Spatiotemporal aspects of Ca2+ signalling Cyclic ADP-ribose (cADPR) is one of the messengers as-
The use of Ca2+ as a universal signal for cell regulation is sociated with the NAD+ signalling pathways. cADPR has
somewhat paradoxical because this ion can be very toxic attracted considerable attention as a putative messenger to

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Module 2: Figure cADPR metabolism

The generation and metabolism of cyclic ADP-ribose (cADPR).

The same enzyme ADP-ribosyl cyclase is responsible for both forming and degrading cADPR. The precursor NAD+ is converted into cADPR by the
cyclase activity, whereas the hydrolase component inactivates cADPR by hydrolysing it to ADP-ribose (ADPR). Reproduced from Trends Cell Biol.,
Vol. 4, Galione, A. and White, A., Ca2+ release induced by cyclic ADP-ribose, pp. 431–436. Copyright (1994), with permission from Elsevier; see
Galione and White (1994).

regulate the Ca2+ signalling pathway. cADPR generation • Secondly, the cADPR control of Ca2+ release
and metabolism is unusual in that it is carried out by the may occur indirectly through an activation of the
same enzyme that possesses both synthase and hydrolase sarco/endo-plasmic reticulum Ca2+ -ATPase (SERCA)
activity. The cADPR control of Ca2+ release is somewhat pump to increase the uptake of Ca2+ into the endoplas-
controversial, as its precise mode of action is still unclear. mic reticulum/sarcoplasmic reticulum. This increased
A cADPR working hypothesis has been put forward to loading of the internal store will serve to sensitize re-
provide a framework to understand some of the current lease channels such as the RYRs, thus leading to an in-
information on how this messenger appears to operate. A crease in Ca2+ signalling. This messenger should thus
relationship between cADPR and cell regulation has been be considered as a modulator rather than a mediator of
established in a number of different cell types. Ca2+ signalling.

cADPR working hypothesis cADPR generation and metabolism

There is sufficient evidence to take seriously the possibility The generation and metabolism of cADPR are described
that cADPR functions to regulate Ca2+ signalling. What together because both processes are carried out by the
seems to be in question is exactly how it functions. This same enzyme: the ADP-ribosyl cyclase (Module 2: Fig-
working hypothesis has two main components: ure cADPR metabolism). The hydrolysis of cADPR pro-
duces ADPR, which has been implicated as a messenger
• Firstly, the generation of cADPR is closely coupled to regulating melastatin-related transient receptor potential
cellular metabolism, as described in cADPR generation 2 (TRPM2), which is a Ca2+ channel in the plasma mem-
and metabolism (Module 2: Figure cADPR/NAADP brane (Module 2: Figure cADPR/NAADP function). This
function). The idea is that cADPR may function as a is a highly versatile enzyme. In addition to synthesizing
metabolic messenger responsible for relaying informa- and metabolizing cADPR, it is also responsible for syn-
tion about the state of metabolism to various systems in thesizing NAADP. In mammals, this bifunctional enzyme
the cell, especially those that require a heavy expendit- appears to be the lymphocyte antigen CD38, which is ex-
ure of energy. An example would be Ca2+ signalling pressed widely and is located both in the plasma membrane
and the downstream elements regulated by Ca2+ . When and at internal sites. With regard to the former location, its
energy is abundant, an increase in cADPR will set the enzymatic region is located on the outside. This is unusual
stage for Ca2+ signalling to occur. because its substrate and its site of action are on the inside

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Module 2: Figure cADPR/NAADP function

AGONISTS
Ca 2+ Ca 2+
VOC TRPM2

Ca 2+ Cellular Ca 2+
metabolism +
Ca 2+
+ ATP
RYR NADH
ADPR ?
NAD
NADP
+ +
+ Ca
2+
H S
ADP ribosyl cyclase
SERCA
2+ cADPR
Ca +
TCP1/2
+ NAADP
2+ Phosphatase
Ca
Lysosome-related
+ NAAD
+ H organelle
H

Synthesis and mode of action of cADPR and NAADP.

The enzyme ADP-ribosyl cyclase is a bifunctional enzyme that has a synthase (S) component that synthesizes cADPR and NAADP from the
precursors NAD+ and NADP respectively, but it also has a hydrolase (H) activity that converts cADPR into ADPR. This hydrolase is sensitive to
metabolism because it is inhibited by either ATP or NADH. The cADPR may act by stimulating the sarco/endo-plasmic reticulum Ca2+ -ATPase
(SERCA) pump to increase the uptake of Ca2+ into the endoplasmic reticulum. NAADP acts on a channel to release Ca2+ from a lysosome-related
organelle.

of the cell. It has been suggested that the enzyme might the hydrolase activity (Module 2: Figure cADPR/NAADP
use NAD+ derived from dying cells at sites of infections function).
to generate cADPR, which is then transported into the In CD38−/ − mice, there is a decrease in the amount
cell. of oxytocin (OT) released from hypothalamic neurons.
The intracellular enzyme is more likely to be the one Such mice show defects in maternal nurturing and in social
that functions in most cells. Just how the cyclase is activ- behaviour.
ated is still unclear. One suggestion is that the formation of
cADPR and NAADP is sensitive to cellular metabolism cADPR control of Ca2+ release
(Module 2: Figure cADPR/NAADP function). In other One of the major uncertainties about cADPR is its mode of
words, cADPR and NAADP might be metabolic messen- action in controlling the release of Ca2+ . There have been
gers that are capable of relaying information about the suggestions that it is a Ca2+ -mobilizing second messenger
state of cellular metabolism to the Ca2+ signalling path- that acts by stimulating the ryanodine receptors (RYRs) to
ways. Such a notion is supported by the fact that cADPR release Ca2+ . However, direct evidence for this assertion is
metabolism by the hydrolase is inhibited by either ATP not particularly convincing. Early single channel record-
or NADH. Another suggestion is that it might be ac- ings seemed to provide such evidence by showing that
tivated by agonists acting through cell-surface receptors, cADPR could open RYRs in lipid membranes, but these
but the coupling mechanism remains to be established observations were challenged on the basis that the cADPR
(Module 2: Figure cADPR/NAADP function). This ab- was acting through the ATP-binding site. When cADPR is
sence of a coupling mechanism might be explained by the injected into cells, it usually fails to release Ca2+ , but after
cADPR working hypothesis if the external agonist en- a period of time, it can begin to enhance the sensitivity of
hanced cADPR formation indirectly by first increasing the RYRs. An example of such an effect is shown in heart
cellular metabolism. Such a mechanism could explain the cells, where there is a gradual increase in spark frequency
ability of β-adrenergic agents to increase cADPR levels in following addition of cADPR (Module 2: Figure cADPR
heart. Likewise, the glucose-dependent increase in cADPR action in heart cells). This observation on cardiac cells
in β-cells can be directly linked to the metabolism of gluc- forms the basis of the second part of the cADPR working
ose, with the resulting increase in ATP acting to reduce hypothesis, which argues that cADPR acts indirectly as a

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Module 2: Figure cADPR action in heart cells

Effect of cADPR on spark frequency in heart cells.

When permeabilized rat ventricular myocytes were treated with 5 µM cADPR, there was a gradual increase in spark frequency that recovered to
the resting level when it was washed off (wo). There was no change in spark amplitude. This increase in spark frequency was shown to depend
upon sensitization of the RYRs due to an increase in the luminal load of Ca2+ resulting from an increase in the activity of the sarco/endo-plasmic
reticulum Ca2+ -ATPase (SERCA) pump. Reproduced from Lukyanenko, V., Györke, I., Wiesner, T.F. and Györke, S. (2001) Potentiation of Ca2+ release
by cADP-ribose in the heart is mediated by enhanced SR Ca2+ uptake into the sarcoplasmic reticulum. Circ. Res. 89:614–622, with permission from
Lippincott Williams & Wilkins (http://lww.com); see Lukyanenko et al. 2001.

modulator of Ca2+ release (Module 2: Figure cADPR/ Nicotinic acid–adenine dinucleotide

NAADP function) by stimulating the sarco/endo-plasmic phosphate (NAADP) signalling
reticulum Ca2+ -ATPase (SERCA) pump to increase the NAADP is one of the messengers associated with the
load of Ca2+ within the lumen of the store. This increase NAD signalling pathways. NAADP has attracted consid-
in luminal Ca2+ then sensitizes the RYRs so that they either erable attention as a Ca2+ -mobilizing second messenger.
begin to open spontaneously to give Ca2+ sparks (Module NAADP generation and metabolism is unusual in that it
2: Figure cADPR action in heart cells) or begin to produce is closely allied to that of cADPR in that the same en-
larger Ca2+ transients when activated by a pulse of trig- zyme synthesizes both messengers. The NAADP control
ger Ca2+ as occurs in neurons (Module 2: Figure cADPR of Ca2+ release depends upon this messenger acting on a
action in neurons). store that is different from that controlled by the inositol
1,4,5-trisphosphate receptors (InsP3 Rs) or the ryanodine
cADPR and cell regulation receptors (RYRs). A relationship between NAADP and
A role for cADPR has been implicated in a number of cell regulation seems to depend upon its interaction with
different cell types: the other Ca2+ -mobilizing messenger systems.
• Insulin-secreting β-cells (Module 7: Figure β-cell sig- NAADP generation and metabolism
nalling) NAADP (Module 2: Figure NAADP structure) is an-
• Hypothalamic neurons: in CD38−/ − mice, there is a other member of the NAD signalling pathways. Its syn-
decrease in the amount of oxytocin (OT) released from thesis from NADP is closely linked to that of cADPR in
hypothalamic neurons. Such mice show defects in ma- that they both share the same enzyme: the ADP-ribosyl
ternal nurturing and in social behaviour. cyclase (Module 2: Figure cADPR/NAADP function).

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Module 2: Figure cADPR action in neurons

cADPR potentiates Ca2+ transients induced by action potentials in sympathetic neurons.

When sympathetic neurons are stimulated for 10 ms at 20 Hz, there is a distinct transient made up of Ca2+ entering through voltage-operated channels
(VOCs) together with release of Ca2+ from internal stores through a process of Ca2+ -induced Ca2+ release (CICR). The latter component is quite
variable and depends upon the sensitivity of the ryanodine receptors (RYRs). When the neurons are perfused with cyclic ADP-ribose (cADPR), there is
a large increase in the amplitude of the transient (compare responses a and b in panel A). Note how the increase developed slowly over the recording
period. There was no augmentation when the neurons were perfused with control solution (panel B). Reproduced from Neuron, Vol. 12, Hua, S.-Y.,
Tokimasa, T., Takasawa, S., Furuya, S., Nohmi, M., Okamoto, H. and Kuba, K., Cyclic ADP-ribose modulates Ca2+ release channels for activation by
physiological Ca2+ entry in bullfrog sympathetic neurons, pp. 1073–1709. Copyright (1994), with permission from Elsevier; see Hua et al. 1994.

Module 2: Figure NAADP structure

The structure of NAADP.

The space-filling model on the right illustrates the molecular organization of the different components of NAADP. Reproduced from Curr. Biol., Vol. 13,
Lee, H.C., Ca2+ signalling: NAADP ascends as a new messenger, pp. R186–R188. Copyright (2003), with permission from Elsevier; see Lee 2003.

This enzyme uses NAD+ to make cADPR through a cyc- NAADP control of Ca2+ release
lization reaction, or it can use NADP as a substrate to pro- NAADP functions as a Ca2+ -mobilizing messenger to
duce NAADP through a base-exchange reaction during release Ca2+ from an internal store (Module 2: Fig-
which the nicotinamide group is exchanged for nicotinic ure cADPR/NAADP function). This NAADP-sensitive
acid. store, which is distinct from the endoplasmic retic-
Unlike cADPR, which is hydrolysed by the same en- ulum/sarcoplasmic reticulum store that is regulated by the
zyme, NAADP is degraded to NAAD by phosphatases inositol 1,4,5-trisphosphate receptors (InsP3 Rs) and the
such as alkaline phosphatase. ryanodine receptors (RYRs), is still being defined. On the

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basis of studies carried out in sea urchin eggs, this novel second messengers to activate protein kinases in the PtdIns
store appears to reside in a lysosome-related organelle. The 3-kinase signalling pathway. In addition to being a pre-
receptors for NAADP appear to be a family of two-pore cursor for these various signalling pathways, PtdIns4,5P2
channels (TCP), with TCP1 being located on the endo- can also function as a messenger for the PtdIns4,5P2 sig-
somal membranes, whereas TCP2 is on lysosomal mem- nalling cassette, where localized synthesis or hydrolysis of
branes. this lipid functions to regulate a number of cellular sys-
tems such as the actin cytoskeleton, membrane trafficking,
NAADP and cell regulation exocytosis, ion channels and exchangers.
The amount of Ca2+ released by NAADP is relatively Alterations in the activity of this phosphoinositide
small and is unlikely to have a direct role in Ca2+ sig- lipid signalling pathway have been implicated in various
nalling. However, there are suggestions that it may act diseases such as manic-depressive illness, Lowe’s oculo-
indirectly to trigger the release of Ca2+ by the other re- cerebrorenal (OCRL) syndrome and Cowden’s disease.
lease channels [ryanodine receptors (RYRs) or the inositol
1,4,5-trisphosphate receptors (InsP3 Rs) (Module 2: Figure Phosphoinositide metabolism
cADPR/NAADP function)]. Phosphoinositide metabolism can be separated into two
NAADP has been implicated in the control of insulin re- main components (Module 2: Figure phosphoinositide
lease by β-cells (Module 7: Figure β-cell signalling) and in metabolism):
the control of secretion by pancreatic acinar cells (Module
• Inositol lipid metabolism concerns the pathways re-
7: Figure control of pancreatic secretion).
sponsible for converting PtdIns into a variety of
ADP-ribosyl cyclase phosphoinositide lipid signalling molecules.
ADP-ribosyl cyclase is the enzyme responsible for syn- • Inositol phosphate metabolism is responsible for creat-
thesizing the Ca2+ mobilizing messengers cADPR and ing a large array of inositol phosphates, some of which
NAADP. During cADPR generation and metabolism it function in the multipurpose inositol polyphosphate
uses NAD+ to make cADPR through a cyclization reac- signalling pathway. This metabolic pathway also forms
tion (Module 2: Figure cADPR/NAADP function). The the inositol that is used to resynthesize the lipid pre-
same enzyme is also responsible for the hydrolysis of cursor PtdIns.
cADPR to ADPR. This enzyme can also use NADP as a These two pathways are connected by the processes of
substrate to produce NAADP through a base-exchange re- lipid hydrolysis and lipid synthesis. The hydrolysis occurs
action during which the nicotinamide group is exchanged when external signals activate phospholipase C (PLC) to
for nicotinic acid. hydrolyse PtdIns4,5P2 to form the second messengers in-
In mammals, this bifunctional enzyme appears to be the ositol 1,4,5-trisphosphate (Ins1,4,5P3 ) and diacylglycerol
lymphocyte antigen CD38, which is expressed widely and (DAG). The Ins1,4,5P3 is one of the major inputs into the
is located both in the plasma membrane and at internal inositol phosphate metabolic pathway that produces the
sites. inositol required for lipid synthesis.
This complex phosphoinositide metabolic network par-
ticipates in a number of highly versatile signalling cas-
Phosphoinositide signalling settes (Module 2: Figure phosphoinositide signalling sys-
Signalling through the phosphoinositide lipids is com- tems):
plex because there are a number of signalling cassettes
associated with both the synthesis and hydrolysis of the • Inositol 1,4,5-trisphosphate (InsP3 )/Ca2+ signalling cas-
phosphoinositides. Phosphoinositide metabolism can be sette
divided into two main parts. Firstly, there is inositol lipid • Diacylglycerol (DAG)/protein kinase C (PKC) sig-
metabolism, which describes the way in which the parent nalling cassette
molecule PtdIns is metabolized to form a number of lipid • PtdIns 3-kinase signalling
intermediates, some of which are key elements in differ- • PtdIns4,5P2 signalling cassette
ent signalling cassettes. Secondly, there is inositol phos- • Multipurpose inositol polyphosphate signalling path-
phate metabolism, which is a complex pathway respons- way
ible for metabolizing soluble inositol phosphates. Some
of the inositol phosphates generated by this metabolism Inositol lipid metabolism
have been implicated as messengers operating within the The different phosphoinositide signalling pathways are
multipurpose inositol polyphosphate signalling pathway. derived from the parent molecule phosphatidylinositol
PtdIns4,5P2 is of particular interest because it is (PtdIns) (Module 2: Figure PtdIns structure). Unlike the
the precursor used to generate the second messen- other phospholipids found in cellular membranes, PtdIns
gers inositol 1,4,5-triphosphate (InsP3 ) and diacylglycerol is unique in that the free hydroxy groups at the 3-, 4- and
(DAG) that function in the inositol 1,4,5-trisphosphate 5-positions on the inositol ring can be phosphorylated
(InsP3 )/Ca2+ signalling cassette and the diacylglycerol further to create a family of phosphoinositides (Module 2:
(DAG)/protein kinase C (PKC) signalling cassette re- Figure phosphoinositide metabolism). One of the more
spectively. PtdIns4,5P2 is also a precursor that is metabol- important lipids involved in phosphoinositide signalling
ized to PtdIns3,4P2 and PtdIns3,4,5P3 , which function as is PtdIns4,5P2 , which is a nodal point for a number of

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r34

Module 2: Figure phosphoinositide metabolism

Inositol lipid metabolism

PtdIns3P 4 PtdIns3,5P 2
1 6
11

2 5 8
PtdIns PtdIns4P PtdIns3,4P 2 PtdIns3,4,5P 3
7

3 8 10 9

PtdIns5P 6 PtdIns4,5P 2

Lipid resynthesis PLC

DAG Ins1,4,5P3
G-6-P

Inositol phosphate
Inositol synthase Inositol metabolism

Summary of phosphoinositide metabolism.

The metabolism of phosphoinositides is separated into inositol lipid metabolism and inositol phosphate metabolism. In the former case, the parent
molecule phosphatidylinositol (PtdIns) (Module 2: Figure PtdIns structure) undergoes a series of phosphorylation reactions to create various poly-
phosphoinositides. One of these is PtdIns4,5P2 , which is of key importance as it can function both as a messenger and as a precursor for the formation
of other phosphoinositide lipid signalling molecules such as PtdIns3,4,5P3 . In addition, it can be hydrolysed to form the second messengers inositol
1,4,5-trisphosphate (Ins1,4,5P3 ) and diacylglycerol (DAG). The Ins1,4,5P3 is the major input into a complex pathway of inositol phosphate metabolism
(Module 2: Figure inositol phosphate metabolism). One of the outputs of this metabolism is inositol that is used for the resynthesis of PtdIns. Cells
also have a 1-l-myo-inositol-1-phosphate synthase that can convert glucose 6-phosphate (G-6-P) into inositol.

Module 2: Figure PtdIns structure

Phosphatidylinositol (PtdIns)

C C
O O
C C C O P
H2 H H2
O OH
HO 1

OH
HO HO
3 5
4

Molecular structure of PtdIns.

The backbone of the molecule consists of a glycerol moiety. Two of the carbons carry fatty acids that insert into the hydrophobic domain of the
membrane, whereas the remaining carbon is attached to a phosphate group that is linked to an inositol ring that projects out into the cytosol. The
hydroxy groups on the 3-, 4- and 5-positions can be phosphorylated further (see Module 2: Figure phosphoinositide metabolism).

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Module 2: Figure PI 3-K family

Class IA
p85α, p85β SH3 P BCR P SH2 p110 binding SH2

p55α, p50α, p55γ P SH2 p110 binding SH2

PIP3
PIP2

Regulatory
subunits
p110α, p110β, p110δ p85 Ras C2 Helical Kinase
Catalytic subunits

p101, p84 Homolgy I Homolgy II

Class IB
Regulatory
subunits
p110γ Ras C2 Helical Kinase
Catalytic subunit

Class II
PI3KC2α
PI3KC2β P P Ras C2 Helical Kinase PX C2
PI3KC2γ Catalytic subunits

Heat domains Wd40 domains CaM-binding Class III

p150 domain Accessory
Kinase
domain
Regulatory PIK3C3 (hVPS34) C2 Kinase
subunit
Catalytic subunit

The regulatory and catalytic subunits of the PtdIns 3-kinase family.

The family of PtdIns 3-kinase is divided into three classes. The kinase domain (shown in yellow) phosphorylates PtdIns4,5P2 (PIP2 ) to form the lipid
messenger PtdIns3,4,5P3 (PIP3 ). The domain structures of the regulatory and catalytic subunits are described in the text. Information for this figure
was taken from Hawkins et al. 2006.

signalling systems (Module 2: Figure phosphoinositide sig- 6. PtdInsP kinase II adds a phosphate to the 4-position
nalling systems). of PtdIns3P to form PtdIns3,4P2 . The same enzyme
The metabolism of PtdIns is carried out by a collection can add a phosphate to the 4-position of PtdIns5P to
of lipid kinases and lipid phosphatases. The numbers below form PtdIns4,5P2 .
refer to the different reactions shown in Module 2: Figure 7. Inositol polyphosphate 5-phosphatase such as Src ho-
phosphoinositide metabolism: mology 2 (SH2) domain-containing inositol phos-
phatase (SHIP) removes a phosphate from the 5-
position of PtdIns 3,4,5P3 to form PtdIns3,4P2 .
1. The Class III PtdIns 3-kinase (PtdIns 3-K) adds
8. A PtdIns4P 5-kinase (PtdIns4P 5-K) adds a phosphate
a phosphate to the 3-position of PtdIns to form
to the 5-position of PtdIns4P to form PtdIns4,5P2 .
PtdIns3P.
The latter can also be formed by Steps 6 and 9.
2. PtdIns 4-kinase (PtdIns 4-K) adds a phosphate to
PtdIns4P5-K can also phosphorylate PtdIns3,4P2 to
the 4-position of PtdIns to form PtdIns4P. It can
form PtdIns3,4,5P3 .
also be formed by removal of the 3-phosphate from
9. Phosphatase and tensin homologue deleted on chro-
PtdIns3,4P2 by phosphatase and tensin homologue
mosome 10 (PTEN) removes a phosphate from the
deleted on chromosome 10 (PTEN).
3-position of PtdIns3,4,5P3 .
3. PtdInsP kinase III, which has not been fully charac-
10. A Class I PtdIns 3-kinase adds a phosphate to the
terized, adds a phosphate to the 5-position of PtdIns
3-position of PtdIns4,5P2 to form PtdIns3,4,5P3 .
to form PtdIns5P.
PtdIns3,4,5P3 , which functions as one of the second
4. The Type 1 PtdIns4P 5-kinase adds a phosphate to
messengers in the PtdIns 3-kinase signalling pathway,
the 5-position of PtdIns3P to form PtdIns3,5P2 . The
can also be formed by the addition of a phosphate
level of PtdIns3,5P2 changes in cells following osmotic
to the 5-position of PtdIns3,4P2 by the PtdIns4P
stress.
5-kinase (PtdIns4P 5-K) (i.e. the same enzyme used
5. Types I and II PtdIns 3-kinase (PtdIns 3-K) add
for Step 8).
a phosphate to the 3-position of PtdIns4P to form
11. The myotubularins remove the 3-phosphate from
PtdIns3,4P2 . PtdIns3,4P2 has been suggested to func-
PtdIns3P.
tion as a messenger operating within the plasma mem-
brane, where it serves to recruit and activate protein PtdIns 3-kinase (PtdIns 3-K)
kinases such as protein kinase B (PKB). PtdIns3,4P2 The PtdIns 3-kinases (PtdIns 3-Ks) are a family of en-
can also be formed by other enzymes (see Steps 6 zymes that have been classified into three classes (Mod-
and 7). ule 2: Figure PI 3-K family).

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Module 2: Figure phosphoinositide signalling systems

The PtdIns4,5P 2 The PtdIns 3-kinase

signalling cassette signalling cassette

PtdIns PtdIns4P PtdIns4,5P2 PtdIns3,4,5P3

Inositol phosphate InsP3

metabolism
DAG

The multipurpose The diacylglycerol (DAG)/

inositol polyphosphate protein kinase C
signalling cassette signalling cassette

The InsP3 / Ca 2+
signalling cassette

PtdIns metabolism spawns a variety of signalling cassettes.

The metabolism of PtdIns creates a number of lipid and inositol phosphate derivatives that operate a variety of signalling cassettes. PtdIns4,5P2 is a
nodal point for a number of signalling cassettes. It is the precursor that is hydrolysed to generate the second messengers inositol 1,4,5-trisphosphate
(InsP3 ) and diacylglycerol (DAG). It is phosphorylated further to form the lipid second messenger PtdIns3,4,5P3 . The localized turnover of PtdIns4,5P2
also has a signalling role within the PtdIns4,5P2 signalling cassette that regulates a variety of processes such as the cytoskeleton, control of the
processes of membrane trafficking and exocytosis and the regulation of ion channels and exchangers. Finally, the InsP3 enters a complex pathway
of inositol phosphate metabolism that generates components of the multipurpose inositol polyphosphate signalling cassette.

Class I PtdIns 3-kinases • The Src homology 3 (SH3) domain can bind to proline-
The primary function of the Class I PtdIns 3-kinases is to rich regions of Shc, Cbl and dynamin.
phosphorylate PtdIns4,5P2 to form the lipid second mes- • The proline-rich regions can bind to the SH3 domains
senger PtdIns3,4,5P3 (Module 2: Figure PtdIns 3-kinase of Abl, Src, Lck, Lyn, Fyn and growth factor receptor
signalling). These Class I enzymes are divided into two bound protein 2 (Grb2).
groups.
The Class IA enzymes are heterodimers that are formed
from five regulatory subunits (p85α, p85β, p55α, p50α and The Class IB enzyme has a different activation mechan-
p55γ), which are typical adaptor proteins. The p85 regu- ism in that it is stimulated by heterotrimeric G proteins
latory subunit contains an Src homology 3 (SH3) domain, (Module 2: Figure PtdIns 3-kinase signalling). It has a
a breakpoint-cluster-region homology (BH) domain, two p110γ catalytic subunit and two regulatory subunits (p101
proline-rich regions (P) on either side of the BH domain and p84) (Module 2: Figure PI 3-K family). These regulat-
and two Src homology (SH2) domains that are separated ory subunits may mediate the translocation of the p110γ
by a p110-binding domain, enabling them to interact with a catalytic subunit to the membrane by binding to the Gβγ
p85-binding domain located on the three catalytic subunits subunit.
(p110α, p110β and p110δ). The different binding domains The Class I enzymes interact strongly with the onco-
on the regulatory subunits enhance the versatility of the gene Ras (Module 2: Figure PtdIns 3-kinase signalling) and
Class IA enzyme by enabling the catalytic subunit to be this can have important consequences for both upstream
activated by interacting with a variety of signalling mo- and downstream events, depending on the cellular context.
lecules: With regard to the former, the binding of Ras can enhance
enzyme activation and the generation of lipid messengers.
• The Src homology (SH2) domains of the regulatory sub- On the other hand, the association with Ras might mediate
unit enable the enzyme to associate with phosphotyr- some of the downstream effects of the PtdIns 3-kinases.
osine residues on the cytoplasmic domains of recept- Class I PtdIns 3-kinases are strongly inhibited by the
ors such as the platelet-derived growth factor receptor fungal metabolite wortmannin (an irreversible inhibitor)
(PDGFR) (Module 1: Figure PDGFR activation). and by LY294002 (a reversible inhibitor).

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Class II PtdIns 3-kinase PtdIns4P 5-kinase Iα

The Class II PtdIns 3-kinase has a more restricted sub- This enzyme functions at multiple locations in the cell. It is
strate range in that it phosphorylates just PtdIns and the major enzyme responsible for forming PtdIns4,5P2 at
PtdIns4P. There are no regulatory subunits, and the three the plasma membrane. It is also found within the nucleus
catalytic subunits (PI3KC2α, PI3KC2β and PI3KC2γ) at nuclear speckles that are sites of mRNA processing.
are characterized by having a C-terminal phox homology
(PX) domain and a C2 domain (Module 2: Figure PI 3-K PtdIns4P 5-kinase Iβ
family). This isoform is found on membranes surrounding the
nucleus.
Class III PtdIns 3-kinase
The Class III PtdIns 3-kinase has a restricted substrate Ins4P 5-kinase Iγ
range in that it phosphorylates just PtdIns. The prototype The PtdIns4,5P2 function in focal adhesions depends upon
of this class is vacuolar protein sorting 34 (Vps34) pro- a spliced form of this enzyme, which is targeted to focal
tein found in Saccharomyces cerevisiae of which there is adhesions by interacting with talin (Module 6: Figure in-
a human homologue (hVps34). The activity of hVps34 is tegrin signalling). Talin contains a FERM domain, which
regulated by Ca2+ and this contributes to the regulation is used to bring PtdIns4P 5-kinase Iγ into the adhesion
of the target of rapamycin (TOR), which functions in cell complex.
growth control (see steps 4 and 5 in Module 9: Figure target This Type I enzyme can be regulated by two separate
of rapamycin signalling). There is a Ca2+ -dependent CaM- mechanisms. Firstly, its activity is stimulated by phospha-
binding domain located between residues 318 and 334 in tidic acid (PA) that is formed either by the phospholipase
the accessory domain (Module 2: Figure PI3-K family). D (PLD) signalling pathway where phosphatidylcholine
(PC) is hydrolysed by phospholipase D (Module 2: Fig-
PtdIns 4-kinase (PtdIns 4-K) ure PLD signalling) or by the phosphorylation of diacyl-
PtdIns 4-kinase catalyses phosphorylation of PtdIns on glycerol (DAG) by diacylglycerol kinase (DAG) kinase
the 4-position of the inositol ring to produce PtdIns4P (Module 2: Figure InsP3 /DAG recycling). Secondly, en-
(Step 2 in Module 2: Figure phosphoinositide metabol- zyme activity can be enhanced by small monomeric G
ism). Enzyme activity is found in most cellular membranes proteins such as Rho (Module 2: Figure Rho signalling).
(plasma membrane, endoplasmic reticulum, Golgi, nuclear During its activation, PtdIns4P 5-kinase Iα translocates to
membrane and secretory vesicles). Cells have two classes the plasma membrane through an activation mechanism
of PtdIns 4-K: Type II and Type III. The latter has α- that is dependent upon Rac and Rho. This G protein reg-
and β-isoforms and is sensitive to the drug wortmannin. ulation results in the localized synthesis of PtdIns4,5P2 ,
Neither the PtdIns 4-KIIIα found in the endoplasmic re- which then acts as a second messenger for the PtdIns4,5P2
ticulum nor the PtdIns 4-KIIIβ found in the cytosol and signalling cassette. For example, it can influence a variety
Golgi seem to be responsible for producing the PtdIns4P of proteins to regulate processes such as actin remodelling,
in the plasma membrane. It is the Type III PtdIns 4-K iso- exocytosis, the PLD signalling pathway (Module 2: Figure
form that is activated by the neuronal Ca2+ sensor-1 pro- PLD signalling) and the permeability of ion channels.
tein (NCS-1) to enhance exocytosis. The Type II PtdIns The PtdIns4P 5-Kα has an additional role in that
4-K also contains α- and β-isoforms that are insensitive it can phosphorylate PtdIns3,4P2 to form the lipid
to wortmannin and may be responsible for generating the second messenger PtdIns3,4,5P3 (Step 8 in Mod-
lipid substrates in the membrane that are used for sig- ule 2: Figure phosphoinositide metabolism).
nalling. The enzymatic activity of PtdIns 4-kinase is greatly
enhanced in various cancer cells. PtdInsP kinase II
Originally, it was considered that PtdIns4P functioned This enzyme, which is found in the cytosol, endoplas-
solely as a precursor for the formation of PtdIns4,5P2 , but mic reticulum and nucleus, but not in the plasma mem-
there is increasing evidence for a role as part of a PtdIns4P brane, is a PtdInsP 4-kinase capable of phosphorylat-
signalling cassette. ing both PtdIns3P and PtdIns5P to produce PtdIns3,4P2
and PtdIns4,5P2 respectively (Steps 6 in Module 2: Figure
PtdIns phosphate kinases phosphoinositide metabolism).
There is a family of PtdIns phosphate kinases that are di-
vided into three subfamilies (Types I–III) that are localized PtdInsP kinase III
to different compartments and function to phosphorylate Despite being classified as a PtdInsP kinase, the main func-
different phosphoinositides. tion of PtdInsP kinase III is to phosphorylate PtdIns on
the 5-position to form PtdIns5P. One of the isoforms has
PtdIns4P 5-kinase (PtdIns4P 5-K) a FYVE domain to target it to endomembranes.
This versatile family of Type I PtdIns phosphate kinases is
composed of three isoforms (α, β and γ) that are located Phosphatase and tensin homologue deleted on
in different cellular regions such as the plasma membrane, chromosome 10 (PTEN)
focal adhesions, Golgi and nucleus. Their primary role is to PTEN (phosphatase and tensin homologue deleted on
phosphorylate PtdIns4P to PtdIns4,5P2 (Step 8 in Module chromosome 10) is a lipid phosphatase that is mutated
2: Figure phosphoinositide metabolism). in many human cancers. Its gene is a tumour suppressor,

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r38

which functions in cell migration, proliferation and sur- and this may be of particular significance for the relation-
vival. PTEN inactivates the 3-phosphorylated lipid second ship between K+ channels and cell proliferation.
messengers that operate within the PtdIns 3-kinase sig- These enzymes are of interest because mutations
nalling cassette. It functions to remove the phosphate from in MTM1 are responsible for X-linked recessive
the 3-position of PtdIns3,4P2 and PtdIns3,4,5P3 (Mod- myotubular myopathy and MTMR2 is mutated in
ule 2: Figure phosphoinositide metabolism). Charcot-Marie-Tooth disease 4B.
PTEN is a redox-sensitive enzyme, which may contrib-
ute to a positive-feedback loop that enhances redox sig- Inositol phosphate metabolism
nalling (Module 2: Figure plasma membrane ROS form- The Ca2+ -mobilizing messenger function of inositol 1,4,5-
ation). It is inhibited by H2 O2 , which induces Cys-124 trisphosphate (InsP3 ) is terminated through its metabol-
in the active site to form a disulphide bond with Cys-71. ism by a complex inositol phosphate metabolic pathway
This disulphide bond formation is specifically reversed by (Module 2: Figure inositol phosphate metabolism) that has
thioredoxin. A reversible inactivation of PTEN may thus two main functions. Firstly, it produces free inositol that
contribute to the accumulation of PtdIns3,4,5P3 (Mod- is resynthesized to PtdIns, which can be reused for further
ule 2: Figure plasma membrane ROS formation), which signalling. Secondly, it generates an assortment of inositol
may thus help to promote proliferation by switching off phosphates, some of which contribute to the multipurpose
the metabolism of the 3-phosphorylated lipid second mes- inositol polyphosphate signalling pathway.
sengers. This reversible inactivation of PTEN contrib- The Ins1,4,5P3 that enters the metabolic pathway is
utes to the accumulation of PtdIns3,4,5P3 by setting up metabolized via two pathways. It is dephosphorylated by
a positive-feedback loop, since the formation of this lipid Type I inositol polyphosphate 5-phosphatase (step 1) to
messenger is responsible for stimulating the production of form Ins1,4P2 or it can be phosphorylated by InsP3 3-
H2 O2 (Module 2: Figure plasma membrane ROS forma- kinase to form Ins1,3,4,5P4 (step 4). Both Ins1,4P2 and
tion). Ins1,3,4,5P4 are putative messengers in the multipurpose
There are additional enzymes that hydrolyse the 3- inositol polyphosphate signalling pathway. The Ins1,4P2
phosphorylated position. Transmembrane phosphatase is sequentially dephosphorylated to free inositol. The
with tensin homology (TPTE) is localized to the plasma Ins1,3,4,5P4 is dephosphorylated to Ins1,3,4P3 , which oc-
membrane, but appears not to have phosphatase activity. cupies an important position in the metabolic pathway
TPTE and PTEN homologous inositol lipid phosphatase in that either it is dephosphorylated down to inositol,
(TPIP) occurs as α and β isoforms. The TPIPα isoform as part of an inositol recycling pathway, or it can be
hydrolyses PtdIns3,4,5P3 , PtdIns3,5P2 , PtdIns3,4P2 and phosphorylated further to form additional inositol poly-
PtdIns3P. It has N-terminal transmembrane domains that phosphates. The inositol phosphates in the pink boxes in
appear to localize the protein to the ER. Module 2: Figure inositol phosphate metabolism have been
PTEN is a potent tumour suppressor that is frequently implicated as intracellular messengers. InsP3 mobilizes in-
inactivated in many different cancers, e.g. endometrial, ternal Ca2+ and the putative functions of the others are
prostate, mammary carcinomas, melanomas and thyroid described in the multipurpose inositol polyphosphate sig-
tumours. When PTEN is inactivated in neurons, there is a nalling pathway.
progressive increase in cell size and increased phosphoryla- Metabolism of the inositol phosphates is carried
tion of protein kinase B (PKB) that create cerebellar ab- out by a large number of inositol phosphate kinases
normalities resembling those seen in human Lhermitte– and phosphatases (Module 2: Figure inositol phosphate
Duclos disease (LDD). metabolism).
Germline mutations in the PTEN gene have been im-
plicated in the development of Cowden’s disease and Inositol polyphosphate 5-phosphatase
Bannayan–Zonana syndrome, where there is an increased Step 1 in Figure 2 inositol phosphate metabolism. This
risk of breast and thyroid cancers. An increase in the ex- large family of enzymes, which are products of multiple
pression of PTEN by the tumour suppressor p53 may genes and splice variants, removes the 5-phosphate group
contribute to p53-induced apoptosis. from inositol lipids and, in some cases, can also act on
inositol phosphates. All members of the family share a
Myotubularins central conserved catalytic domain:
There is a large family of myotubularins that function
as lipid phosphatases that specifically dephosphorylate • 5-Phosphatase I. Functions to hydrolyses Ins1,4,5P3
PtdIns3P. They may also act on PtdIns3,5P2 . Mammalian and Ins1,3,4,5P4 . If this enzyme is deleted, there
cells express 14 myotubularins. The founding member is is an increase in the level of InsP3 , and this
MTM1 and the remainder are MTMR2–MTMR13. These was associated with a transformed phenotype.
enzymes have a phosphatase domain, and a GRAM do- This enzyme is inhibited following phosphoryla-
main, which associates with membranes and a C-terminal tion by Ca2+ /calmodulin-dependent protein kinase II
coiled-coil domain that links these enzymes to other pro- (CaMKII). Type I, which hydrolyses the inositol phos-
teins. One of the isoforms, MTMR6, appears to have a phates, is directed to the membrane by isoprenylation
specific role in the PtdIns3P signalling cassette. Hydro- of the C-terminal region. If these membrane attachment
lysis of PtdIns3P may control the activity of the Ca2+ - sites are removed, allowing the enzyme to disperse into
activated intermediate-conductance (IK) channel (KCa 3.1), the cytosol, InsP3 metabolism appears to be severely

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r39

curtailed. It seems that much of the InsP3 is metabol- and type B are activated by Ca2+ /calmodulin, with the
ized close to its site of action at the plasma membrane. type B being the most sensitive. This enzyme is also sens-
• 5-Phosphatase II. A 75 kDa protein that hydrolyses itive to other signalling pathways in that both forms are
inositol phospholipids. inhibited by PKC. On the other hand, protein kinase A
• OCRL. A gap-domain-containing enzyme that can (PKA) activates type A but inhibits type B.
hydrolyses both inositol phosphates (Ins1,4,5P3 and
Ins1,3,4,5P4 ) and the phosphoinositides PtdIns4,5P2 Inositol polyphosphate 4-phosphatase
and PtdIns3,4,5P3 . A deficiency of this enzyme is re- Steps 5 in Module 2: Figure inositol phosphate metabol-
sponsible for Lowe’s oculocerebrorenal (OCRL) syn- ism. A multifunctional phosphatase capable of dephos-
drome. phorylating both Ins3,4P2 and Ins1,3,4P3 , as well as the
• SHIP. The Src homology 2 (SH2) domain-containing inositol lipid PtdIns3,4P2 . This enzyme might be a regu-
inositol phosphatases (SHIP1 and SHIP2) hydrolyse lator of cell proliferation, since it is absent in hyperprolif-
Ins1,3,4,5P4 and PtdIns3,4,5P3 . The SHIPs, which come erative megakaryocytes that lack the transcription factor
in different forms, are found in haematopoietic cells, GATA-1.
where they play a role in inhibiting signalling. In B
cells, for example, the FcγRIIB receptor has an im- Inositol polyphosphate 3-phosphatase
munoreceptor tyrosine-based inhibitory motif (ITIM) Steps 6 in Module 2: Figure inositol phosphate metabol-
to which SHIPs attach causing a reduction in Ca2+ sig- ism. A multifunctional phosphatase capable of dephos-
nalling by hydrolysing PtdIns3,4,5P3 and thereby re- phorylating both Ins1,3P2 and PtdIns3P.
ducing the stimulatory effect of the Tec tyrosine kinase
family on phospholipase Cγ (Module 2: Figure ROS ef- Ins1,3,4P3 6-kinase
fects on Ca2+ signalling). SHIPs perform a similar func- Step 7 in Module 2: Figure inositol phosphate metabolism.
tion in mast cells (Module 11: Figure mast cell inhibit-
ory signalling). SHIP1 functions to localize PIP3 at the Ins1,3,4,6P4 5-kinase
front of the cell during neutrophil chemotaxis (Mod- Step 8 in Module 2: Figure inositol phosphate metabolism.
ule 11: Figure neutrophil chemotaxis).
• Synaptojanin-1. Ins3,4,5,6P4 1-kinase
• Synaptojanin-2. Step 9 in Module 2: Figure inositol phosphate metabolism.
• Proline-rich inositol polyphosphate 5-phosphatase This enzyme is responsible for inactivating the Ins3,4,5,6P4
(PIPP). that regulates chloride channels. It is an unusual enzyme
• Skeletal muscle and kidney enriched inositol phos- in that it has alternative positional specificity (5/6-kinase
phatase (SKIP). activity against Ins1,3,4P3 ). The enzyme is the same as the
Ins1,3,4,5,6P5 1-phosphatase.
Inositol polyphosphate 1-phosphatase
Steps 2 in Module 2: Figure inositol phosphate meta- Ins1,3,4,5,6P5 1-phosphatase
bolism. This enzyme dephosphorylates both Ins1,3,4P3 Step 10 in Module 2: Figure inositol phosphate metabol-
and Ins1,4P2 . This enzyme has been implicated in the ism. The enzyme is equivalent to the Ins 3,4,5,6P4 1-kinase.
signalling pathways of compensatory hypertrophy and is
up-regulated in human colorectal cancer. Ins1,4,5,6P4 3-kinase
Step 11 in Module 2: Figure inositol phosphate meta-
bolism.
Inositol monophosphatase
Steps 3 in Module 2: Figure inositol phosphate metabol- Ins1,3,4,5,6P5 /InsP6 3-phosphatase
ism. The free inositol that is formed by this enzyme is Steps 12 in Module 2: Figure inositol phosphate metabol-
then used to resynthesize PtdIns, which is returned to ism. This enzyme is also known as the multiple inositol
the plasma membrane, where it can be reused to function phosphate phosphatase (MIPP). Its function is somewhat
as the precursor for the phosphoinositide signalling lip- unclear because it is located within the lumen of the ER,
ids (Module 2: Figure phosphoinositide metabolism). This apparently insulated from its two substrates.
inositol monophosphatase is inhibited by lithium (Li+ ),
which thus acts to reduce the supply of inositol (Module Ins1,3,4,5,6P5 2-kinase
2: Figure InsP3 /DAG recycling). This ability of Li+ to Step 13 in Module 2: Figure inositol phosphate metabol-
lower the level of inositol is the basis of an inositol de- ism. The production of InsP6 occurs primarily through the
pletion hypothesis to account for its action in controlling phosphorylation of Ins1,3,4,5,6P5 by a 2-kinase.
manic-depressive illness.
InsP6 kinase
Ins1,4,5P3 3-kinase Steps 14 in Module 2: Figure inositol phosphate meta-
Step 4 in Module 2: Figure inositol phosphate metabolism. bolism. This kinase adds an additional phosphate to that
This kinase is one of the major pathways for metabolizing already present on the 5-position to produce PP-InsP5
the second messenger Ins1,4,5P3 . There are three isoforms (InsP7 ). This kinase can run in reverse and is thus poten-
of this enzyme (InsP3 3-kinase A, B and C). Both type A tially capable of reforming ATP. The same enzyme may act

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r40

on Ins1,3,4,5,6P5 to produce PPInsP4 . There are three iso- of the inositol ring is phosphorylated to form PtdIns4P.
forms (InsP6 K1–InsP6 K3). InsP6 K2 has been implicated In the next step, the PtdIns4P is phosphorylated further
in the action of interferons on cell growth and apoptosis. to yield the precursor PtdIns4,5P2 . The next step is the
agonist-dependent hydrolysis of PtdIns4,5P2 to generate
Diphosphoinositol phosphate phosphohydrolase InsP3 and diacylglycerol (DAG).
(DIPP)
Steps 15 in Module 2: Figure inositol phosphate metabol- Hydrolysis of PtdIns4,5P2 to generate InsP3 and
ism. This enzyme dephosphorylates both PP-InsP5 (InsP7 ) diacylglycerol (DAG)
and [PP]2 -InsP4 (InsP8 ), during which it removes the β- External stimuli (e.g. hormones, neurotransmitters and
phosphate from the diphosphate groups. growth factors) gain access to this signalling pathway by
activating cell-surface receptors. The latter fall into two
PP-InsP5 kinase main classes, the G protein-coupled receptors (GPCRs)
Step 16 in Module 2: Figure inositol phosphate metabol- and the protein tyrosine kinase-linked receptors (PTKRs).
ism. This kinase adds a further phosphate to PP-InsP5 to During the transduction process, the precursor lipid
form [PP]2 -InsP4 , but the positional specificity of the lat- PtdIns4,5P2 is hydrolysed by phospholipase C (PLC) to
ter remains to be established. Like the InsP6 kinase, this produce both InsP3 and DAG. The family of PLCs can be
enzyme is capable of forming ATP by running in a reverse distinguished by the way they are coupled to cell-surface
mode. receptors. In general, the GPCRs use the PLCβ isoforms,
whereas the receptor tyrosine kinases (RTKs) are coupled
Mammalian inositol phosphate multikinase (mIPMK) to the PLCγ isoforms (Module 2: Figure PLC structure
Steps 17 in Module 2: Figure inositol phosphate metabol- and function).
ism. This mammalian inositol phosphate multikinase (mI- DAG functions to activate the diacylglycerol
PMK) is a multifunctional kinase capable of the following (DAG)/protein kinase C (PKC) signalling cassette,
phosphorylations: whereas the InsP3 diffuses into the cytosol to activate the
• Ins4,5P2 → Ins1,4,5P3 InsP3 receptors to release Ca2+ stored in the endoplasmic
• Ins1,4,5P3 → Ins1,3,4,5P4 reticulum. The signalling function of this bifurcating sig-
• Ins1,3,4,5P4 → InsP5 nalling pathway is curtailed by the metabolism of InsP3
• InsP5 → PP-InsP4 and the resynthesis of PtdIns.

Inositol 1,4,5-trisphosphate (InsP3 )/Ca2+ Metabolism of InsP3 and DAG and the resynthesis of
signalling cassette PtdIns
This signalling cassette uses the second messenger inos- The metabolism of InsP3 and DAG and the resynthesis
itol 1,4,5-trisphosphate (InsP3 ) to mobilize Ca2+ from in- of PtdIns are the OFF reactions that terminate the ac-
ternal stores. When external stimuli engage receptors on tions of these two messengers. There are two pathways
the cell surface, they activate the enzyme phospholipase C for diacylglycerol (DAG) metabolism. Similarly, there are
(PLC), which hydrolyses PtdIns4,5P2 to yield InsP3 and two separate mechanisms of inositol 1,4,5-trisphosphate
diacylglycerol (DAG) (Module 2: Figure InsP3 and DAG (InsP3 ) metabolism.
formation). This is a bifurcating signalling pathway in that
InsP3 generates a Ca2+ signal, whereas DAG functions to
Diacylglycerol (DAG) metabolism
stimulate protein kinase C (PKC). The InsP3 released from
The second messenger DAG is metabolized via two separ-
the membrane diffuses into the cytosol where it engages
ate pathways. It can be phosphorylated by diacylglycerol
the InsP3 receptors (InsP3 Rs) to release Ca2+ from the
(DAG) kinase to form phosphatidic acid (PA) or it is hy-
endoplasmic reticulum.
drolysed by diacylglycerol (DAG) lipase. The PA is trans-
The operation of this InsP3 /Ca2+ signalling cassette can
ferred to the endoplasmic reticulum, where it interacts
be divided into separate components:
with CTP to form the CDP/DAG complex, which is a
• PtdIns conversion into the precursor lipid PtdIns4,5P2 precursor in the resynthesis of PtdIns (Module 2: Figure
• Hydrolysis of PtdIns4,5P2 to generate InsP3 and DAG InsP3 /DAG recycling).
• Metabolism of InsP3 and DAG and the resynthesis of
PtdIns Diacylglycerol (DAG) kinase
• Inositol 1,4,5-trisphosphate (InsP3 ) and Ca2+ release Diacylglycerol (DAG) kinase α (DAGKα), which is one
of a family of nine mammalian isotypes, has a number
PtdIns conversion into the precursor lipid PtdIns4,5P2 of domains, including Ca2+ -binding EF-hand motifs and
The precursor PtdIns4,5P2 , which is hydrolysed to give an N-terminal recoverin homology domain that is related
InsP3 , is part of the complex inositol lipid metabolic path- to the recoverin family of neuronal Ca2+ sensors. These
way (Module 2: Figure phosphoinositide metabolism). The two domains appear to function as a unit during Ca2+ -
synthesis of the lipid precursor PtdIns4,5P2 from PtdIns is induced activation of DAGKα. In response to an increase
controlled by two substrate cycles involving lipid kinases in Ca2+ , DAGKα translocates to the membrane, where
and phosphatases (Module 2: Figure InsP3 and DAG form- it phosphorylates DAG to phosphatidic acid (PA) (Mod-
ation). In the first step, the hydroxy group at the 4-position ule 2: Figure InsP3 /DAG recycling).

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r41

Module 2: Figure inositol phosphate metabolism

[PP] InsP (InsP8 )

2 4
ATP
16 15

PPInsP5 (InsP7 )
ADP + Pi
14 15

ATP InsP6 PP-InsP4

17
14
13 12

Ins1,3,4,5,6P5

10 11 12
17 8 9
Inositol lipid signalling

Ins1,3,4,5P4 Ins1,3,4,6P4 Ins3,4,5,6P Ins1,4,5,6P

4 4

4 1 7 +
Ins1,4,5P3
-
Ins1,3,4P3
2
17
1 5

Ins1,4P2 Ins4,5P2 Ins1,3P2 Ins3,4P2

-
Cl
2 6 5

Ins4P Ins1P Ins3P

3 3 3

Inositol Inositol lipid

synthesis

Inositol phosphate metabolism.

The major input into this metabolic system is the Ins1,4,5P3 released from the membrane following receptor-activated inositol lipid signalling. One of
the functions of this pathway is to form free inositol that is used for inositol lipid synthesis. The pathway also generates additional inositol phosphates
(highlighted in pink) that have putative signalling functions. See the text for further details of the enzymes responsible for Steps 1–17.

Diacylglycerol (DAG) lipase This InsP3 /Ca2+ signalling system controls many different
A diacylglycerol (DAG) lipase is responsible for removing cellular processes in a large number of different cell types
one of the fatty acid tails from the sn-position of DAG (Module 2: Figure InsP3 /Ca2+ signalling functions).
to form monoacylglycerol. This hydrolysis of DAG may
represent the primary mechanism for removing the DAG Inositol 1,4,5-trisphosphate (InsP3 ) metabolism
that is produced following the hydrolysis of inositol lipids. The inositol 1,4,5-trisphosphate (InsP3 ) that is formed
during signalling enters the pathways of inositol phos-
Inositol 1,4,5-trisphosphate (InsP3 ) and Ca2+ signalling phate metabolism from which it emerges as free inositol.
The primary function of inositol 1,4,5-trisphosphate It is dephosphorylated by Type I inositol polyphos-
(InsP3 ) is to function as a second messenger to release phate 5-phosphatase (Steps 1 in Module 2: Figure inositol
Ca2+ from the internal stores. The 1,4,5-trisphosphate re- phosphate metabolism) to form Ins1,4P2 or it can be phos-
ceptors (InsP3 Rs) located on the endoplasmic reticulum phorylated by InsP3 3-kinase to form Ins1,3,4,5P4 (Step
respond to InsP3 by releasing puffs of Ca2+ (Module 3: 4). These two products then enter a complex metabolic
Figure InsP3 R activation). These InsP3 Rs are sensitive to pathway that plays an important role in recycling the inos-
both InsP3 and Ca2+ and can thus function as co-incident itol headgroup. Cells have access to three separate sources
detectors (Module 2: Figure Ca2+ -induced Ca2+ release). of inositol: recycling the second messenger InsP3 , de novo

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r42

Module 2: Figure InsP3 and DAG formation

Agonist

Receptor
PtdIns PtdIns4P PtdIns4,5P 2 Diacylglycerol

Plasma
membrane

Gq/11 PLC PKC

P P P OH
PtdIns4P PtdIns4,5P
2
Pase Pase
P P P

Protein
PtdIns PtdIns4P phosphorylation
4-kinase 5-kinase P

InsP P P
3

Calcium signalling

Agonist-dependent formation of the second messengers inositol 1,4,5-trisphosphate (InsP3 ) and diacylglycerol (DAG).
The inositol lipids that function in signalling are embedded in the inner leaflet of the plasma membrane. The precursor lipid is phosphatidylinositol
(PtdIns), which is successively phosphorylated, first on the 4-position to form PtdIns4P and then on the 5-position to form PtdIns4,5P2 . Activated
cell-surface receptors are coupled through the G protein Gq/11 to phospholipase C (PLC) that hydrolyses PtdIns4,5P2 to generate inositol 1,4,5-
trisphosphate (InsP3 ). InsP3 activates Ca2+ signalling, and diacylglycerol (DAG) stimulates protein kinase C (PKC) to initiate protein phosphorylation
(Module 2: Figure PKC structure and activation). An animated version of this figure is available.

Module 2: Figure InsP3 /Ca2+ signalling functions

Proliferation
Metabolism T cell Fertilization
Contraction Liver cells Mesangial cell
Smooth muscle Exocytosis
Smooth muscle
Mesangial cell b-Cell
Brown fat cell
Modulation of L Cell
ventricular and Mast cell,
atrial cell contraction Macrophage
Parathyroid gland
Fluid secretion Piuitary cells
Intestinal cell 2+ Astrocytes
Parietal cell InsP3 /Ca Renin-producing
Pancreas granular cells
Salivary gland
Sweat gland Chemotaxis
Neutrophils
Aggregation
Osteoclast
Blood platelets
precursors (?)
Neuronal synaptic Ion channel Aldosterone
plasticity Differentiation
opening secretion Osteoclasts
Purkinje neurons Astrocytes Glomerulosa cell Brown fat cells
Hippocampal neurons T Cells

InsP3 /Ca2+ signalling functions.

The mobilization of Ca2+ by inositol 1,4,5-trisphosphate (InsP3 ) functions in the control of many different cellular processes in a wide range of cell
types.

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Module 2: Figure InsP3 /DAG recycling

Stimulus Plasma inositol

Na +
SMIT1
PtdIns PtdIns4P PtdIns4,5P PLC DAG DAG DAG MAG
2 kinase lipase
1 2
InsP3
6
G-6-PO4
Valproate InsP3 InsP 3 PA
- Inositol Pase kinase
synthase de novo
PITP synthesis
InsP 2 InsP4
InsP1 3
Lithium
Recycling
- - Inositol
monophosphatase Dietary
PITP inositol
PtdIns
Inositol
CTP
5 PtdIns CDP.DAG
PtdIns 4 CDP.DAG PA
synthetase synthetase

Endoplasmic reticulum

Inositol 1,4,5-trisphosphate (InsP3 ) and diacylglycerol (DAG) recycling.

The cell has access to three sources of inositol. It can be synthesized de novo from glucose 6-phosphate (G-6-PO4 ) by an inositol synthase, it
can come into the cell from the plasma using a sodium-dependent myo-inositol cotransporter-1 (SMIT1) or it can be obtained by recycling InsP3 .
Agonist-dependent inositol lipid metabolism occurs through a series of steps as outlined in the text.

synthesis from glucose 6-phosphate or uptake of di- by a CDP/DAG synthetase. The inositol, which can
etary inositol circulating in the plasma (Module 2: be produced through three mechanisms (de novo syn-
Figure InsP3 /DAG recycling). Drugs such as lithium thesis, recycling or uptake of dietary inositol), is at-
and valproate may control manic-depressive illness by tached to CDP/DAG by the PtdIns synthetase [cytid-
reducing the supply of inositol by inhibiting the inositol ine diphosphate (CDP)/diacylglycerol (DAG):myo-
monophosphatase and inositol synthase respectively. The inositol 3-phosphatidyltransferase] located on the en-
supply of free inositol is one of the essential precursors doplasmic reticulum (ER) to form PtdIns.
for the synthesis of PtdIns. 5. The PtdIns is then transported from the ER back to the
plasma membrane by a PtdIns transfer protein (PITP).
Synthesis of PtdIns Cells express two PITPs, an α and a β isoform, pro-
The two second messengers that are formed during phos- duced by separate genes. The latter appears to be the
phoinositide signalling can be recycled back to the pre- housekeeping isoform that is essential for cell survival,
cursor lipid PtdIns4,5P2 through a series of steps (Mod- whereas the α isoform has a more specialized function.
ule 2: Figure InsP3 /DAG recycling): When PtdIns is added back to the plasma membrane, it
can once again be converted through the two phos-
1. Conversion of PtdIns into the precursor lipid phorylation reactions to maintain the supply of the
PtdIns4,5P2 . PtdIns4,5P2 that is the precursor for the phosphoin-
2. The agonist-dependent activation of phospholipase C ositide signalling pathway.
(PLC), which hydrolyses PtdIns4,5P2 to generate the 6. Cells can also use plasma inositol, which is taken up
second messengers inositol 1,4,5-trisphosphate (InsP3 ) by a sodium-dependent myo-inositol cotransporter-1
and diacylglycerol (DAG). (SMIT1).
3. The InsP3 is recycled back to free inositol by a complex
metabolic pathway (Module 2: Figure inositol phos-
phate metabolism). DAG is phosphorylated by DAG Phospholipase C (PLC)
kinase to form phosphatidic acid (PA) or it is hy- Phospholipase C (PLC) hydrolyses the lipid precursor
drolysed by DAG lipase to form monoacylglycerol PtdIns4,5P2 to produce both InsP3 and DAG. It is made
(MAG). up of five subclasses, which have variable isoforms PLCβ
4. The PA is transferred from the plasma membrane to the (β1–β4), PLCδ (δ1–δ4), PLCγ (γ1 and γ2), PLCε and
ER where it interacts with CTP to form CDP/DAG PLCζ (Module 2: Figure PLC structure and function).

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Module 2: Figure PLC structure and function

EF hands
PLCβ(1 - 4) PH X Y C2

PLCγ(1, 2) PH X SH2 SH2 SH3 Y

PLCδ(1 - 4) PH X Y C2

PLCε RasGEF PH X Y C2 RA RA
EF hands
PLCζ X Y C2

GPCR PLCβ PLCγ PTKR PLCδ PLCε

Gα PH C2 Gβγ PH PH C2 RA PH C2
EF EF SH2 P P

BLNK
Gq EF XY + RA EF XY
XY P P
XY SH2 RasGEF
P Ras
Gα Gβγ Translocation +
IP IP IP IP
3 3 Ca 2+ 3 3
+
Ca 2+ PH Ca 2+ Ca 2+ Ca 2+
EF SH2
XY SH2

The domain structure and activation mechanisms of PLC isoforms.

The pleckstrin homology (PH) domain, four EF-hand domains, the catalytic X and Y domains, and the C2 domain are a common feature of most of
the PLC isoforms. The two PLCγ isoforms differ from the others by lacking a C2 domain and by having additional domains such as the Src homology
2 (SH2) and Src homology 3 (SH3) domains located between a split PH domain. The black triangles indicate the sites of tyrosine phosphorylation
that are important for the activation of PLCγ. PLCε also has additional domains related to its activation by Ras, such as the Ras association motifs
(RA) and the Ras guanine nucleotide exchange factor (RasGEF). The bottom panel illustrates how the various PLC isoforms are activated by different
mechanisms. Not included in this figure is the action of PLCζ, which is unusual in that it is activated at fertilization following its injection into the oocyte
(Module 8: Figure mammalian fertilization).

All forms of the enzyme have an absolute requirement PLCβ isoform distribution and function
for Ca2+ that plays a critical role in the catalytic site. The
PLCβ1 is highly concentrated in brain (pyramidal cells of
PtdIns4,5P2 is cleaved through two sequential reactions:
hippocampus, Purkinje cells and granule cells). The knock-
first, the phosphodiester bond is cleaved to DAG and in-
out phenotype is characterized by seizures leading to sud-
ositol 1,2-cyclic phosphate, the latter is then hydrolysed to
den death. These seizures are similar to those seen dur-
give the acyclic InsP3 that is released into the cytoplasm.
ing epilepsy. Many of the defects are found in the central
The domain structure of the different PLC isoforms re-
nervous system (CNS). Activation of PLC by muscarinic
veals a number of common structural features related to
receptors is suppressed in the temporal lobe, cerebellum
the way the enzyme associates with the membrane and
and hippocampus, and this could decrease the inhibitory
functions to hydrolyse PtdIns4,5P2 . The catalytic domain
tone, leading to the seizures.
is made up from the X and Y regions. They have at least
PLCβ2 is mainly expressed in cells of the immune sys-
two potential lipid-binding domains: the pleckstrin homo-
tem. Knockout mice show some disruption of chemokine
logy (PH) domain and the C2 domain (Module 6: Figure
signalling. For example, neutrophils fail to respond to
modular lipid-binding domains). In the case of PLCδ1, the
the chemoattractant fMet-Leu-Phe with the usual changes
enzyme may first associate with the membrane through its
in PLC activation, Ca2+ release and superoxide radical
PH domain, which has a high affinity for PtdIns4,5P2 . Fur-
(O2 −• ) production. However, the response to lipopolysac-
ther interactions may then occur through the C2 domain,
charide (LPS) is normal. Despite this absence of PLC activ-
which has an extensive interface with the catalytic domain
ation in leucocytes, chemotactic responses are enhanced,
and may thus enable the catalytic site to integrate itself into
suggesting that this signalling pathway may normally ant-
the membrane to hydrolyse the lipid. The main difference
agonize the signalling pathways normally used to control
between these PLC isoforms concerns the way in which
chemotaxis.
they are activated.
PLCβ3 is found mainly in brain, parotid, smooth
Phospholipase Cβ (PLCβ) muscle and liver. Disruption of this gene is lethal, with
The PLCβ isoforms are mainly activated by G protein- the mice dying by day 2.5. The embryos are highly dis-
coupled receptors (GPCRs). PLCβ1 and PLCβ3 are fairly organized and have low cell numbers, suggesting a role
ubiquitous, whereas PLCβ2 and PLCβ4 have more lim- for this isoenzyme in cell division. This isoform has two
ited tissue distributions. sites (Ser-26 and Ser-105) that are phosphorylated by cyclic

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r45

GMP-dependent protein kinase (PKG) resulting in a de- Modulation of PLCβ by other signalling pathways
crease in enzyme activity (Module 7: Figure smooth muscle In general, the phosphorylation of PLCβ, particularly iso-
cell cyclic GMP signalling). forms 2 and 3, by protein kinase A (PKA) results in inhib-
PLCβ4 is found in cerebellum and granule cells. Knock- ition of the enzyme.
out mice appear to have defects in the cerebellum, result-
ing in poor motor co-ordination due to a decrease in PLC Phospholipase Cγ (PLCγ)
stimulation through metabotropic glutamatergic and mus- PLCγ functions predominantly in early development and
carinic receptors. There also are defects in the processing in the signalling pathways that control cell proliferation.
of visual information. PLCγ1 knockout mice die at embryonic day 9, by which
The primary activation mechanism of the PLCβ iso- time the embryos appear normal, albeit somewhat smaller.
forms is through the Gq family of heterotrimeric G pro- PLCγ is characterized by having a large insert between
teins (Gq , G11 , G14 , G15 and G16 ) (Module 2: Figure PLC the X and Y domains (Module 2: Figure PLC structure and
structure and function). While Gq and G11 are found function). This insert contains an additional pleckstrin ho-
in most tissues, the other three are restricted to cells of mology (PH) domain, which is itself split by another insert
haematopoetic origin. The function of these G proteins containing two Src homology 2 (SH2) domains and a single
is complicated because the PLCβ isoforms are sensitive Src homology 3 (SH3) domain. The SH2 domains play a
to both the α and βγ components of the heterotrimeric critical role in the activation of PLCγ because they provide
complex. PLCβ1 and PLCβ4 are most sensitive to stim- a docking module that enables the enzyme to translocate to
ulation through α subunits, whereas βγ is more effective the membrane to dock to phosphorylated tyrosine residues
at activating PLCβ2 and PLCβ3. This sensitivity to βγ on activated receptors or associated scaffolding proteins.
subunits may explain the pertussis-toxin-sensitive stimu- This tyrosine phosphorylation occurs during activation of
lation of PLC by the Gi family of G proteins. For example, protein tyrosine kinase-linked receptors (PTKRs) or the
the adenosine A1 , muscarinic M2 , somatostatin and µ-, δ- non-receptor protein tyrosine kinases (e.g. Src, Syk, Btk,
and κ-opioid receptors can couple to PLC through Gi and Lck, Fyn). This sequence of events is well illustrated by
Go . Of the five Gβ and 11 Gγ subunits, there appears to the activation of PLCγ1 during activation of the T cell re-
be little specificity with regard to the activation of PLC. ceptor (Module 9: Figure TCR signalling) or PLCγ2 fol-
In general, Gβγ is less efficacious than Gα in stimulating lowing stimulation of the B cell receptor (BCR) (Module 2:
PLC. Figure ROS effects on Ca2+ signalling). This translocation
The two G protein subunits interact with PLCβ at dif- to the cell surface has two important consequences for the
ferent sites. The α subunit interacts with a region within activation of the enzyme. Firstly, it brings the enzyme close
the long C-terminal tail, whereas the βγ subunit interacts to its substrate in the membrane. Secondly, by interacting
with a site within the Y region of the catalytic site (Module with tyrosine kinases, PLCγ1 is itself phosphorylated on
2: Figure PLC structure and function). tyrosine residues located at positions 771, 783 and 1254
PLCβ also contains GAP (GTPase-activating protein) (black triangles in Module 2: Figure PLC structure and
activity that stimulates the intrinsic GTPase activity of the function). Phosphorylation at Tyr-783 is particularly im-
α subunit. When an agonist binds to the GPCRs, it induces portant for switching on enzyme activity. The PLCγ2 iso-
a conformational change that is transmitted to the under- form is activated by phosphorylation of tyrosine residues
lying G protein, causing it to dissociate. At this time the 753 and 759, mainly by the tyrosine kinase Btk (Module
GDP bound to the α subunit is exchanged for GTP. The 2: Figure ROS effects on Ca2+ signalling).
active GTP/α subunit complex can then activate PLCβ The enzymatic activity of PLCγ is also stimulated by
and this activation process is terminated when the intrinsic the PtdIns3,4,5P3 produced by the PtdIns 3-kinase sig-
GTPase activity of the α subunit hydrolyses GTP back nalling pathway and thus represents a major point of in-
to GDP allowing the GDP/α subunit to once again bind teraction between these two signalling pathways. This in-
to the βγ subunit to form the inactive complex. The in- teraction is particularly important during lymphocyte ac-
trinsic GTPase activity of the α subunit is rather low, but tivation, where the Tec tyrosine kinase family are activ-
is greatly enhanced by two mechanisms. There is a GAP ated by PtdIns3,4,5P3 to phosphorylate and activate both
activity associated with the long C-terminal tail of PLCβ. PLCγ1 and PLCγ2. Both isoforms bind strongly to this
Therefore PLCβ plays a direct role in terminating its own highly charged lipid through the N-terminal PH domain,
activity. In addition, there are regulators of G protein sig- and this interaction enhances enzymatic activity.
nalling (RGS) proteins. There are about 20 of these RGS The Src homology 3 (SH3) domain, which binds to
proteins, of which RGS2–RGS4 seem particularly effect- proline-rich sequences, may enable PLCγ to bind to other
ive in interacting with Gαq . The GTPase activity of Gαq components, such as the cytoskeleton and the protein
is enhanced 25-fold by RGS4. There is considerable dis- dynamin.
crimination with regard to how individual RGS proteins
bind to specific receptor/Gαq complexes enabling them Phospholipase Cδ (PLCδ)
to exert an agonist-specific inhibitory mechanism. This Less is known about the activation of PLCδ. Unlike PLCβ
mechanism could explain the different types of Ca2+ sig- and PLCγ, PLCδ appears not to be regulated by receptors
nals observed in hepatocytes following stimulation with directly. Once PLCδ1 has associated with the membrane,
different agonists. it appears to be activated by an elevation in cytosolic Ca2+ .

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r46

This sensitivity to changes in intracellular Ca2+ is thought The activation of these different PKC families is com-
to depend on the C2 domain (Module 2: Figure PLC struc- plex in that it depends on a number of processes such
ture and function). as priming, translocation and association with scaffolding
Crystallographic studies of PLCδ1 have begun to reveal proteins. The priming process is driven by a sequence of
how PLC enzymes function to cleave their lipid substrates multisite phosphorylation events that have to occur before
that are embedded in the plasma membrane. the enzyme can perform its signalling function. The newly
synthesized proteins are inactive and undergo a prim-
Phospholipase Cε (PLCε) ing process that begins when phosphoinositide-dependent
Phospholipase Cε (PLCε) differs from the other isoforms protein kinase 1 (PDK1), which is part of the PtdIns 3-k-
by having two Ras-association domains (RA) at its C-ter- inase signalling pathway, phosphorylates a site in the C4
minus (Module 2: Figure PLC structure and function). domain (Module 2: Figure PKC structure and activation).
It binds specifically to the GTP-bound forms of Ha-Ras This is then followed by intramolecular autophosphoryla-
and Rap1A. The enzyme is targeted to the membrane tion reactions of two further sites in the C-terminal tail.
through its ability to bind to these G proteins. It also has The primed enzyme is now ready to perform its signalling
a Cdc25 homology domain that is a guanine nucleotide function by responding to both diacylglycerol (DAG)
exchange factor (GEF) motif for Rap1, which enables it to and Ca2+ . The cPKCs and most of the nPKCs are lipid-
translocate to the perinuclear region following epidermal sensitive enzymes in that they are activated by DAG that
growth factor (EGF) stimulation, where it can activate the binds to the C1 domain. This C1 domain is also respons-
mitogen-activated protein kinase (MAPK) signalling path- ible for binding the tumour-promoting phorbol esters. In
way. There is evidence that cyclic AMP acting through the the case of the cPKCs, Ca2+ also plays an important role
exchange protein activated by cAMP (EPAC) can stimu- as a cofactor that binds to the C2 domain to increase its
late Rap1 to activate PLCε (Module 2: Figure cyclic AMP affinity for acidic phospholipids and is thus responsible for
signalling). inducing the translocation of cPKCs to the plasma mem-
brane.
Phospholipase Cζ (PLCζ)
The activation of the cPKC isoforms follows a set
Phospholipase Cζ (PLCζ) has a restricted expression in
sequence of events that begins with the hydrolysis of
that it is only found in mammalian sperm. PLCζ lacks
PtdIns4,5P2 to form DAG and inositol 1,4,5-trisphosphate
the N-terminal PH domain (Module 2: Figure PLC struc-
(InsP3 ). The InsP3 releases Ca2+ , which initiates the activa-
ture and function), which means that there is no obvious
tion process by binding to the C2 domain to induce a con-
mechanism for it to bind to phospholipids. PLCζ has a
formational change that greatly enhances the membrane
sensitivity to Ca2+ that is 100-fold greater than that of
affinity of cPKC and thus promotes its translocation to
PLCδ1. Both the C2 and the tandem EF-hands are neces-
the membrane where it is activated when it makes contact
sary for PLCζ to hydrolyse PtdIns4,5P2 to generate the
with DAG. The pseudosubstrate (PS) domain swings away
InsP3 that plays a central role in sperm-induced oocyte ac-
from the active site, which is now free to phosphorylate its
tivation during fertilization (Module 8: Figure mammalian
substrates.
fertilization).
The nPKCs, which are also activated by DAG, have
Diacylglycerol (DAG)/protein kinase C (PKC) a much slower activation process because the Ca2+ -
signalling cassette dependent facilitation of membrane translocation is
The diacylglycerol (DAG) that is formed when absent.
PtdIns4,5P2 is hydrolysed by phospholipase C (PLC) re-
mains within the plane of the membrane where it acts as a Protein kinase Cζ (PKCζ)
lipid messenger to stimulate some members of the protein This is one of the atypical PKC isoforms (aPKC),
kinase C (PKC) family (Module 2: Figure InsP3 and DAG which fails to respond to diacylglycerol (DAG) or Ca2+ ,
formation). This PKC family contains a number of iso- but is sensitive to low levels of ceramide (Module 2:
forms with diverse structures and activation mechanisms. Figure sphingomyelin signalling). PKCζ is also activ-
The PKC signalling function is equally diverse, and it has ated following phosphorylation by kinases from other
proved difficult to pin down its precise function in the signalling pathways. For example, PKCζ is activated
regulation of specific cellular processes. by phosphoinositide-dependent kinase 1/2 (PDK1/2)
(Module 2: Figure PtdIns 3-kinase signalling). In some
Protein kinase C (PKC) cells, it is responsible for activating nuclear factor κB (NF-
The protein kinase C family has been divided into three κB). PKCζ can also phosphorylate the glucose transporter
subgroups (Module 2: Figure PKC structure and activa- in skeletal muscle (Module 7: Figure skeletal muscle E-C
tion): coupling).

• Conventional PKCs (cPKCs: α, β1, β2 and γ) contain Protein kinase Cθ (PKCθ)

C1 and C2 domains Protein kinase Cθ (PKCθ) is one of the novel PKCs
• Novel PKCs (nPKCs: δ, ε, η and θ) contain a C1 and a (nPKCs) that plays an important role in lymphocyte ac-
C2-like domain tivation where it is concentrated in the central region of
• Atypical PKCs (aPKCs: ζ, ι and λ) contain a truncated theimmunological synapse (Module 9: Figure immunolo-
C1 domain gical synapse structure).

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Module 2: Figure PKC structure and activation

DAG 2+ ATP PDK1

Ca
PS P PP Conventional (cPKC)
C1A C1B C2 C3 C4 (α, βΙ, βΙΙ, γ)
PS P PP
Novel (nPKC)
C2-like C1A C1B C3 C4
(δ, ε, θ, η)
PS P P
Atypical (aPKC)
C1 C3 C4
(ζ, ι/λ)
AGONIST

Plasma membrane
R
PLC
DAG C1 C2
PtdIns4,5P Translocation C4
2
InsP3 P P
P

C1 C2 Substrate
P
C2

C4
C4
2+ PS P P P
Ca
C1

P P P
Substrate Substrate
cPKC

Structure of PKC isoforms and activation mechanism of the conventional protein kinase C (cPKC) isoforms.
The nine genes that code for protein kinase C (PKC) fall into three families. Conventional PKCs (cPKCs) have a domain structure that has an N-terminal
pseudosubstrate (PS) domain followed by regulatory domains (C1 and C2), which are responsible for binding the diacylglycerol (DAG) and Ca2+
responsible for membrane targeting, an ATP-binding C3 domain and the C4 catalytic domain. The C1 region is present as a tandem repeat (C1A
and C1B). The PS domain has an autoinhibitory function in that it associates with the catalytic site on C4, thus preventing it from phosphorylating its
substrates. Novel PKCs (nPKCs) have a similar domain structure, except that they have a C2-like domain that does not bind Ca2+ . Atypical (aPKCs)
also lack a C2 domain, and the C1 domain is small and fails to bind DAG. The illustration at the bottom indicates how cPKC is activated, as described
in the text.

PKC signalling functions cassette, the PtdIns4P signalling cassette and the PtdIns5P
The different PKC isoforms function to regulate a wide signalling cassette.
range of cellular processes:

• PKCε functions to control the N-type Ca2+ channel PtdIns4,5P2 signalling cassette
(Module 3: Figure CaV 2 channel family). The phosphoinositide PtdIns4,5P2 is widely distributed
• PKC is one of the kinases responsible for phosphorylat- throughout the cell. Not only is it found in the plasma
ing the transcription factor p53 (Module 4: Figure p53 membrane, but also it occurs in the Golgi, endosomes,
domains). endoplasmic reticulum (ER) and within dense structures
• PKC functions to regulate the calmodulin (CaM)- within the nucleus. This lipid has two main roles. Firstly, it
binding properties of proteins such as neuromodulin is hydrolysed to form InsP3 and diacylglycerol (DAG) or it
and neurogranin. can be phosphorylated further to form the lipid messenger
• Protein kinase C functions in the modulation of CaV 1.2 PtdIns3,4,5P3 . Secondly, PtdIns4,5P2 can also function as a
L-type channels from heart muscle (Module 3: Figure lipid messenger (Module 2: Figure PtdIns4,5P2 signalling).
CaV 1.2 L-type channel). Such a signalling role is supported by the observation that
cell stimulation can result in highly localized increases in
PtdIns4,5P2 . The localized formation of this lipid messen-
Phosphoinositide lipid signalling molecules ger can be activated by the monomeric G proteins and in
Some of the phosphoinositides derived from the metabol- particular the family of Rho proteins. For example, Rac
ism of PtdIns (Module 2: Figure phosphoinositide meta- and Rho can function to target PtdIns4P 5-kinase I to the
bolism) have important signalling functions that are often plasma membrane. In addition, Rho-GTP can activate this
located in specific regions of the cell. One of the most kinase directly to form PtdIns4,5P2 (Module 2: Figure Rho
versatile systems is the PtdIns4,5P2 signalling cassette. signalling).
Another important system is PtdIns 3-kinase signalling, There is increasing evidence that a variety of cellular
where the formation of PtdIns3,4,5P3 functions as a mes- processes are activated in response to a change in the level
senger to activate a great variety of cellular processes. There of this lipid:
is increasing evidence that some of the other lipids may also
play signalling roles as described in the PtdIns3P signalling • PtdIns4,5P2 regulation of actin remodelling

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r48

Module 2: Figure PtdIns4,5P2 signalling

DAG + InsP 3 2+
Ca

PtdIns3,4,5P3
Rac Rho Arf PA
PLD PA SPHK S1P

Cytoskeleton
PtdIns PtdIns4P PtdIns4,5P2
Phagocytosis 2+
Ca

Exocytosis
(Primes vesicles)

Endocytosis
(Recruits AP-2 to
form clathrin-coated pits)

Ion channels
(Agonist-induced hydrolysis of
PtdIns4,5P2 activates TRPV1
but inhibits potassium channels)

Multiple signalling functions of PtdIns4,5P2 .

PtdIns4,5P2 has multiple signalling functions in the plasma membrane. It is the lipid precursor used for the generation of second messengers such as
InsP3 , diacylglycerol (DAG) and PtdIns3,4,5P3 . It functions in the regulation of the phospholipase D (PLD) signalling pathway. In addition, PtdIns4,5P2
can function as a messenger to control a whole variety of cellular systems, including the cytoskeleton, phagocytosis, exocytosis, endocytosis and ion
channels. All of these processes are sensitive to changes in the membrane level of this lipid.

• PtdIns4,5P2 regulation of membrane trafficking and en- stimulate members of the Rho family of G proteins by
docytosis catalysing the exchange of GDP for GTP. The active G
• PtdIns4,5P2 regulation of exocytosis proteins can then have a number of functions. For example,
• PtdIns4,5P2 activation of phospholipase D one function of Rac-GTP is to activate actin polymeriz-
• PtdIns4,5P2 regulation of ion channels and exchangers ation (Module 2: Figure Rac signalling). Similarly, Cdc42
• PtdIns4,5P2 function in focal adhesions also stimulates actin remodelling (Module 2: Figure Cdc42
• PtdIns4,5P2 regulation of phagocytosis signalling). The consequence of this remodelling varies de-
pending on which G protein is operating. For example,
the Cdc42 and Rac polymerization of actin results in the
PtdIns4,5P2 regulation of actin remodelling formation of filopodia and ruffles respectively (Module 4:
There are a number of cellular processes, such as chemo- Figure actin remodelling).
taxis, locomotion, phagocytosis, shape change and cy- The formation of PtdIns4,5P2 contributes to actin
tokinesis, where there are rapid changes in the cytoskel- remodelling by acting on a number of the mol-
eton in response to external stimuli such as growth factors ecules that control actin polymerization such as the
and cytokines. Phosphoinositides play a significant role in Wiskott-Aldrich syndrome protein (WASP) and the re-
transmitting information from cell-surface receptors to the lated Wiskott-Aldrich syndrome protein (WASP) verpro-
battery of proteins that are responsible for actin remodel- lin homologous (WAVE) proteins. WASP and WAVE relay
ling. Its main actions are to uncap the barbed end of actin information from the upstream signalling elements to the
filaments and to facilitate actin nucleation and polymeriz- downstream cytoskeletal regulators. Perhaps the most im-
ation by controlling a variety of actin regulatory proteins. portant example of the latter is the actin-related protein
A critical component of the signalling network is the Rho 2/3 (Arp2/3) complex (containing seven strongly associ-
family of monomeric G proteins (Rho, Cdc42 and Rac) ated subunits of which two are the actin-related proteins
that relay information from various signalling pathways Arp2 and Arp3) that is responsible for catalysing the poly-
such as the PtdIns 3-kinase signalling pathway to the ef- merization of actin. PtdIns4,5P2 appears to have two ac-
fector molecules that are directly responsible for remodel- tions. It binds to and activates WASP and to Scar and
ling the actin network. One of the signalling molecules is thus contributes to the relay of information to Arp2/3. In
PtdIns4,5P2 . addition, it can alter the activity of various proteins that
The signalling cascade begins when external signals act- modify the structure of actin. For example, it is an indirect
ing through guanine nucleotide exchange factors (GEFs) activator of Cdc42 and it may loosen the gelsolin caps on

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r49

the end of the barbed ends, opening up new sites for actin PtdIns4,5P2 regulation of ion channels and exchangers
polymerization. The PtdIns4,5P2 may also induce a con- A number of ion channels and exchangers appear to be
formational change in vinculin that enables it to bind to regulated by PtdIns4,5P2 . In some cases, the hydrolysis of
talin during the formation of the focal adhesion complex PtdIns4,5P2 results in channel opening as occurs for the
(Module 6: Figure vinculin function). transient receptor potential (TRP) vanilloid 1 (TRPV1)
channel, whereas the TRPC6 channel, TRP melastatin 4
PtdIns4,5P2 function in focal adhesions (TRPM4), TRP melastatin 7 (TRPM7) and TRP melast-
The Iγ isoform of PtdIns4P 5-kinase (PtdIns4P 5-K) is atin 8 (TRPM8) channels are closed (Module 2: Figure
highly localized at the focal adhesion complex, where it PtdIns4,5P2 regulation of TRP channels). In the case of
is tightly bound to talin to regulate the local formation of the inward rectifying K+ channels, lipid hydrolysis results
PtdIns4,5P2 (Module 6: Figure integrin signalling). in channel opening (Module 2: Figure PtdIns4,5P2 regula-
tion of K+ channels). Modulation of the TRPV1 channel is
particularly important in hyperalgesia (Module 10: Figure
PtdIns4,5P2 regulation of exocytosis
nociception).
The synthesis of PtdIns4,5P2 has been implicated in the
Much of the information on this regulatory function
ATP-dependent processes of priming vesicles as part of
of PtdIns4,5P2 has emerged from studies on K+ chan-
the exocytotic mechanism. The priming process seems to
nels. Stimulation of phospholipase C (PLC) by G protein-
depend upon a number of steps that begin with the PtdIns
coupled receptors (GPCRs) causes a reduction in the level
transfer protein carrying PtdIns to the vesicle membrane
of PtdIns4,5P2 , which results in a reduction in the activity
where it is phosphorylated to PtdIns4P by PtdIns 4-kinase
of some of the voltage-dependent K+ (Kv ) channels:
(PtdIns 4-K) and then to PtdIns4,5P2 by the PtdIns4P
5-kinase (PtdIns4P 5-K). It is still not clear exactly how this
PtdIns4,5P2 functions to prime the vesicle for exocytosis. • The human ether-a-go-go-related (HERG) K+ channel
The ability of the neuronal Ca2+ sensor-1 (NCS-1) to (Kv 11.1 channel)
facilitate exocytosis may depend on its ability to activate • The Kv 7.1 channel, which is coded for by the KCNQ1
the PtdIns 4-kinase (PtdIns 4-K). gene, is regulated by PtdIns4,5P2 . This inositol lipid
keeps the channel open, but when PtdIns4,5P2 is hy-
drolysed by angiotensin II, the channel closes. Such
PtdIns4,5P2 regulation of membrane trafficking and lipid regulation is seen in adrenal zona glomerulosa cells
endocytosis (Module 7: Figure glomerulosa cell signalling) and also
PtdIns4,5P2 plays a role in endocytosis by targeting in the heart. Mutation of KCNQ1 causes long QT) syn-
clathrin-associated proteins to endocytic vesicles, leading drome (LQT).
to the formation of clathrin-coated pits. • The heteromultimer KCNQ2/3 is responsible for the
The PtdIns4P 5-kinase (PtdIns4P 5-K) Iβ is recruited to M channel in neurons and has been linked to a form of
the plasma membrane, where it phosphorylates PtdIns4P epilepsy known as benign familial neonatal convulsions.
to PtdIns4,5P2 , which recruits the adaptor protein AP-2 • KCNQ4, which codes for Kv 7.4 (Module 3: Table
to form clathrin-coated pits. voltage-dependent K+ channels), is the delayed rectifier
that controls K+ efflux from sensory hair cells of the in-
PtdIns4,5P2 regulation of phagocytosis ner ear. KCNQ4 mutations cause autosomal dominant
When macrophages engulf foreign particles, PtdIns4P 5-k- nonsyndromic deafness type 2 (DFNA2).
inase (PtdIns4P 5-K) Iα is recruited to the plasma mem-
brane at the phagosome cup, where it induces a local pulse
of PtdIns4,5P2 , which is then rapidly degraded by phos-
PtdIns 3-kinase signalling
The PtdIns 3-kinase signalling cassette, which generates
pholipase C (PLC) to leave behind diacylglycerol (DAG).
the lipid second messenger phosphatidylinositol 3,4,5-
This local formation of PtdIns4,5P2 seems essential for the
tetrakis phosphate (PtdIns3,4,5P3 ), has multiple functions
early process of phagocytosis.
in regulating a wide range of cellular processes such as the
The localized transient increase in PtdIns4,5P2 may
control of glycogen metabolism, lipid synthesis, protein
function in the initial recruitment of actin to the phagocyte.
synthesis, gene transcription and cell growth, inflamma-
tion, cytoskeletal rearrangement and apoptosis. With re-
PtdIns4,5P2 activation of phospholipase D gard to the latter, the PtdIns 3-kinase signalling cassette has
Phospholipase D (PLD), which catalyses the hydrolysis a special function in cell survival through its contribution
of phosphatidylcholine (PC) to generate phosphatidic acid to the hormonal modulation of apoptosis and by enhan-
(PA), exists as two isoforms, PLD1 and PLD2. The PLD1 cing the activity of the target of rapamycin (TOR). PtdIns
is primarily located on vesicles associated with the en- 3-kinase signalling is one of the major pathways used by
dosomal/lysosomal pathway, whereas PLD2 is mainly the insulin receptor to regulate energy uptake and storage.
found on the plasma membrane. An interaction between This lipid signalling pathway has a special relation-
PtdIns4P 5-kinase α (PtdIns4P 5-Kα) and the PLDs en- ship to Ca2+ signalling in that it can regulate the way in
sures that there is a local generation of PtdIns4,5P2 that which external signals can maintain information flowing
plays an important role in regulating PLD activity (Module through the inositol 1,4,5-trisphosphate (InsP3 )/Ca2+ sig-
2: Figure PLD signalling). nalling cassette.

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Module 2: Figure PtdIns4,5P2 regulation of K+ channels

AGONIST
Channel open + Channel closed
membrane K membrane
hyperpolarized depolarized

R
PtdIns PLC
PtdIns4P
4-Kinase 5-Kinase PtdIns4,5P
PtdIns PtdIns4P 2

InsP3
DAG

DAG
Inositol Kinase
phosphate
metabolism
PtdIns
Inositol
Phosphatidic
PtdIns acid
synthase

Regulation of K+ channels by agonist-dependent hydrolysis of PtdIns4,5P2 .

A number of different K+ channels are activated when they bind to PtdIns4,5P2 . Upon stimulation by agonists that are coupled to phospholipase C
(PLC), this lipid is hydrolysed to InsP3 and diacylglycerol (DAG), and this removal causes the channel to shut. The channel will open again when
another PtdIns4,5P2 molecule associates with the lipid-binding site. The supply of PtdIns4,5P2 is maintained by the resynthesis of PtdIns and its
rephosphorylation by PtdIns 4-kinase and then by PtdIns4P 5-kinase (Module 2: Figure InsP3 /DAG recycling).

Module 2: Figure PtdIns4,5P2 regulation of TRP channels

AGONIST Cations
TRPC6
TRPM4 TRPM4
TRPM7 TRPM7
TRPV1 TRPM8
TRPM8
opens closes
TRPV1
R
PLC
PtdIns PtdIns4P
4-Kinase 5-Kinase PtdIns4,5P
PtdIns PtdIns4P 2

InsP3

DAG
Inositol
phosphate
metabolism DAG
Kinase
PtdIns
Inositol
PtdIns Phosphatidic
synthase acid

Regulation of TRP channels by agonist-dependent hydrolysis of PtdIns4,5P2 .

A number of transient receptor potential (TRP) channels are opened (TRPM4, TRPM7 and TRPM8) or closed (TRPV1) when they are bound to
PtdIns4,5P2 . Upon stimulation by agonists that are coupled to phospholipase C (PLC), this lipid is hydrolysed, and its removal causes the channel to
open (TRPV1) or closed (TRPM4, TRPM7 and TRPM8). The channel returns to the resting state when PtdIns4,5P2 re-associates with the lipid-binding
site. The supply of PtdIns4,5P2 is maintained by the resynthesis of PtdIns and its sequential phosphorylation by PtdIns 4-kinase and PtdIns4P 5-kinase.
The operation of this signalling system is particularly important in nociception (Module 10: Figure nociception).

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Module 2: Figure PtdIns 3-kinase signalling

Growth factor

PtdIns3,4,5P3 GPCR
PTKRs
Ras
P P Ras
p110γ
p101/ β/γ
P P p85 p110 p84
PtdIns- α,β,δ PtdIns-
P P Class IB
4,5P2 Class IA PI 3-K 4,5P2
PI 3-K
PDK1/2 Rac
Rho Superoxide
formation
Cdc42
Btk PKB
Itk

Cytoskeletal
rearrangement
PLCγ
PKCs
δ,ζ S6K TOR BAD GSK3 FOXO
IP DAG
3
Protein Cell Glycogen Gene
transcription
Ca 2+ PKCs synthesis survival metabolism
α,β

The PtdIns 3-kinase signalling pathway.

The precursor lipid PtdIns4,5P2 is phosphorylated on the 3-position by Class I PtdIns 3-kinases (PtdIns 3-Ks) to generate the lipid second messenger
PtdIns3,4,5P3 . The Class 1A enzyme has regulatory subunits such as p85 that attaches the catalytic p110 subunits to the phosphorylated tyrosine
residues on the cytoplasmic domains of activated growth factor receptors. The Class 1B enzymes (p110γ), which are activated by G protein-coupled
receptors (GPCRs), translocate to the membrane by binding to the Gβγ subunit. When brought into the vicinity of the membrane, these PtdIns 3-kinases
form the 3-phosphorylated lipid messenger PtdIns3,4,5P3 , to regulate a large number of processes. It binds to other signalling components such
as Btk and PLCγ (shown in pink). It stimulates phosphoinositide-dependent kinase 1/2 (PDK1/2) and protein kinase B (PKB), which activate a large
number of downstream targets (yellow). It also activates monomeric G proteins (Rac, Rho and Cdc42) to stimulate both cytoskeletal rearrangement
and superoxide radical (O2 −• ) formation (shown in green).

Operation of the PtdIns 3-kinase signalling cassette can phatases or by phosphatase and tensin homologue deleted
be considered in two parts: on chromosome 10 (PTEN).

• Generation and metabolism of the 3-phosphorylated

lipid messengers Mode of action of the 3-phosphorylated lipid
• Mode of action of the 3-phosphorylated lipid messen- messengers
gers The lipid messengers PtdIns3,4P2 and PtdIns3,4,5P3 act
One of the important functions of this pathway is to reg- within the plane of the plasma membrane by binding to a
ulate cell proliferation. Since a number of human cancers great variety of target proteins (Module 2: Figure PtdIns
are found to have mutations in certain components of this 3-kinase signalling). Most of these downstream targets are
pathway, there is considerable interest in the relationship soluble proteins that translocate to the membrane by bind-
between PtdIns 3-kinase signalling and cancer. ing to the lipid messengers through various lipid-binding
domains [e.g. pleckstrin homology (PH), Phox homology
Generation and metabolism of the 3-phosphorylated (PX), C2 domains and basic amino acid regions] (Mod-
lipid messengers ule 6: Figure modular lipid-binding domains). Once drawn
Cells contain a number of phosphoinositides carrying a on to the membrane, these target proteins are activated and
phosphate on the 3-position (Module 2: Figure phosphoin- function as effectors to control a large number of cellular
ositide metabolism). One of these is PtdIns3,4,5P3 , which processes.
is the main second messenger operating in the PtdIns
3-kinase signalling pathway. This highly phosphorylated • Contribution to liver cell signalling mechanisms to con-
lipid is generated in cells following activation of either trol glycogen metabolism (Module 7: Figure liver cell
G protein-coupled or tyrosine kinase-coupled receptors signalling).
(Module 2: Figure PtdIns 3-kinase signalling). • Functions in the insulin control of skeletal muscle gly-
These 3-phosphorylated lipid messengers are metabol- cogen synthesis (Module 7: Figure skeletal muscle E-C
ized either by the type II inositol polyphosphate 5-phos- coupling).

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r52

• Functions in white fat cells to control lipid metabolism (TSC1/2), which integrate a number of inputs that control
(Module 7: Figure lipolysis and lipogenesis). the activity of the target of rapamycin (TOR) (Module
• Functions in the control of cell proliferation (Module 9: 9: Figure target of rapamycin signalling). PKB can also
Figure growth factor signalling). translocate into the nucleus, where it phosphorylates the
• PtdIns 3-kinase signalling in cardiac hypertrophy is an Forkhead box O (FOXO) transcription factors (Module 4:
example where this signalling system carries out mul- Figure FOXO control mechanisms) that play an important
tiple roles in the same cell (i.e. inhibits apoptosis, ac- role in regulating the cell cycle.
tivates protein synthesis and facilitates a programme
of foetal gene transcription (Module 12: Figure hyper-
Ribosomal S6 protein kinase 1 (S6K1)
trophy signalling mechanisms).
This kinase plays an important role in regulating protein
• Modulation of InsP3 -induced Ca2+ release by phos-
synthesis by phosphorylating the S6 ribosomal protein
phorylation of the InsP3 receptor, and this could provide
(a component of the 40S ribosomal subunit), which then
a possible mechanism for the action of insulin in liver
enhances the translation of those mRNA transcripts that
cells (Module 7: Figure liver cell signalling). 
contain a polypyrimidine tract at the 5 transcriptional start
• PtdIns3,4,5P3 activates the monomeric G proteins Rac
site. The control of S6K1 is complex in that it depends upon
(Module 2: Figure Rac signalling), Rho (Module 2: Fig-
a priming step that is followed by series of phosphoryla-
ure Rho signalling) and Cdc42 (Module 2: Figure Cdc42
tion events at multiple sites that are sensitive to a number of
signalling).
kinases. The priming step depends on Ca2+ , which appears
• Formation of osteoclast podosomes (Module 7: Figure
to act by opening up the enzyme so that it becomes sensit-
osteoclast podosomes).
ive to phosphorylation by different kinases. One of these is
• Contributes to the amplification of the early polarity
phosphoinositide-dependent kinase 1 (PDK1), which car-
signalling during neutrophil chemotaxis (Module 11:
ries out the wortmannin-sensitive phosphorylation. In ad-
Figure neutrophil chemotactic signalling).
dition there are rapamycin-sensitive sites phosphorylated
• Functions as a regulator of autophagy (Module 11: Fig-
by the target of rapamycin (TOR) (Module 9: Figure target
ure autophagy).
of rapamycin signalling).
Phosphoinositide-dependent kinase 1 (PDK1)
One of the main functions of phosphoinositide-dependent Glycogen synthase kinase-3 (GSK-3)
kinase 1 (PDK1) is to phosphorylate protein kinase B One of the primary functions of insulin is to act through
(PKB), which translocates to the membrane, where it binds glycogen synthase kinase-3 (GSK-3) to stimulate the con-
to PtdIns3,4,5P3 . This binding alters the conformation of version of glucose into glycogen by increasing the activity
PKB so that critical sites become available to PDK1. Like of glycogen synthase (Module 7: Figure skeletal muscle
PKB, PDK1 has pleckstrin homology (PH) domains that E-C coupling). GSK-3 is a proline-directed protein kinase
also bind to the 3-phosphorylated lipid messengers that that usually requires a priming kinase to add a phosphate
serve to activate their kinase activity. PDK1 is also re- to its substrate before it can carry out further phosphoryla-
sponsible for phosphorylating and activating other sig- tions. Casein kinase I (CKI) often acts as the priming kinase
nalling molecules such as ribosomal S6 protein kinase 1 as it does for β-catenin in the Wnt signalling pathway
(S6K1) and atypical protein kinase Cζ (PKCζ). (Module 2: Figure Wnt canonical pathway).
GSK-3 also has a number of other signalling func-
Protein kinase B (PKB) tions:
Protein kinase B (PKB), which is also known as Akt, is
a serine/threonine protein kinase. PKB has three mem-
bers (PKBα, PKBβ and PKBγ) that are activated through • It phosphorylates nuclear factor of activated T cells
a two-stage process. Firstly, it translocates to the mem- (NFAT) and thus contributes to the NFAT shuttle
brane by binding to either PtdIns3,4P2 or PtdIns3,4,5P3 (Module 4: Figure NFAT activation).
through pleckstrin homology (PH) domains. The latter • It contributes to the Wnt signalling pathway (Mod-
appears to be particularly important when cells are stud- ule 2: Figure Wnt canonical pathway).
ied in vivo. The next stage depends upon its interaction • It functions in dorsoventral specification during devel-
with phosphoinositide-dependent kinase 1 (PDK1), which opment (Module 8: Figure dorsoventral specification).
then completes the activation process by phosphorylat- • It is one of the kinases that phosphorylates the tran-
ing PKB on Thr-308. In addition, a DNA-dependent scription factor p53 (Module 4: Figure p53 domains).
protein kinase (previously called PDK2) phosphorylates • It contributes to Myc degradation.
Ser-473. The activated PKB then functions to stimulate • It phosphorylates the neuron-specific microtubule-
a variety of molecular targets, including glycogen syn- associated protein tau, which forms tangles in neurons
thase kinase-3 (GSK-3) (which mediates the effect of during the onset of Alzheimer’s disease (Module 12:
insulin on glycogen metabolism) and the pro-apoptotic Figure amyloid plaques and tangles).
factor Bad. PKB also plays a role in redox signalling in • It plays a significant role in cardiac gene transcription,
apoptosis. where it has an important role in the NFAT shuttle
PKB is a key player in cell growth control, where it (Module 12: Figure hypertrophy signalling mechan-
functions by phosphorylating tuberous sclerosis 1 and 2 isms).

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Module 2: Figure insulin receptor

α 1
Cysteine-rich domain
L1

Fibronectin III repeat

L2 Insulin Tyrosine kinase domain

β
SS SS SS SS
SS SS

PtdIns4,5P PtdIns3,4,5P
2 3
972 P 5
P IRS
P p85
1158 P P P P
1162 PI 3-K
P P
1163 P 2 P
PDK1/2
3
1328
P P
6
1334 P P 4
PKB

IRS Glycogen Glucose

synthesis entry
p85 Lipid Protein
PI 3-K synthesis synthesis

Activation of the PtdIns 3-kinase signalling pathway by the insulin receptor.

The insulin receptor is a homodimer that is connected together by disulphide bonds. Each monomer consists of an α-chain, which is extracellular
and has the insulin-binding region. The β-chain has a single membrane-spanning region with a large intracellular region that contains the tyrosine
kinase domain that is critical for the process of signal transduction that proceeds through a sequence of events as described in the text.

Tec tyrosine kinase family Inducible T cell kinase (Itk)

All members of this tyrosine kinase family are cytoso- As for Bruton’s tyrosine kinase (Btk) in B cells, inducible
lic, but they translocate rapidly to the membrane through T cell kinase (Itk) has a similar function in controlling
pleckstrin homology (PH) domains that are particularly the maturation of T cells by activating phospholipase Cγ1
sensitive to PtdIns3,4,5P3 . Once bound to the mem- (PLCγ1) (Module 9: Figure TCR signalling).
brane, these enzymes are also phosphorylated by Src
family tyrosine kinases such as Lyn and Src. Activa-
tion thus requires both translocation to the membrane
Insulin receptor
Insulin has a major role to play in regulating a variety of
and phosphorylation on tyrosine residues. One of the
cellular processes, with particular emphasis on the regula-
functions of these Tec tyrosine kinases is to enhance
tion of energy uptake and storage. The action of insulin is
Ca2+ signalling by maintaining the activity of phosphol-
carried out by the insulin receptor, which is a disulphide-
ipase Cγ (PLCγ). This enhancement of PLCγ activity
linked homodimer that belongs to the large family of re-
by the Tec kinases represents a major point of interac-
ceptors that have tyrosine kinase domains (Module 1: Fig-
tion between the inositol 1,4,5-trisphosphate (InsP3 )/Ca2+
ure tyrosine kinase-linked receptors). An important com-
signalling cassette and the PtdIns 3-kinase signalling
ponent of the signal transduction mechanism used by the
cassette.
insulin receptor is the insulin receptor substrate (IRS),
which is a classical scaffolding protein (Module 6: Figure
Bruton’s tyrosine kinase (Btk) IRS domain structure).
Bruton’s tyrosine kinase (Btk) is one of the non-receptor The function of the insulin receptor depends upon the
protein tyrosine kinases (Module 1: Figure non-re- activation of the PtdIns 3-kinase signalling pathway that
ceptor tyrosine kinases) that plays an important role occurs through the sequence of events shown in Mod-
in B cell maturation. One of the main functions of ule 2: Figure insulin receptor:
Btk is to activate PLCγ2 in B cells (Module 2: Fig-
ure ROS effects on Ca2+ signalling). It has a similar 1. Insulin binds to the extracellular domain to induce a
mode of action in the mast cell FcεRI signalling path- conformational change in the receptor resulting in the
way (Step 7 in Module 11: Figure FcεRI mast cell activation of the intracellular tyrosine kinase domains.
signalling). 2. Once activated, the tyrosine kinase domains undergo
Inactivation of Btk results in Bruton’s type X-linked autophosphorylation whereby they phosphorylate up
agammaglobulinaemia. to six tyrosine residues on the opposite β chain.

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r54

3. The insulin receptor substrate (IRS), which is a scaf- activating protein 1 (AP-1) to regulate the trafficking of
folding domain (Module 6: Figure IRS domain struc- clathrin-coated vesicles through the trans-Golgi network.
ture), attaches itself to juxtamembrane phosphotyr-
osine residue 972. Once it is drawn into the vicinity
PtdIns5P signalling cassette
of the receptor, the tyrosine kinase domain also phos-
PtdIns5P has been implicated in several signalling events.
phorylates IRS on multiple residues.
It may modulate the PtdIns 3-kinase signalling pathway
4. The phosphotyrosine residues on IRS then function as
by interfering with the metabolism of the lipid messenger
docking sites for Class IA PtdIns 3-kinase (PI 3-K).
PtdIns3,4,5P3 . It may also have a signalling role within the
5. The PI 3-K then phosphorylates PtdIns4,5P2 to form
nucleus to control the response to DNA damage. PtdIns5P
the lipid second messenger PtdIns3,4,5P3 .
located on chromatin may provide an anchor to bind in-
6. The PtdIns3,4,5P3 then activates the PtdIns 3-kinase
hibitor of growth family, member 2 (ING2), which then
signalling pathway (Module 2: Figure PtdIns 3-kinase
results in acetylation of the p53 tumour suppressor.
signalling) that controls a number of cellular processes:
7. It stimulates lipogenesis in white fat cells (Module 7:
Figure lipolysis and lipogenesis). Multipurpose inositol polyphosphate signalling
8. It stimulates glucose uptake and glycogen synthesis in pathway
skeletal muscle cells (Module 7: Figure skeletal muscle The process of inositol phosphate metabolism generates
E-C coupling). a large number of inositol phosphates (Module 2: Figure
9. It stimulates glycogen synthesis in liver cells (Mod- inositol phosphate metabolism). Many of these are meta-
ule 7: Figure liver cell signalling). bolic intermediates, but some have been implicated in a
variety of control functions.
The onset of diabetes (i.e. Type 2 diabetes) begins when
cells become resistant to the action of insulin in energy
uptake and storage. Ins1,4P2
There is some evidence to suggest that Ins1,4P2 may func-
tion within the nucleus to activate DNA polymerase. This
PtdIns3P signalling cassette inositol phosphate has also been implicated in Ca2+ sig-
PtdIns3P, which is formed by the phosphorylation of nalling and cardiac hypertrophy.
PtdIns by the type III PtdIns 3-kinase, has a specific role
to play in controlling a number of cellular processes:
Ins1,3,4P3
• One of the main functions of PtdIns3P is to regulate This inositol phosphate has an important signalling func-
intracellular vesicle trafficking. Most of the PtdIns3P tion as a negative regulator of the Ins1,3,4,5,6P5 1-phos-
is found on internal membranes and particularly those phatase (Module 2: Figure inositol phosphate metabolism)
that function in vesicle trafficking. This function is par- that controls the level of Ins3,4,5,6P4 , which is an inhibitor
ticularly evident in phagosome maturation (Module 4: of Ca2+ -sensitive Cl− channels.
Figure phagosome maturation).
• Formation of PtdIns3P also has a role to play in activat-
Ins1,3,4,5P4
ing the isolation membrane responsible for initiating the
This inositol phosphate, which is formed by phosphorylat-
events of autophagy (Module 11: Figure autophagy).
ing Ins1,4,5P3 , has been implicated in the control of Ca2+
• PtdIns3P plays a role in regulating the activity of the
entry into cells. However, its mode of action is unknown.
intermediate conductance (IK) channel, particularly as
Some clues concerning its action may come from the iden-
part of the relationship between K+ channels and cell
tification of GAP1IP4BP , which is one of the GTPase-
proliferation.
activating protein (GAP) family (Module 2: Table mono-
PtdIns3P levels can be reduced through the action of meric G protein toolkit). GAP1IP4BP normally associates
the myotubularin family of 3-phosphatases (MTM1 and with the plasma membrane. When Ins1,3,4,5P4 binds to
MTMR1–MTMR8). These enzymes are of interest be- the membrane-anchoring domain of GAP1IP4BP , it causes
cause mutations in MTM1 are responsible for X-linked this GAP to come off the membrane. A closely related
recessive myotubular myopathy and MTMR2 is mutated GAP1m is located on the ER.
in Charcot-Marie-Tooth disease 4B.
Ins3,4,5,6P4
PtdIns4P signalling cassette Ins3,4,5,6P4 functions as an inhibitor of the Ca2+ -sensitive
For a long time, PtdIns4P was considered to be only a pre- Cl− channels (CLCAs) in epithelial cells, which regulate
cursor of PtdIns4,5P2 , but now there are indications that salt and fluid secretion, cell volume homoeostasis and elec-
it might have a signalling role to regulate trafficking at the trical excitability in neurons and smooth muscle cells. It
Golgi, where it is formed by at least two PtdIns 4-kinases appears to act by preventing Ca2+ /calmodulin-dependent
(PtdIns 4-Ks). The small GTPase ADP-ribosylation factor protein kinase II (CaMKII) from activating the channel.
Arf-1 recruits the type III PtdIns 4-kinase to the Golgi. In Ins3,4,5,6P4 is formed by an Ins1,3,4,5,6P5 1-phos-
addition, the Golgi also has a PtdIns 4-kinase α that pro- phatase, which is activated by Ins1,3,4P3 (Module 2: Figure
duces the PtdIns4P that has a specific role to associate with inositol phosphate metabolism).

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r55

InsP6 PP-InsP5
There are a number of suggestions concerning the possible This diphosphoinositide may function as an energy store
messenger role of InsP6 . Cells contain high levels of InsP6 as does [PP]2 -InsP4 .
(approximately 15–100 µM), and much of this is prob-
ably not in solution, but is probably attached to the phos-
pholipids in membranes through electrostatic interactions [PP]2 -InsP4
with bivalent cations. The level of InsP6 does not change This diphosphoinositide may function as a high-energy
much during acute stimulation, but its level can vary over store in that it can donate a high-energy phosphate to
the long term, as occurs during the cell cycle and during ADP to form ATP.
cellular differentiation. The various proposed messenger
functions outlined below may thus depend upon highly
localized fluctuations in specific cellular compartments. Nitric oxide (NO)/cyclic GMP signalling pathway
Nitric oxide (NO) is a highly diffusible messenger, which
Trafficking of vesicles passes rapidly through cell membranes. It can act as a
InsP6 may inhibit clathrin cage assembly by binding to ad- second messenger within its cell of origin or it can dif-
aptor protein (AP)-2 and -3. Such a role in membrane traf- fuse across membranes to act on neighbouring cells as a
ficking is also consistent with the observation that InsP6 paracrine signalling agent. NO synthesis is carried out
binds to synaptotagmin by competing with the inositol by nitric oxide synthase (NOS), which comes in three
lipid-binding site. It therefore seems that InsP6 may func- different forms: neuronal nitric oxide synthase (nNOS),
tion as a negative regulator of endocytic vesicle traffic. inducible nitric oxide synthase (iNOS) and endothelial
Such a possibility may explain the observation that nitric oxide synthase (eNOS). These different isoforms
GRAB, which is a guanine nucleotide exchange factor that share a similar NO synthetic reaction mechanism, which
acts on Rab3A, interacts with InsP6 kinase which converts uses L-arginine as a substrate and O2 and NADPH as co-
InsP6 into InsP7 . substrates to form NO. NOS regulation is very different
for the three isoforms and is partly dependent on the way
they are located in different parts of the cell. The action
Endocytosis
of NO is complex in that it can transmit information in
In insulin-secreting β-cells, InsP6 appears to promote dy-
markedly different ways. One of its actions is mediated
namin I-mediated endocytosis through a mechanism that
through the cyclic GMP signalling pathway, where it stim-
seems to depend upon protein kinase C (PKC), which may
ulates soluble guanylyl cyclase to produce the cyclic GMP
act to inhibit the phosphoinositide phosphatase synapto-
that can modify the properties of ion channels, protein
janin, thereby raising the level of PtdIns4,5P2 that has been
phosphatases or cyclic nucleotide phosphodiesterase. NO
implicated in vesicle dynamics.
can also act through the reactive nitrogen species (RNS)
signalling pathways, whereby the NO alters the activity
Regulation of Ca2+ channels
of a variety of protein targets through a nitrosylation re-
InsP6 is present in many brain regions and appears to be el-
action. This diverse NO/cyclic GMP signalling pathway
evated following neural activity. It may act by stimulating
operates to control the following cellular processes:
adenylyl cyclase to produce cyclic AMP, which then in-
creases the activity of L-type Ca2+ channels. Alternatively,
it may activate L-type Ca2+ channels through an inhibition • NO/cyclic GMP and smooth muscle relaxation
of protein phosphatases. In vascular smooth muscle cells, • NO/cyclic GMP and synaptic plasticity
InsP6 appears to act through a protein kinase C (PKC)- • NO/cyclic GMP and cardiac hypertrophy
dependent pathway.

Regulation of gene transcription NO synthesis

Studies on yeast have revealed that ARG82, which is an Nitric oxide (NO) synthesis is carried out by NO syn-
Ins1,4,5P3 6-kinase that phosphorylates Ins1,4,5P3 within thase (NOS), of which there are three isoforms named
the nucleus to form Ins1,4,5,6P4 , functions as a transcrip- either after the tissues where they were first discovered,
tional regulator. i.e. neuronal nitric oxide synthase (nNOS) and endothelial
nitric oxide synthase (eNOS) or by the way in which they
are controlled, i.e. inducible nitric oxide synthase (iNOS)
Regulation of mRNA export from the nucleus
(Module 2: Figure NO and cyclic GMP signalling). The
Studies on yeast indicate that InsP6 formed by an inositol
expression of these enzymes is not as restricted as their
polyphosphate kinase located on the nuclear pores may
names imply, but are widely expressed and can coexist in
facilitate the export of mRNA from the nucleus.
many cell types. Even though these isoforms are regulated
differently and have different cellular locations, they all
PP-InsP4 seem to use the same NO synthetic reaction mechanism.
This diphosphorylated inositol phosphate has been con- The excessive production of NO can have patholo-
sidered as an orphan signal, as its function is unknown. Its gical consequences and has been linked to various disease
level declines in response to either cyclic AMP or cyclic states such as Huntington’s disease, Alzheimer’s disease
GMP. and hypertension.

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Module 2: Figure NO and cyclic GMP signalling

Atrial natriuretic factor (ANP) C-type natriuretic factor (CNP)

Brain natriuretic factor (BNP) Guanylin

Ca 2+
NO
pGC CNGC

Ca 2+ eNOS
iNOS GCKGC
nNOS

cGK I Protein
NO GC cGMP targets
cGK II
sGC
O2-
ROS 2+
GSH Mn
Signalling
PDE5 GMP

2
O GS-NO Mn -+
O-N NO
ON
Cy-SH S-Nitrosylation Cy-SNO

Nitric oxide (NO) and cyclic GMP (cGMP) signalling pathways.

The nitric oxide (NO) signal can either diffuse in from other cells as a paracrine signal or it can be generated within the cell by different NO synthases
(NOSs). The NO has two main actions. It can stimulate soluble guanylyl cyclase (sGC) to form the messenger cyclic GMP (cGMP), which can act
through cyclic nucleotide-gated channels (CNGCs) to promote Ca2+ entry or it can activate cyclic GMP-dependent protein kinase (cGK). The other
main action is through reactive oxygen species (ROS) signalling mechanisms. Cyclic GMP is also formed by a plasma membrane guanylyl cyclase
(pGC), which is part of the single membrane-spanning receptor activated by a variety of peptides such as atrial natriuretic peptide (ANP), brain type
natriuretic factor (BNP), C-type natriuretic factor (CNP) and guanylin.

NO synthetic reaction mechanism The NO released from endothelial cells diffuses out to
The different nitric oxide synthase (NOS) enzymes func- regulate smooth muscle cell contraction and hence controls
tion as homodimers, which are arranged in a head-to-head blood pressure, smooth muscle cell proliferation, aggrega-
orientation with the N-terminal oxidase domain of one tion of blood platelets and leucocyte adhesion. Studies on
monomer lined up alongside the C-terminal reductase do- NO/cyclic GMP and cardiac hypertrophy have revealed
main of its neighbour. The substrates for the enzymatic that expression of eNOS in endothelial cells can inhibit
reaction mechanism are L-arginine, oxygen and NADPH, hypertrophy in neighbouring cardiac cells. Given its cent-
which combine to form citrulline with the liberation of ral role in regulating so many cellular processes, alterations
NO (Module 2: Figure NO synthase mechanism). NOS in the endothelial production of NO have been implicated
regulation is complicated because each isoform appears to in many disease states, such as hypertension, diabetes and
be regulated by different mechanisms. hypercholesterolaemia.

Endothelial nitric oxide synthase (eNOS) Inducible nitric oxide synthase (iNOS)
As its name implies, endothelial nitric oxide synthase Inducible nitric oxide synthase (iNOS), which is also
(eNOS) was first described in endothelial cells, where it known as immunocyte NOS, was first described in mac-
generates NO in response either to agonists such as acet- rophages, where its expression is up-regulated by inflam-
ylcholine and bradykinin that elevate Ca2+ or to blood matory mediators. Although macrophages are the main
flow-induced shear stress. It is now evident that eNOS is cells that express iNOS, it is also found in other cell types
expressed in many different cell types (lung epithelial cells, (cardiac cells, vascular smooth muscle cells and glial cells).
blood platelets, cardiac myocytes and hippocampal neur- Unlike the other isoforms, the activation of iNOS does
ons). It has a complex regulation, which is very dependent not require an elevation of Ca2+ . However, iNOS does
on its attachment to caveolin, one of the proteins in cave- bind calmodulin (CaM), which is essential for its activa-
olae (Module 6: Figure caveolae molecular organization), tion. Since the enzyme is constitutively active, its primary
where it contributes to their signalling function. One of regulation depends upon its induction by inflammatory re-
the key regulators of eNOS is Ca2+ , which acts through ceptors such as those that respond to interferon-γ (IFN-γ)
calmodulin (CaM) to stimulate the enzyme to release NO or lipopolysaccharide (LPS). As large amounts of enzyme
(Module 2: Figure eNOS activation). are produced, this is a high-output pathway capable of

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Module 2 : Figure NO synthase mechanism

Reductase domain

O
2
Oxidase domain CAM
Arginine
Oxidase domain

Haem BH4
CAM FMN FAD NADPH
Reductase domain

Ca 2+ S 1177

Citrulline NO

Nitric oxide synthase (NOS) reaction mechanism.

The two nitric oxide synthase (NOS) monomers are lined up alongside each other so that the reductase domain of one functions together with
the oxidase domain of its neighbour. The enzyme dimer also functions as a scaffold to organize the other components of the reaction mechanism
such as the bound cofactors [flavin–adenine dinucleotide (FAD), flavin adenine mononucleotide (FMN), haem and tetrahydrobiopterin (BH4 )] and
the tightly bound prosthetic group calmodulin (CaM). The formation of NO is driven by an NADPH-dependent electron flux that passes from the
reductase towards the oxidase domain. The attached haem is the terminal electron acceptor, which binds the oxygen that is inserted into arginine to
form the hydroxyarginine that decays to release NO. One of the important regulators of NOS is calmodulin, which is constitutively active in inducible
NOS (iNOS), but requires an elevation of Ca2+ for both neuronal NOS (nNOS) and endothelial NOS (eNOS). One consequence of increasing the
concentration of Ca2+ in cells is therefore to increase the formation of NO.

delivering NO for prolonged periods as part of the cells cyclase (GC). The latter comes in two different forms:
defence against invading micro-organisms. As such, it does there are the soluble GC (sGC) and membrane-bound
not strictly function as a messenger. However, the large particulate guanylyl cyclases (pGCs). Many of the sig-
production of NO at the sites of inflammation will spill nalling functions of cyclic GMP are carried out by cyclic
over to affect neighbouring cells. The large up-regulation GMP-dependent protein kinase (cGK). In addition, cyc-
of iNOS may account for the fall in blood pressure during lic GMP can act directly to open cyclic nucleotide-g-
endotoxic shock. ated channels. Cyclic GMP hydrolysis is carried out by a
cyclic GMP-specific phosphodiesterase (PDE5). Through
these different signalling pathways, cyclic GMP func-
Neuronal nitric oxide synthase (nNOS)
tions to regulate a diverse collection of cellular processes.
Neuronal nitric oxide synthase (nNOS) was first described
There are those where cyclic GMP mediates the action
in neurons, but has since been found in other cell types such
of NO:
as skeletal muscle, which has an alternatively spliced vari-
ant that has a 34-amino-acid insert between exons 16 and
• NO/cyclic GMP and smooth muscle relaxation
17. In both neurons and muscle, nNOS is closely associated
• NO/cyclic GMP and synaptic plasticity
with the plasma membrane where it binds to various pro-
• NO/cyclic GMP and cardiac hypertrophy
teins through its PDZ domains. In neurons, nNOS binds
to the postsynaptic density (PSD) proteins such as PSD-
93 and PSD-95 (Module 10: Figure postsynaptic density), In those cases where cyclic GMP is formed by the pGC,
whereas in skeletal muscle it interacts with α1-syntrophin cyclic GMP acts independently of NO to regulate cellular
(a binding partner of dystrophin). processes such as phototransduction (Module 10: Figure
phototransduction overview). Certain strains of Escheri-
chia coli, which secrete the STa toxin, increase intestinal se-
Cyclic GMP signalling pathway cretion and cause diarrhoea by activating the cyclic GMP
The cyclic GMP signalling pathway (Module 2: Figure signalling pathway by stimulating the particulate guanylyl
NO and cGMP signalling) is governed by the second cyclase C (pGC-C) receptor that is normally activated by
messenger cyclic GMP, which is synthesized by guanylyl guanylin (Module 7: Figure intestinal secretion).

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Module 2: Figure eNOS activation

Myristic
eNOS acid
Inactive
Caveolin Palmitic
CAM FAD BH4
acid
FMN Heme

Calcium-induced
NOS activation

eNOSActive
CAM FAD BH4
Caveolin
FMN Haem

Arginine Citrulline
NADPH NADP
2+ O2
Ca NO

Ca2+ -dependent activation of endothelial nitric oxide synthase (eNOS).

An important feature of eNOS is its location on the membrane of caveolae (Module 6: Figure caveolae molecular organization). Its membrane
localization is facilitated by an N-terminal myristic acid and by two palmitic acid residues attached to two cysteine residues (Cys-15 and Cys-26),
whereas its association with the caveolae depends upon its attachment to caveolin, which is responsible for keeping the enzyme inactive under
resting conditions. There appears to be a competition between caveolin and calmodulin (CaM) for the caveolin-binding site (amino acids 350–358)
on eNOS. In the absence of Ca2+ , caveolin dominates, but when Ca2+ increases and binds to CaM, the latter relieves the inhibitory effect of caveolin,
and the enzyme is activated to generate NO from arginine using oxygen and NADPH as co-substrates.

NO/cyclic GMP and synaptic plasticity a group of single membrane-spanning receptors that use
The enzymes responsible for NO formation and its action an enzyme to transduce information. In this case, it is the
are richly expressed in the nervous system. Although the guanylyl cyclase region of the cytoplasmic domain that
precise function of NO is still debated, there are indica- functions both as a transducer and an amplifier to generate
tions that it might function in synaptic plasticity by con- the second messenger cyclic GMP.
tributing to cerebellar cell long-term potentiation (LTP) at
the parallel fibre/Purkinje cell synapse. Cyclic GMP hydrolysis
The enzyme that reverses the second messenger action of
cyclic GMP by hydrolysing it to GMP is the cyclic GMP-
Guanylyl cyclase (GC) specific phosphodiesterase (PDE5), which can be activated
Guanylyl cyclase (GC) is the enzyme that synthesizes cyc- by cGKI, thus setting up a negative-feedback loop. Excess-
lic GMP from ATP (Module 2: Figure NO and cyclic ive signalling through the cyclic GMP signalling pathway
GMP signalling). It comes in two main forms, soluble GC will thus be curtailed through this ability of cyclic GMP
(sGC), which is activated by NO, and membrane-bound to enhance its own hydrolysis. PDE5 is of considerable
particulate guanylyl cyclases (pGCs). The latter belongs to interest as it is the target of Viagra.

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Cyclic GMP-dependent protein kinase (cGK) NO + GSH → GS-NO

There are two cyclic GMP-dependent protein kinases NO + Cys → Cys-NO
(cGKs): cGKI and cGKII. The cGKI comes in two al-
ternatively spliced forms, cGKIα and cGKIβ. These cGKs Nitrosylation reaction
are serine/threonine protein kinases that exist as homodi- The S-nitrosylation reaction depends upon the transfer
mers that are held together by leucine zippers in their N- of NO from one of the reactive nitrogen species (RNS)
terminal domains, which fold over to inhibit the catalytic to a peptidyl cysteine thiol group of target proteins (RS)
domain. The binding of cyclic GMP to a regulatory site (Module 2: Figure NO and cGMP signalling):
induces a conformational change that relieves this inhibi-
tion, thus enabling the catalytic domain to phosphorylate RS + ONO-NO → RS-NO
its substrates. RS + Mn+ -NO → RS-NO
RS + GS-NO → RS-NO
cGK targets RS + Cys-NO → RS-NO
These cGKs are targeted to specific sites in the cell. The
N-terminal domain is responsible for targeting cGKIα and Specificity is determined by the fact that the target pro-
cGKIβ to specific cellular regions as is particularly import- teins (RS) have hyperreactive cysteine groups where the
ant for smooth muscle relaxation mediated by nitric oxide thiol moiety exists as a thiolate anion due to the presence
(NO) and cyclic GMP (Module 7: Figure smooth muscle of positively charged amino acids in the immediate vicinity
cell cGMP signalling). of the protein chain.
The cGKII has an N-terminal Gly-2 myristic acid
residue that serves to target it to the plasma membrane. Redox signalling
Cells have evolved a sophisticated mechanism of intracel-
cGK protein substrates
lular signalling based on localized changes in the oxidation
The cGMP-sensitive target proteins regulated by cGKs
state of specific proteins. The internal environment of cells
include inositol 1,4,5-trisphosphate receptor-associated
is normally highly reduced. Certain forms of stress are as-
cGKI substrate (IRAG), large conductance (BK) chan-
sociated with an increase in the oxidative state, and this
nels, cGMP-specific phosphodiesterase (PDE5), cerebellar
can induce apoptosis. It is also well known that certain
G substrate, vesicle-associated membrane protein (VASP)
phagocytic cells, such as neutrophils, can rapidly generate
and telokin.
superoxide radical (O2 −• ) and hydrogen peroxide (H2 O2 )
that are used to kill other cells during inflammatory re-
Inositol 1,4,5-trisphosphate receptor-associated cGKI
sponses. In addition to these pathological effects, there is
substrate (IRAG)
increasing evidence that the redox system has been adapted
Inositol 1,4,5-trisphosphate receptor (InsP3 R)-associated
to perform a variety of signalling functions and can mod-
cGKI substrate (IRAG) is located on the endoplasmic
ulate the activity of other signalling pathways. As such,
reticulum (ER), where it appears to associate with the
they can control many cellular processes, including cell
InsP3 R. When it is phosphorylated by cGKIβ, IRAG
proliferation, apoptosis and cellular senescence. Some of
acts to inhibit channel opening (Module 7: Figure smooth
these effects are exerted through a two-way interaction
muscle cell cGMP signalling).
with Ca2+ signalling. For example, redox signalling can
help to promote the tyrosine phosphorylation events that
Reactive nitrogen species (RNS) signalling
generate many signalling cascades and it can modulate the
One of the ways by which NO functions in cells is through
activity of the ryanodine receptors (RYRs) and inositol
an S-nitrosylation reaction. This covalent modification
1,4,5-trisphosphate receptors (InsP3 Rs) that release Ca2+ .
results from the addition of NO to reactive cysteine
Conversely, Ca2+ can stimulate redox signalling, particu-
residues on specific target proteins. NO does not react
larly within the mitochondrion, indicating that there are
directly with these proteins, but is first of all converted
dynamic interactions operating between these signalling
into reactive nitrogen species (RNS) are then respons-
pathways.
ible for carrying out the nitrosylation reaction. These
There are two main types of redox signalling. The first
nitrosylation-sensitive signalling pathways carry out many
type is reactive oxygen species (ROS) signalling, which
of the functions of NO, and there is growing evidence for
depends on the formation of ROS. The second type is
nitrosylation dysfunction in disease.
reactive nitrogen species (RNS) signalling, which is carried
out by RNS and is linked to the nitric oxide (NO)/cyclic
Reactive nitrogen species (RNS) GMP signalling pathway.
The reactive nitrogen species (RNS) that carry out the
nitrosylation reaction are formed when NO interacts
Reactive oxygen species (ROS) signalling
with various acceptors such as superoxide radical (O2 −• ),
ROS signalling has all the hallmarks of a classical signalling
cysteine (Cys), glutathione (GSH) or transition metal ions
mechanism. The second messengers are the reactive oxy-
(Mn+ , e.g. Fe3+ or Cu2+ ) (Module 2: Figure NO and cGMP
gen species (ROS) formed in response to many agonists.
signalling):
Reactive oxygen species (ROS) formation depends on the
NO + O2 − → ONO-NO stimulation of a NADPH oxidase that removes an electron
NO + Mn+ → Mnn+ -NO from NADPH and adds it to oxygen to create superoxide

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r60

Module 2: Figure summary of redox signalling

REDOX
SIGNAL SIGNALLING
PATHWAY

_
Messenger
formation

Messenger
SOD Catalase
O2 H O2 H O metabolism
2 2

Messenger
OH OH action
Oxidation
SH
S
SH
S Recovery

Reduction

Oxidation-sensitive
processes
Cellular
response

Summary of the main features of redox signalling.

The sequential processes that constitute redox signalling begin with an external signal activating a receptor (R) that then generates reactive oxygen
species, such as superoxide radical (O2 −• ), which is then converted into hydrogen peroxide (H2 O2 ) by superoxide dismutase (SOD). O2 −• and H2 O2
diffuse into the cell where they exert their messenger action by stimulating the oxidation of specific proteins, sometimes forming an internal disulphide
bond. Recovery is carried out by various enzyme systems that return the target protein to its reduced state. The oxidized protein acts to stimulate a
variety of cellular processes.

radical (O2 −• ), which is one of the ROS found in cells. cellular responses such as gene activation, modulation of
Superoxide dismutase (SOD) rapidly converts the O2 −• ion channels and the activity of other signalling pathways
into hydrogen peroxide (H2 O2 ), which is one of the main [mitogen-activated protein kinase (MAPK) cascade and
messenger molecules used by the redox signalling pathway Ca2+ signalling]. It is therefore not surprising to find a
(Module 2: Figure summary of redox signalling). role for redox signalling in proliferation and cancer.
Rapid reactive oxygen species (ROS) metabolism en-
sures that H2 O2, like other intracellular messengers, has a Reactive oxygen species (ROS)
short half-life. This metabolism of H2 O2 is carried out by Reactive oxygen species (ROS) is a collective term
a range of enzymes, including catalase, glutathione peroxi- that refers to those oxygen species [superoxide (O2 −• ),
dase (GPx) and peroxiredoxin (Prx). Since the inside of the hydrogen peroxide (H2 O2 ) and the hydroxyl radical
cell is a highly reducing environment, the last two enzymes (OH• )] that are more reactive than ground-state oxygen
can draw upon a large reservoir of reducing equivalents in [Module 2: Figure reactive oxygen species (ROS)]. These
order to metabolize the ROS. The cell has a large redox are sometimes considered synonymous with free radicals,
buffer capacity in the form of glutathione, which func- but not all ROS are free radicals. The latter are defined
tions to maintain the redox balance in the cell. The fact as atoms or molecules that contain one or more unpaired
that H2 O2 is metabolized so rapidly means that its site of electrons. With regard to signalling, it is the O2 −• and
action is highly localized close to its site of production. H2 O2 that appear to be the most important messengers.
The reactive oxygen species (ROS) messenger action of Also, there are indications that these two messengers may
H2 O2 depends upon its ability to react with the cysteine perform different functions.
residues of a certain group of target proteins. The latter are There are two important sources of reactive oxygen spe-
marked out by virtue of having hyperreactive thiol groups cies (ROS): one is at the plasma membrane and the other
that are rapidly oxidized to form a disulphide bond. The is within the mitochondria (Module 2: Figure sites of ROS
recovery from this oxidized state back to a fully reduced formation).
thiol group is carried out by the glutaredoxin and/or the
thioredoxin systems. Superoxide (O2 −• )
Finally, the oxidized target proteins activate a number of The superoxide radical (O2 −• ) that is formed by the
oxidation-sensitive processes that bring about a number of one-electron reduction of O2 is short-lived (half-life of

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10−6 s) in that it rapidly dismutates into hydrogen peroxide (ROS), including cytokine receptors (tumour necrosis
(H2 O2 ). This conversion can occur spontaneously, but the factor α, interleukin-1 and interferon-γ), growth factor
reaction is greatly accelerated by the enzyme superoxide receptors [platelet-derived growth factor (PDGF), epi-
dismutase (SOD). dermal growth factor (EGF) and basic fibroblast growth
factor (bFGF) receptors] and G protein-coupled receptors
Hydrogen peroxide (H2 O2 ) (GPCRs) (5-hydroxytryptamine, bradykinin, angiotensin
Much attention is focused on hydrogen peroxide (H2 O2 ) II, thrombin and endothelin receptors). Just how all these
because it appears to be the primary messenger molecule receptors are coupled to the formation of ROS is still some-
functioning in the redox signalling pathway. Since it has no what uncertain, but there is increasing evidence that the
unpaired electrons, H2 O2 is not a free radical and thus is mechanism might resemble that found in phagocytic cells.
not a particularly powerful oxidizing agent. This means In phagocytes, external signals acting through cell-
that it can function as a messenger by diffusing away surface receptors stimulate the PtdIns 3-kinase signalling
from its site of action to interact with more distant tar- pathway to form the lipid messenger PtdIns3,4,5P3
gets. However, its sphere of influence is restricted by its (Module 2: Figure PtdIns 3-kinase signalling), which then
short half-life, which is determined by the rapid reactive acts through Rac to stimulate NADPH oxidase. This mul-
oxygen species (ROS) metabolism of H2 O2 . ticomponent enzyme uses NADPH as an electron donor
to carry out the first step of ROS formation i.e. the re-
Hydroxyl radical (OH• ) duction of oxygen to superoxide radical (O2 −• ) (Module
While H2 O2 is relatively benign, it can be converted into 2: Figure plasma membrane ROS formation). In addition
the highly toxic hydroxyl radical (OH• ) through a reduc- to this activation through Rac, it seems that the enzyme
tion process catalysed by transition metals (Fe3+ or Cu2+ ). can also be regulated through diacylglycerol (DAG) and
OH• has a half life of 10−9 s indicative of its very high Ca2+ acting through protein kinase C (PKC), which phos-
reactivity in that it reacts immediately and indiscrimin- phorylates p47phox , one of the cytoplasmic components of
ately with the first molecule it finds. Much of the oxidative NADPH oxidase, to bring about the assembly of the mul-
damage cause by ROS is mediated by OH• . ticomponent complex. Non-phagocytic cells that generate
ROS for signalling use a similar, but genetically distinct,
Superoxide dismutase (SOD) NADPH oxidase called Nox1, which plays a significant
A family of metalloproteinases that converts superox- role in redox signalling in proliferation and cancer.
ide radical (O2 −• ) into hydrogen peroxide (H2 O2 ) (Mod- In phagocytes, the O2 −• is released to the outside of the
ule 2: Figure plasma membrane ROS formation): cell, where it is able to attack invading micro-organisms,
whereas, in non-phagocytic cells, it functions as a mes-
2O2 −• + 2H+ → H2 O2 + O2
senger to activate a signalling cascade on the inside of
There are four families of SOD enzymes: the cell (Module 2: Figure plasma membrane ROS form-
ation). Superoxide dismutase (SOD) rapidly converts the
• Copper/zinc-containing superoxide dismutases
O2 −• into hydrogen peroxide (H2 O2 ), which appears to
(CuZnSODs)
be the primary messenger molecule of this redox signalling
• Manganese-containing superoxide dismutases (MnS-
pathway.
ODs)
• Copper-containing superoxide dismutases (CuSODs) NADPH oxidase
• Iron-containing superoxide dismutases (FeSODs) The NADPH oxidase (NOX/DUOX) family (Module 2:
Table redox signalling components) consists of a number
Expression of MnSOD is regulated by the FOXO3a
of enzymes with different cellular locations.
transcription factor (Module 4: Figure FOXO control
NOX2, also known as gp91phox , has been described best
mechanisms).
for phagocytes, where it is made up of a number of sub-
Mutation of SOD is the cause of amyotrophic lateral
units. The catalytic component is cytochrome b558 , which
sclerosis (ALS), which is a debilitating and progressive
is a heterodimer formed from gp91phox and p22phox . In
neurological disease.
addition to this heterodimer, which is located in the mem-
brane, there are cytoplasmic components (e.g. p47phox ,
Reactive oxygen species (ROS) formation
p67phox , Rap1A and Rac) that play a role in regulating
Reactive oxygen species (ROS) are formed at two main
enzyme activity. The DUOX (dual oxidase) enzymes are
sites: there is plasma membrane reactive oxygen species
sensitive to Ca2+ and play an important role in interacting
(ROS) formation and mitochondrial reactive oxygen spe-
with the Ca2+ signalling pathway (Module 2: Figure ROS
cies (ROS) formation (Module 2: Figure sites of ROS
effects on Ca2+ signalling).
formation). This production of ROS appears to be highly
localized suggesting the existence of reactive oxygen spe- Mitochondrial reactive oxygen species (ROS)
cies (ROS) microdomains. formation
Most of the electrons that enter the electron transport
Plasma membrane reactive oxygen species (ROS) chain are transferred to oxygen in an orderly manner, but
formation there is always a 1–2% leakage during which an electron is
A large number of receptors responding to external sig- transferred directly to oxygen to form superoxide (O2 −• )
nals stimulate the formation of reactive oxygen species and this is the source of mitochondrial reactive oxygen

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Module 2: Table redox signalling components due to formation of a MTP and a concomitant increase in
The major components of the redox signalling pathway intrinsic ROS production, i.e. a process of ROS-induced
Component Comment ROS release (RIRR). This RIRR often occurs synchron-
NADPH oxidases (NOXs)
NOX1 Inducible enzyme found in colon ously and reversibly among long chains of adjacent mito-
and smooth muscle chondria, suggesting a co-operative mechanism.
NOX2 (gp91phox ) Major NOX in phagocytes
NOX3 Foetal kidney
NOX4 Widespread Redox balance
NOX5 Brain, spleen and sperm The redox balance of the cell is maintained by energy meta-
DUOX1 (dual oxidase 1) A Ca2+ -sensitive isoform bolism, primarily the pentose phosphate shunt that feeds
DUOX2 (dual oxidase 2) A Ca2+ -sensitive isoform
NOX/DUOX regulatory factors reducing equivalents into the cell in the form of NADPH.
p47phox The latter is then used to maintain redox buffers such as
p67phox glutathione (GSH) (Module 2: Figure GSH/GSSG couple)
p40phox
p22phox
and thioredoxin (Trx) in their reduced forms. The concen-
NOXO 1 tration of GSH in the cytoplasm lies within the 1–10 mM
NOXA 1 range, with over 99% existing as the reduced GSH form.
Rac1/Rac2
ROS metabolism
The 2GSH/GSSG redox couple is thus a measure of the
Superoxide dismutase (SOD) redox balance in the cell. A similar balance exists for the
Catalase Localized in peroxisomes oxidized and reduced forms of thioredoxin (Trx). The re-
Glutathione peroxidase (GPx) Localized in cytosol and
mitochondria
duced GSH and Trx are used for a number of reductive
Peroxiredoxin (Prx) processes such as the metabolism of hydrogen peroxide
Prx I–IV (2-Cys) I and II in cytosol; III in by glutathione peroxidase (GPx) (Module 2: Figure H2 O2
mitochondria; IV in endoplasmic
reticulum
metabolism) or as a source of reducing equivalents for the
Prx V (atypical 2-Cys) glutaredoxin system (Module 2: Figure recovery of protein
Prx VI (1 Cys) oxidation). The GSSG (the oxidized form of glutathione)
Thiol-containing
proteins/peptides
is converted back into GSH by glutathione reductase.
Glutathione (GSH) Similar redox control enzymes and buffers are located in
Glutaredoxin (Grx) both the mitochondria and within the lumen of the endo-
Thioredoxin (Trx)
Trx-1
plasmic reticulum (ER). Like the cytoplasm, the mitochon-
Trx-2 Mitochondria-specific drial matrix maintains a reducing environment and uses
Reductases similar enzymatic mechanisms to control the ROS eman-
Glutathione reductase
Glutaredoxin reductase
ating from the electron transport chain. The ER, however,
Thioredoxin reductase (TrxR) is somewhat different in that the GSH/GSSG ratio is close
Sulphiredoxin (Srx) Catalyses reduction of to 1 and this more oxidizing environment is necessary for
hyperperoxidized proteins
the formation of the disulphide bonds that are an integral
component of the extracellular proteins that are processed
and packaged within the ER.
species (ROS) (Module 5: Figure mitochondrial Ca2+ sig-
nalling). This orderly electron transfer to oxygen occurs Since the cytoplasm and the ER lumen have different
redox potentials, there is a redox potential gradient across
during energy metabolism, where oxygen is reduced to
the ER membrane and this might be used to modulate
water by accepting four electrons (e− ) from cytochrome c
oxidase: Ca2+ signalling by altering the activity of the ion channels
that release Ca2+ .
O2 + 4e− + 4H+ → 2H2 O
Reactive oxygen species (ROS) metabolism
Mitochondrial energy metabolism is inherently danger- Like all other intracellular signalling molecules, ROS are
ous, because the electron transport chain is somewhat metabolized rapidly. Superoxide dismutase (SOD) rapidly
leaky in that some of the molecular oxygen is diverted converts superoxide radical (O2 −• ) into hydrogen perox-
into the formation of the superoxide radical (O2 −• ), which ide (H2 O2 ), which is then metabolized by a number of
is then converted into hydrogen peroxide (H2 O2 ) and the enzyme systems including catalase, glutathione peroxidase
hydroxyl radical (OH• ). These mitochondrial ROS may (GPx) and peroxiredoxin (Prx) (Module 2: Figure H2 O2
play an important role in apoptosis by acting synergist- metabolism).
ically with Ca2+ to stimulate the formation of the mito- With so many enzyme systems co-operating to meta-
chondrial permeability transition pore (MTP) (Module 5: bolize H2 O2 , it is likely that this messenger will have a
Figure mitochondrial Ca2+ signalling). The formation of highly restricted sphere of influence localized to its site of
mitochondrial ROS appears to be highly localized in that production either at the plasma membrane or within the
small superoxide flashes have been recorded in single mi- mitochondrion.
tochondria. This is another example of reactive oxygen
species (ROS) microdomains. Catalase
The generation of ROS by mitochondria might be a Catalase is a haem-containing protein that decomposes
regenerative process in that a local release of ROS in car- hydrogen peroxide (H2 O2 ) to water and oxygen (Mod-
diac myocytes causes a rapid mitochondrial depolarization ule 2: Figure H2 O2 metabolism).

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Module 2: Figure reactive oxygen species (ROS)

OO O2 Oxygen

e- REACTIVE OXYGEN
SPECIES (ROS)

OO O2
-. Superoxide

e-
H OO H H2 O2 Hydrogen
peroxide

e-

O H OH . Hydroxyl
radical

Paired electrons
Unpaired electrons
Formation and metabolism of the reactive oxygen species (ROS).
To understand the properties of reactive oxygen species (ROS) and how they are formed, it is best to begin with oxygen. Oxygen is a strong oxidizing
agent in that it has two unpaired electrons (e− ; red dots), which have parallel spins (i.e. they spin in the same direction as indicated by the two black
arrows) and occupy separate π-antibonding orbitals. Given that they have these unpaired electrons, oxygen qualifies as a free radical. However,
oxygen is relatively inert because, in order to react with another molecule, it has to accept a pair of electrons with antiparallel spins to fit into the empty
spaces in the π orbitals. Because of this restriction, oxygen accepts electrons (e− ) one at a time and this leads to the formation of the different ROS.

Most of the catalase in cells is found in peroxisomes, 3. The Cys-SOH can then interact with the conserved
thus restricting the role of the enzyme in dealing with the Cys-SH on the C-terminal regions of the neighbouring
H2 O2 generated during the redox signalling mechanism at dimer to form two intermolecular disulphides.
the plasma membrane. 4. The Prx disulphide is converted back into the reduced
form by Trx, which is regenerated by thioredoxin re-
Glutathione peroxidase (GPx)
ductase (Module 2: Figure recovery of protein oxida-
The glutathione peroxidase (GPx) family uses the reducing
tion).
power of glutathione to convert H2 O2 into water (Mod-
5. The sulphenic residues formed by Reaction 2 can un-
ule 2: Figure H2 O2 metabolism).
dergo hyperperoxidation by interacting with further
Peroxiredoxin (Prx) molecules of H2 O2 to form the sulphinic acid residues.
The peroxiredoxins (Prxs) are a family of small antioxidant 6. This hyperperoxidation reaction can be reversed by a
proteins that function to metabolize hydrogen peroxide reaction that requires ATP catalysed by the enzyme
(H2 O2 ) to water, thus curtailing its messenger action. The sulphiredoxin (Srx).
operation of the catalytic cycle goes through the following 7. The phosphorylated intermediate is reduced back to
steps (Module 2: Figure peroxiredoxin catalytic cycles): the reduced form of Prx by the Trx system.

1. H2 O2 is generated near the plasma membrane when Mammals express five Prxs which contain catalytic
the PtdIns3,4,5P3 (PIP3 ) formed by receptor activation cysteine residues and use Trx as an electron donor (Mod-
stimulates NADPH oxidase. ule 2: Figure peroxiredoxin catalytic cycles).
2. H2 O2 interacts with the reduced cysteine residues The reducing equivalents derived from thioredoxin
(Cys-SH) in the N-terminal regions of the thioredoxin (Trx) are used to regenerate Prx-(SH)2 :
(Trx) dimers to form two oxidized sulphenic residues
(Cys-SOH). Prx-S2 + Trx-(SH)2 → Prx-(SH)2 + Trx-S2

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Module 2: Figure sites of ROS formation

Agonist

NADPH oxidase
complex on the
plasma membrane
NADH
ROS
NAD+

ROS Leakage of electrons

from mitochondrial
electron transport
O
2 chain

HO
2

The two main sites of reactive oxygen species (ROS) formation.

There are two main sites of ROS formation in cells. One is at the level of the plasma membrane, where an NADPH oxidase complex is activated by
cell signalling pathways. The other is at the mitochondria, where ROS are produced as a result of electron leakage from the electron transport chain.
As indicated, this production of ROS appears as microdomains indicating that ROS signalling might be highly localized in cells.

Module 2: Figure plasma membrane ROS formation

Growth
Cytokines factors Hormones

NADPH
oxidase

PIP2 PI 3-K PIP 3 Rac NADPH

Cyt b
+
NADP
PTEN
O2
O2
PIP2
Superoxide
- dismutase
Tyrosine
phosphatase
H2 O2
Channel
modulation
Oxidation
SH S Gene
S transcription
SH
Reduction
Apoptosis

Receptor-dependent reactive oxygen species (ROS) formation at the plasma membrane.

The formation of reactive oxygen species (ROS) occurs in response to many external signals such as cytokines, growth factors and hormones. In
many cases, these signals activate receptors coupled to PtdIns 3-kinase (PI 3-K) which produces PtdIns3,4,5P3 (PIP3 ) that then acts through Rac
to stimulate NADPH oxidase at the plasma membrane. An electron is removed from NADPH and transferred to oxygen to form superoxide radical
(O2 −• ). This O2 −• is then transformed by the addition of further electrons by superoxide dismutase (SOD) to form hydrogen peroxide (H2 O2 ). There is
a positive-feedback loop that will amplify the redox signalling pathway because one of the actions of H2 O2 is to inhibit the enzyme phosphatase and
tensin homologue deleted on chromosome 10 (PTEN) that hydrolyses PIP3 .

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Module 2: Figure GSH/GSSG couple

GSSG
Reduction
GSH Glu Cys Gly

Glu Cys Gly S

S
SH Glu Cys Gly

Oxidation

H 2O2 GSH Grx-S2

S *
SH

S
H2O GSSG Grx-(SH) S
2

The GSH/GSSG redox couple.

GSH is a tripeptide consisting of glutamic acid, cysteine and glycine. In its oxidized state, two molecules of GSH are joined together through a
disulphide bond to form GSSG. This is the most abundant redox couple in the cell. The state of this couple can be determined by measuring the
half-cell reduction potential (E-hc ). Under normal reducing conditions, this potential is high, i.e. −240 mV, and this seems to be associated with cell
proliferation. Differentiation seems to occur at lower potentials (−200 mV), whereas still lower potentials of −170 mV favour apoptosis. At these lower
potentials, where there is an alteration in the redox balance, the build-up of GSSG within the cell can reverse the operation of the glutaredoxin system
that functions normally in the recovery of oxidation-sensitive processes. GSSG interacts with reduced glutaredoxin [Grx-(SH)2 ] to form oxidized Grx-S2 ,
and this disulphide bond can be transferred to oxidize target proteins.

When Prx1 in mice is knocked out, animals develop 5. The sulphenic acid residue can undergo hyperperoxid-
haemolytic anaemia and malignant cancers. ation by interacting with another molecule of H2 O2 to
form a sulphinic acid intermediate (Cys-SO2 H).
6. The sulphinic acid intermediate undergoes further hy-
Reactive oxygen species (ROS) messenger action
perperoxidation to form the sulphonic acid intermedi-
The primary action of hydrogen peroxide (H2 O2 ) is to
ate (Cys-SO3 H).
reversibly oxidize a variety of target proteins with a high
7. The sulphinic acid group (Cys-SO2 H) can be reduced
degree of specificity (Steps 1–7 in Module 2: Figure revers-
by a reaction that requires ATP and is catalysed by the
ible and irreversible ROS oxidations):
enzyme sulphiredoxin (Srx).

1. The primary action of H2 O2 is to oxidize the hyperre- The various oxidated intermediates can be converted
active cysteine to form a sulphenic acid group (-SOH), back into the initial reduced state by either the thioredoxin
which can be metabolized further along a number of (Trx) or the glutaredoxin (Grx) system (Module 2: Figure
pathways. recovery of protein oxidation).
2. The sulphenic acid residue can interact with nitrogen on These oxidation processes can occur through a num-
a neighbouring serine residue to form an intramolecular ber of mechanisms. What is remarkable about this process
cyclic sulphenyl amide as occurs during the oxidation of is its specificity. Only a subset of proteins are modified,
protein tyrosine phosphatases (Module 2: Figure ROS and within these there is a high degree of specificity in
formation and action). that only certain thiols are modified. How is it that an
3. The sulphenic acid residue can be converted into an oxidizing agent such as H2 O2 is able to seek out and se-
intramolecular disulphide bond with the elimination of lectively modify specific target proteins? The answer lies
water. in the fact that proteins vary considerably in their sensit-
4. The sulphenic acid residue can interact with glutathione ivity to mild oxidizing agents, such as H2 O2 . Most of the
(GSH) to form an intermolecular disulphide bond. cysteine residues in proteins have a high acidic constant

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Module 2: Figure H2 O2 metabolism

Catalase
2 H 2O 2 2 H 2O + O2

GSH
peroxidase
H 2O 2 + 2GSH 2 H 2O + GSSG

Peroxiredoxin
H 2O 2 + Prx(SH) 2 H2 O + PrxS 2
2

Hydrogen peroxide (H2O2) metabolism by different enzyme systems.

Hydrogen peroxide (H2 O2 ) can be metabolized by three main mechanisms. The enzyme catalase, which is restricted to peroxisomes, converts H2 O2
into water and oxygen. GSH peroxidase uses the reducing power of glutathione (GSH) to convert H2 O2 into water with the formation of GSSG. The
peroxiredoxin (Prx) family is a major player in the metabolism of H2 O2 through a series of catalytic cycles (Module 2: Figure peroxiredoxin catalytic
cycles).

Module 2: Figure peroxiredoxin catalytic cycles

Growth
Cytokines factors Hormones

PI 3-K PIP3 NOX/DUOX

1

H 2 O2
O
H O H O
Prx

-SH
Prx

-SH
2 2 O S -SH 6
O ATP
S
-SH -SH
-SH S
Trx O
S Srx
2 5
Prx

-SOH -SH O
4
Prx

P S -SH
-SH -SOH -SH
3 SH S P
Trx --S
Prx

SH S S O
Trx --S H
H S S 7
H O
2
Prx

S -SH -SH
Trx
S
-SH
-SH

Metabolism of H2O2 by peroxiredoxin.

Peroxiredoxin (Prx) plays a major role in restricting the microdomain of hydrogen peroxide (H2 O2 ) that forms beneath the plasma membrane by rapidly
removing this messenger through a series of catalytic reactions as described in the text. Information adapted from Curr. Opin. Cell Biol., Vol. 17, Rhee,
S.G., Kang, S.W., Jeong, W., Chang, T.-S., Yang, K.-S. and Woo, H.A., Intracellular messenger function of hydrogen peroxide and its regulation by
peroxyredoxins, pp. 183–189. Copyright (2005), with permission from Elsevier; see Rhee et al. 2005.

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r67

Module 2: Figure reversible and irreversible ROS oxidations

Protein
substrate
NH
*
S
N
Grx and Trx
systems
SH S 8
H O
H2 O 2 2
1 SH
H 2O 2
NH NH
Sulphenic H O
acid SOH 2
3
S
SH S
H2 O 2
5 GSH 4 NH
H 2O
SSG
NH H O
Sulphinic 2
acid SO H SH
2
7
SH
Srx NH O
H2 O 2
S
6 ATP PO4
H 2O
NH SH
Sulphonic
acid SO H
3
SH

Reactive oxygen species (ROS) messenger action through the reversible oxidation of target proteins.
The target protein (blue) undergoes a variety of oxidative covalent modifications, some of which induce the conformational changes responsible for
mediating a variety of oxidation-sensitive processes. The target proteins that are modified by reactive oxygen species (ROS) have a hyperreactive
cysteine thiolate anion (asterisk) that is highly sensitive to oxidation by H2 O2 to initiate a cascade of reactions as described in the text.

(i.e. pK a values of approximately 8.5), which means that amide intermediate protects against further oxidation, and
they are resistant to attack by H2 O2 . However, some of the the enzyme can be reactivated by converting the intermedi-
cysteine residues, particularly those located next to posit- ate back into a thiol group via a mixed disulphide reaction
ively charged amino acids, have pK a values between 4 and involving GSH.
5 and thus exist as a thiolate anion (Cys-S− ), which is very Two of the main signalling molecules whose activities
vulnerable to oxidation, and these have been referred to as are reduced by oxidation are the protein tyrosine phos-
hyperreactive cysteine residues (the S− group marked with phatases and phosphatase and tensin homologue deleted
an asterisk in Module 2: Figure reversible and irreversible on chromosome 10 (PTEN) (Module 2: Figure ROS form-
ROS oxidations). ation and action). An analysis of tyrosine phosphatase
The sulphenic acid intermediate is somewhat unstable structure and function reveals the presence of a hyperre-
and can be converted into a number of intermediates by active cysteine residue in the catalytic domain that is sens-
either eliminating water or causing it to interact with GSH itive to oxidants resulting in inactivation of the enzyme.
(Reactions 2, 3 and 4 in Module 2: Figure reversible and ir- Other examples of proteins that have such hypersensitive
reversible ROS oxidations). In addition, sulphenic acid can residues include the cell cycle regulatory enzyme Cdc25C,
be oxidized further by H2 O2 to sulphinic acid and sulph- the Ca2+ -release channels ryanodine receptors (RYRs) and
onic acid. The formation of sulphinic acid is reversible inositol 1,4,5-trisphosphate receptors (InsP3 Rs). The spe-
through a reaction that requires ATP and sulphiredoxin cificity with which this redox signalling system exerts its
(Srx). However, the final step to form sulphonic acid is effects therefore depends on the fact that H2 O2 will only
irreversible and can result in serious damage and has been modify hyperreactive cysteine residues that exist in these
implicated in the ageing process. One idea is that this ir- target proteins of the different oxidation-sensitive pro-
reversible change may accumulate with time and the pro- cesses.
gressive damage may result in ageing. Recovery of oxidation-sensitive processes
There is a suggestion that the hyperperoxidation reac- Like other signalling pathways, there are mechanisms in
tions that lead to the irreversible oxidation may be avoided place for the recovery of oxidation-sensitive processes
by an internal reaction whereby the sulphenic acid is rap- based on the thioredoxin (Trx) and glutaredoxin (Grx)
idly converted into a sulphenylamide species by interacting systems (Module 2: Figure recovery of protein oxidation).
with the main-chain nitrogen atom of an adjacent serine Although the two systems have much in common, there
residue (Step 2 in Module 2: Figure reversible and irrevers- are some differences, not least of which are their substrate
ible ROS oxidations). The formation of this sulphenyl- specificity and the kinds of disulphide bonds that they

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r68

Module 2: Figure ROS formation and action

Growth
factors
Cytokines Hormones

NOX/DUOX

Protein PI 3-K PIP3 PIP PIP2

kinase 3
P
Signalling Signalling
protein protein SH
H 2 O2 PTEN -S- S
H
2 Trx
S SH 1 S
Trx PTP
S NH
SH
S Trx --S
PTEN H
SH S S
Trx --S PTP
H N
Inactivated PTEN
Inactivated protein
tyrosine phosphatase (PTP)

Formation of H2 O2 and its action to regulate phosphotyrosine phosphatase (PTP) and phosphatase and tensin homologue deleted on chromo-
some 10 (PTEN).
The hydrogen peroxide (H2 O2 ) formed by the NADPH oxidase (NOX)/DUOX (dual oxidase) complex creates a microdomain in which it can inactivate
enzymes involved in various signalling pathways: 1. The phosphotyrosine phosphatase (PTP) that dephosphorylates various signalling proteins is
inactivated following its oxidation by H2 O2 ; 2. The inositol lipid phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which
dephosphorylates the lipid second messenger PtdIns3,4,5P3 , is inactivated following its oxidation by H2 O2 . These two enzymes can be reactivated
following their reduction by the thioredoxin (Trx) system (Module 2: Figure recovery of protein oxidation).

can reduce. For example, Trx is more effective at reducing The level of Trx reductase is greatly increased in various
protein tyrosine phosphatase 1B than is Grx. tumour cells, where it may play an important role in inhib-
Trx and Grx exist in a reduced or oxidized state, and iting apoptosis. Reduced Trx-(SH)2 is known to bind to
it is the former that enables them to reduce their sub- the apoptosis signal-regulating kinase 1 (ASK1), but when
strates. In doing so, they become oxidized and have to be it is oxidized to Trx-S2 , the ASK1 is released, and proceeds
converted back into a reduced state by the Trx and Grx to induce apoptosis. An enhanced level of Trx reductase
systems respectively. In the case of Trx, this is carried out may prevent this process by ensuring the Trx remains
by thioredoxin reductase. The Grx system is somewhat reduced.
more complicated in that it depends upon glutathione that The ability of this thioredoxin system to reverse
is regenerated by a glutathione reductase. redox signalling is inhibited by Ca2+ , which acts to
The level of Trx is markedly elevated during rheumatoid convert a large proportion of the reduced Trx-(SH)2
arthritis and this may influence the rate of secretion of into its oxidized form. Such an action would enhance
matrix metalloproteinases (MMPs). the growth-promoting activity of the redox signalling
system.
Thioredoxin reductase Thioredoxin-2 (Trx-2) is a mitochondria-specific mem-
Thioredoxin (Trx) reductase together with thioredoxin is ber of the Trx family. It functions to regulate the mitochon-
an important oxidoreductase system that has a signific- drial membrane potential and contributes to the inhibition
ant role in regulating the redox state (Module 2: Figure of apoptosis.
recovery of protein oxidation).
Trx reductase is unusual in that it contains seleno-
Glutathione reductase
cysteine (SeCys) located in the C-terminal active site,
Glutathione reductase is responsible for converting oxid-
which has a highly conserved -Gly-Cys-SeCys-Gly- se-
ized GSSG back into the reduced GSH (Module 2: Figure
quence. The N-terminal region contains one mol of flavin–
recovery of protein oxidation):
adenine dinucleotide (FAD). The enzyme operates by
transferring electrons from NADPH to FAD and then
on to the active site in the C-terminus. GSSG + NADPH + H+ → 2GSH + NADP+

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Module 2: Figure recovery of protein oxidation

THIOREDOXIN SYSTEM
Glucose 100 M 1 M 1-20 M
NADPH TrxR-(SH) Trx-(SH)2 S
2
S
Pentose
cycle SH

NADP + TrxR-S 2 Trx-S 2 SH

GLUTAREDOXIN SYSTEM
100 M 1 M 1-10mM 1 - 20 M
NADPH GrxR-(SH) GSH Grx-(SH) S
2 2
S

SH

NADP + GrxR-S 2 GSSG Grx-S2 SH

Recovery of protein oxidation by the thioredoxin and glutaredoxin systems.

These two systems operate through protein disulphide oxidoreductases, which function to reduce the disulphide bond on the oxidized protein (red)
back to the reduced thiol groups (blue). Thioredoxin (Trx) system: the active site on Trx is a -Cys-32-Gly-Pro-Cys-35- motif and it is these two cysteine
residues in the reduced Trx-(SH)2 form that are responsible for reducing disulphide bonds. Upon transferring the two protons to the substrate protein,
the Trx becomes oxidized to Trx-S2 . Before it can operate again, the Trx-S2 must be converted back into Trx-(SH)2 by thioredoxin reductase (TrxR),
which extracts reducing equivalents from the NADPH formed from the pentose cycle. Glutaredoxin (Grx) system: Grx has a -Cys-Pro-Tyr-Cys- motif,
which is the active site for the oxidoreduction reaction. Glutaredoxin can act both as a dithiol–disulphide oxidoreductase and as a GSH–disulphide
oxidoreductase. The latter action enables Grx to reverse mixed protein disulphides (protein-SSG not shown on the figure) formed when proteins
interact with GSH. In order to continue its reducing function, the Grx-S2 or Grx-SSG must be reduced back to Grx-(SH)2 by its interaction with GSH,
which is maintained in a reduced form by glutathione reductase.

Oxidation-sensitive processes Redox signalling in proliferation and cancer

The redox signalling pathway acts to regulate a vari- One of the main actions of redox signalling is to con-
ety of oxidation-sensitive processes (Module 2: Figure trol growth. During the action of many growth factors
plasma membrane ROS formation). The cellular pro- there is an increase in the production of hydrogen peroxide
teins that are sensitive to oxidation are those that contain (H2 O2 ) (Module 2: Figure plasma membrane ROS form-
hyperreactive cysteine residues. One of the main func- ation), which facilitates growth factor signalling by in-
tions of redox signalling is to modulate the activity of hibiting tyrosine phosphatases and the tumour suppressor
other signalling systems that contain protein compon- phosphatase and tensin homologue deleted on chromo-
ents with such hyperreactive cysteine residues, e.g. Ca2+ - some 10 (PTEN). The latter inhibits the hydrolysis of
releasing channels [inositol 1,4,5-trisphosphate receptors PtdIns3,4,5P3 (PIP3 ), which functions in cell migration,
(InsP3 Rs) and ryanodine receptors (RYRs)], transcription proliferation and survival. H2 O2 inactivates PTEN by in-
factors, protein tyrosine phosphatases and by activating ducing a disulphide bond to form between Cys-124 in the
the mitochondrial permeability transition pore (MTP) to active site and Cys-71. This disulphide is specifically re-
induce apoptosis. As so many key signalling functions are versed by thioredoxin. A reversible inactivation of PTEN
being influenced, it is not surprising to find that there is a may thus contribute to the accumulation of PIP3 , which
role for redox signalling in many cellular processes: will thus set up a positive-feedback loop, since the forma-
tion of PIP3 is responsible for stimulating the production
• redox signalling in proliferation and cancer of H2 O2 .
• redox signalling in apoptosis Many cancer cells are known to use ROS to control their
• Redox signalling and DNA damage proliferation. There are five human homologues of Nox1.
• redox signalling in vascular homoeostasis When Nox1 is overexpressed in fibroblasts, there is an
• redox signalling and gene transcription increase in proliferation and tumour formation. The addi-
• redox signalling and modulation of Ca2+ signalling tion of antioxidants can reduce the growth of cancer cells.

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Module 2: Figure thimerosal-induced Ca2+ signalling

Thimerosal-induced Ca2+ oscillations in oocytes.

A. The normal Ca2+ oscillation induced by sperm fusion in a mouse oocyte. B–D. Examples of Ca2+ oscillations induced by addition of the oxidizing
agent thimerosal at three different concentrations: B, 100 µM; C, 10 µM; D, 1 µM. Reproduced from Cheek, T.R., McGuinness, O.M., Vincent, C.,
Moreton, R.B., Berridge, M.J. and Johnson, M.H. (1993) Fertilisation and thimerosal stimulate similar calcium spiking patterns in mouse oocytes but
by separate mechanisms. Development 119, 179–189, with permission from The Company of Biologists; see Cheek et al. 1993.

Many cancer cells, like a number of stem cells, have en- Cells have different ways of suppressing this ROS-
hanced ROS defences in the form of elevated levels of GSH induced apoptosis. One mechanism is carried out by
and thioredoxin, which make them particularly resistant to PtdIns 3-kinase signalling during which protein kinase
apoptosis. Thioredoxin may also play a role in increasing B (PKB) plays a prominent role through the Forkhead
the expression of the hypoxia-inducible factor 1α (HIF- box O 3a (FOXO3a) transcription factor, which acts
1α), resulting in an increase in vascular endothelial growth by increasing the amount of manganese superoxide dis-
factor (VEGF) and tumour angiogenesis. mutase (MnSOD) to provide greater protection against
ROS (Module 4: Figure FOXO control mechanisms).
An overactive redox signalling system may contribute
Redox signalling in apoptosis to the increase in neuronal cell death that characterizes
In addition to playing a role in cell proliferation, there Alzheimer’s disease (Module 12: Figure astrocyte-induced
is a darker side to reactive oxygen species (ROS) in that neuronal death) and Down’s syndrome.
they can also activate apoptosis. One of the actions of
redox signalling is that it contributes to Ca2+ -induced
apoptosis at the level of the mitochondria. The uptake Redox signalling and DNA damage
of Ca2+ and the resulting increase in ROS formation One of the major pathological consequences of excess
act synergistically to open the mitochondrial permeabil- ROS formation is DNA damage. When this damage oc-
ity transition pore (MTP) (Module 5: Figure mitochon- curs during the G1 phase of the cell cycle, specific repair
drial Ca2+ signalling). ROS can also increase apoptosis mechanisms function to repair the damage and they also
by stimulating the acidic sphingomyelinases (SMases) induce the process of G1 checkpoint signalling to DNA
that produce ceramide (Module 2: Figure sphingomyelin double-strand breaks (DSBs) (Module 9: Figure G1 check-
signalling). point signalling).

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Redox signalling in vascular homoeostasis check the protein tyrosine phosphorylation cascade that
Hydrogen peroxide (H2 O2 ) may function as an occurs during B cell receptor (BCR) activation (Module 2:
endothelium-derived hyperpolarizing factor (EDHF) that Figure ROS effects on Ca2+ signalling). This is a reciprocal
diffuses across to relax neighbouring smooth muscle cells. interaction because it is the increase in Ca2+ that activates
This action may be particularly important in regulating the the formation of H2 O2 through a Ca2+ -dependent activa-
tone of cerebral arteries. tion of dual oxidase (DUOX). This is a good example of
the cross-talk that can exist between signalling pathways.
Redox signalling and gene transcription
The redox signalling pathway has been implicated in the
control of gene transcription, particularly with regard to Mitogen-activated protein kinase
the activation of nuclear factor κB (NF-κB). Whether this (MAPK) signalling
activation is due to a direct modulation of the transcription
factor by messengers such as H2 O2 or indirectly through Overview
activation of other signalling pathways remains to be de- The multifunctional mitogen-activated protein kinase
termined. (MAPK) signalling system consists of separate pathways
A large number of other transcription factors [activating that function to control a number of different cellular pro-
protein 1 (AP-1), specificity protein 1 (SP1), c-Myb, p53 cesses such as gene transcription, metabolism, motility, cell
and Egr-1] are redox-sensitive. Many of these have a highly proliferation, apoptosis, synaptic plasticity and long-term
conserved cysteine residue located within their DNA- memory. These different downstream effectors are activ-
binding domains that has to be reduced in order for the ated by the final MAPK components associated with the
factor to bind DNA (Module 4: Figure SRF and AP-1). In three main signalling pathways:
theory, therefore, such factors would be inhibited by ox-
• Extracellular-signal-regulated kinase (ERK) pathway
idation. There is a nuclear redox factor 1 (Ref-1) that func-
• c-Jun N-terminal kinase (JNK) pathway
tions to control transcription by reducing this cysteine.
• p38 pathway
HDAC oxidation is an important mechanism used to
control chromatin remodelling and gene transcription. These different pathways are assembled by combining
components from an extensive mitogen-activated protein
Redox factor 1 (Ref-1) kinase (MAPK) signalling toolkit.
Ref-1 plays a role in the nucleus to promote gene tran- The mitogen-activated protein kinase (MAPK) sig-
scription (Module 4: Figure SRF and AP-1) and to protect nalling properties such as their spatio-temporal control
cells against oxidative stress. There is a possibility that it mechanisms help to explain how they operate to regulate
might also have a function within the cytoplasm to reduce so many cellular processes.
the Rac-1-regulated production of reactive oxygen species The activity of the MAPK signalling pathway is reversed
(ROS). by the mitogen-activated protein kinase (MAPK) phos-
phatases.
Redox signalling and modulation of Ca2+ signalling
There are reciprocal interactions operating between the Mitogen-activated protein kinase (MAPK)
redox and Ca2+ signalling pathways. There are Ca2+ sig- signalling toolkit
nalling effects on redox signalling and there are redox sig- There is an extensive mitogen-activated protein kinase
nalling effects on Ca2+ signalling. (MAPK) signalling toolkit, which can be divided into
different functional components such as the transducers,
Ca2+ signalling effects on redox signalling
the MAPK kinase kinases (MAPKKKs), MAPK kinases
One of the actions of Ca2+ is to enhance redox signalling
(MAPKKs), MAPKs, MAPK scaffolding proteins and
by interfering with the recovery of the oxidation-sensitive
MAPK target proteins (Module 2: Table MAPK signalling
processes. Ca2+ acts by turning down the thioredoxin sys-
toolkit). Specific components from this toolkit are then
tem by inhibiting the thioredoxin reductase that normally
assembled into the different signalling pathways (Mod-
switches off redox signalling.
ule 2: Figure MAPK signalling).
Redox signalling effects on Ca2+ signalling
There are numerous examples of redox signalling acting to Extracellular-signal-regulated kinase (ERK)
enhance Ca2+ signalling. For example, the two Ca2+ release pathway
channels ryanodine receptors (RYRs) and the InsP3 recept- The extracellular-signal-regulated kinase (ERK) pathway
ors (InsP3 Rs) can be activated by oxidation of key cysteine is one of the major signalling cassettes of the mitogen-
residues. In the case of the latter, the oxidizing agent thi- activated protein kinase (MAPK) signalling pathway
merosal faithfully reproduces the Ca2+ transients normally (Module 2: Figure MAPK signalling). It performs a num-
induced by sperm fusion during fertilization (Module 2: ber of important signalling functions, including the control
Figure thimerosal-induced Ca2+ signalling). of cell proliferation and the synaptic plasticity responsible
Another example is that hydrogen peroxide (H2 O2 ) can for learning and memory. The main MAPK/ERK kinase
markedly enhance Ca2+ signalling by inhibiting the Src kinase (MEKK) components are the Raf family members
homology 2 (SH2) domain-containing protein tyrosine Raf-1, A-Raf and B-Raf that phosphorylate two serine
phosphatase-1 (SHP-1), which normally acts to keep in residues on the MAPK/ERK kinase (MEK) components

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Module 2: Figure ROS effects on Ca2+ signalling

Antigen
BCR

PtdIns4,5P
2 DUOX O2
P PLCγ2
P P

BLNK
P P
P P P
P IP3
Lyn

P H2O2

Syk
P
P Btk Ca
2+ +

SHP1
SHP1
SHP1

_ _
_

Reciprocal interaction between the ROS and Ca2+ signalling pathways during B cell receptor (BCR) activation.
Cross-linking the B cell receptor (BCR) with antigen sets up a protein phosphorylation cascade that begins with Lyn phosphorylating both the receptor
and Syk. The latter then binds to the phosphorylated receptor, where it phosphorylates the adaptor protein B cell linker (BLNK) and the tyrosine kinase
Bruton’s tyrosine kinase (Btk). The latter phosphorylates BLNK and phospholipase Cγ2 (PLCγ2), which associates with BLNK and begins to hydrolyse
PtdIns4,5P2 to form inositol 1,4,5-trisphosphate (IP3 ). This phosphorylation cascade is kept in check by the tyrosine phosphatase Src homology 2
(SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1). One of the functions of the Ca2+ released by IP3 is to set up a positive-feedback
loop based on the activation of the Ca2+ -sensitive enzyme dual oxidase (DUOX) that generates hydrogen peroxide (H2 O2 ). The latter feeds back to
inhibit SHP-1, which enables the signalling cascade to work more effectively in generating Ca2+ signals. This formation of H2 O2 is highly localized
as a microdomain in the immediate vicinity of the BCR (Module 2: Figure ROS microdomains). This figure is based on information taken from
Singh et al. 2005.

MEK1/2. The latter are dual-specificity protein kinases face by the scaffolding protein kinase suppressor of Ras
that phosphorylate the tyrosine and threonine residues of 1 (KSR1). Up to this point, all the signal transduction
the characteristic MAPK components ERK1/2 that are re- processes have occurred at the cell surface, and the next
sponsible for stimulating the downstream effectors, many information transfer step depends upon the diffusion of
of which are transcription factors (Module 2: Figure ERK the activated enzyme from the cell surface to the nucleus.
signalling). There is thus a linear transfer of information Once it is phosphorylated, the activated phospho-ERK1/2
through a phospho-relay system based on a sequential leaves the plasma membrane to diffuse into the cytoplasm
series of phosphorylation events. and then into the nucleus, where it phosphorylates and ac-
An important feature of this ERK pathway, which can be tivates a number of transcription factors (Module 2: Figure
activated by both protein tyrosine kinase-linked receptors ERK signalling). Some of these genes code for MAPK sig-
(PTKRs) and by G protein-coupled receptors (GPCRs), is nalling components, such as MAPK phosphatase 1 (MKP-
its spatial organization (Module 2: Figure ERK signalling). 1), which sets up a negative-feedback loop. In addition,
In the case of the PTKRs, growth factors such as phospho-ERK1/2 can also act together with Ca2+ to stim-
platelet-derived growth factor (PDGF) usually cause re- ulate cytoplasmic phospholipase A2 (cPLA2 ). The Ca2+
ceptor dimerization, which allows the cytosolic tyrosine induces the cPLA2 to associate with cytosolic membranes,
kinase domains to come together and to phosphorylate whereas the ERK1/2 phosphorylates Ser-505 and Ser-727
each other (Module 1: Figure PDGFR activation). These to stimulate the enzymatic release of arachidonic acid from
phosphorylated residues then function as docking motifs phospholipid precursors.
to pull in signalling components such as Shc, growth factor There are a number of putative mechanisms for linking
receptor-bound protein 2 (Grb2) and Son-of-sevenless the activation of G protein-coupled receptors (GPCRs) to
(SoS) that then activate the small GTP-binding protein Ras. the ERK pathway. The release of βγ subunits may activ-
Activated Ras then interacts with the protein kinase Raf, ate Src, which can then feed into the processes that ac-
which initiates the phosphorylation cascade of the ERK tivate Ras. The arrestins can also function as scaffolds to
pathway. The three components of this pathway (Raf-1, assemble components of the ERK pathway such as Raf1
MEK1/2 and ERK1/2) are held in place at the cell sur- and ERK1/2. Alternatively, activation of phosphoinositide

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Module 2: Table MAPK signalling toolkit Module 2 Table continued

Mitogen-activated protein kinase (MAPK) signalling toolkit Component Comment
Component Comment MKP-2 MAPK phosphatase-2
Transducers MKP-3 MAPK phosphatase-3, acts
GCK Germinal centre kinase specifically to dephosphorylate
GLK GCK-like kinase ERK2
HPK1 Haematopoietic progenitor kinase 1 MKP-4 MAPK phosphatase-4
MST1 Mammalian Ste20-like protein kinase MKP-5 MAPK phosphatase-5
MAPKKKs VHR VH1-related
ASK1 Apoptosis signal-regulating kinase 1 hVH3/B23
ASK2 Apoptosis signal-regulating kinase 2 hVH5/M3/6
DLK Dual leucine-zipper-bearing kinase PAC1
MEKK1 MAPK/ERK kinase kinase 1 Pyst2
MEKK2 MAPK/ERK kinase kinase 2 STEP Striatal-enriched protein tyrosine
MEKK3 MAPK/ERK kinase kinase 3 phosphatase; dephosphorylates
MEKK4 MAPK/ERK kinase kinase 4 ERK in neurons (Module 10:
MLK1 Mixed lineage kinase 1 Figure medium spiny neuron
MLK2 Mixed lineage kinase 2 signalling).
MLK3 Mixed lineage kinase 3
Mos
Raf-1
B-Raf hydrolysis by Gq to produce inositol 1,4,5-trisphosphate
PAK p21-activated kinase
TAK1 TGFβ-activated protein kinase 1 (InsP3 ) and diacylglycerol (DAG) can access the ERK
Tpl2 Tumour progression locus 2 pathway via two mechanisms. The InsP3 /Ca2+ can act
MAPKKs through proline-rich tyrosine kinase 2 (Pyk2), whereas
MEK1 MAPK/ERK kinase 1
MEK2 MAPK/ERK kinase 2 DAG and Ca2+ act through protein kinase C (PKC).
MKK3 MAPK kinase 3 One of the major functions of the ERK pathway is
MKK4 MAPK kinase 4 to activate a range of different transcription factors such
MEK5 MAPK/ERK kinase 5
MKK6 MAPK kinase 6 as cyclic AMP response element-binding protein (CREB)
MKK7 MAPK kinase 7 (Module 4: Figure CREB activation) and Elk-1 (Module
MAPKs 4: Figure ETS activation).
ERK1 Extracellular-signal-regulated kinase 1
ERK2 Extracellular-signal-regulated kinase 2 This ERK pathway contributes to the control of a large
ERK3-related number of cellular processes:
ERK3 Extracellular-signal-regulated kinase 3
ERK5 Extracellular-signal-regulated kinase 5 • Regulation of cell proliferation such as T cell activation
ERK7 Extracellular-signal-regulated kinase 7 (Module 9: Figure TCR signalling)
ERK8 Extracellular-signal-regulated kinase 8
JNK1 c-Jun N-terminal kinase 1 • Cardiac hypertrophy (Module 12: Figure hypertrophy
JNK2 c-Jun N-terminal kinase 2 signalling mechanisms)
JNK3 c-Jun N-terminal kinase 3 • Synaptic plasticity such as long-term potentiation (LTP)
p38α
p38β in hippocampal neurons
p38γ • Proliferation of endothelial cells during angiogenesis
p38δ (Module 9: Figure VEGF-induced proliferation)
MAPK scaffolding proteins
β-Arrestin-2 • Phosphorylation of the transcription factor p53
JIP1 JNK-interacting protein 1 (Module 4: Figure p53 domains)
JIP2 JNK-interacting protein 2 • Remodelling the ERK signalling pathway may contrib-
JIP3 JNK-interacting protein 3
KSR1 Kinase suppressor of Ras 1 ute to the development of polycystic kidney disease
KSR2 Kinase suppressor of Ras 2 (Module 12: Figure polycystins and polycystic kidney
MP1 MEK partner 1 disease)
MKPX Phosphatase that may also function as
a scaffold • Activation of phospholipase A2 (PLA2 ) in mast cells
SKRP1 Stress-activated protein kinase (SAPK) (Module 11: Figure mast cell signalling)
pathway-regulating phosphatase 1
MAPK target proteins
ATF2 Activating transcription factor 2 c-Jun N-terminal kinase (JNK) pathway
Cytoplasmic PLA2 Cytoplasmic phospholipase A2 The c-Jun N-terminal kinase (JNK) pathway is one of the
ETS
Elk-1 major signalling cassettes of the mitogen-activated pro-
c-Jun tein kinase (MAPK) signalling pathway. It functions in the
Jun-B control of a number of cellular processes, including prolif-
Jun-D
MAPKAP-kinase MAPK-activated protein kinase eration, embryonic development and apoptosis. The path-
MEF2 Myocyte enhancer factor 2 way takes its name from the c-Jun N-terminal kinases 1–3
MSK1 Mitogen- and stress-activated protein (JNK1–JNK3), which are the MAPKs that interact with
kinase 1
MSK2 Mitogen- and stress-activated protein the final effectors (Module 2: Figure MAPK signalling).
kinase 2 They contain the dual phosphorylation motif Thr-Pro-
SAP-1 Stomach cancer-associated protein Tyr, which is phosphorylated following activation of the
tyrosine phosphatase-1
MAPK phosphatases upstream phosphorylation cascade.
MKP-1 MAPK phosphatase-1 (Module 2: The JNK pathway is activated by a bewildering number
Figure ERK signalling) of mechanisms. This complexity is evident by the fact that

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Module 2: Figure ROS microdomains

BCR
Resting
SHP1 DUOX SHP1

Lyn
SHP1

BLNK
SHP1 SHP1 SHP1 SHP1
SHP1 PLC IP3

Syk

Btk
SHP1 SHP1
SHP1 2+ SHP1
SHP1 Ca
SHP1
SHP1 SHP1 SHP1
SHP1 SHP1
SHP1 SHP1 SHP1
SHP1

Antigen

Stimulated
DUOX
SHP1 SHP1
Lyn

SHP1

BLNK
SHP1 SHP1 SHP1 SHP1
SHP1
Syk PLC IP3
SHP1 SHP1

Btk
SHP1 SHP1
SHP1
SHP1 2+
H O microdomain
SHP1
SHP1
2 2 Ca
SHP1
SHP1 SHP1 SHP1 SHP1
SHP1 SHP1
SHP1 SHP1
PROLIFERATION

Localized ROS signalling in a microdomain surrounding the B cell receptor.

Under resting conditions, the protein phosphorylation cascade that operates between Lyn, Syk, Bruton’s tyrosine kinase (Btk) and phospholipase C
(PLC) (Module 2: Figure ROS effects on Ca2+ signalling) is inhibited by the tyrosine phosphatase Src homology 2 (SH2) domain-containing protein
tyrosine phosphatase-1 (SHP-1), which is present at very high levels. When the receptor is stimulated by antigen, there is a Ca2+ -dependent activation
of dual oxidase (DUOX) that creates a microdomain of H2 O2 (yellow oblong) to inhibit the SHP-1 enzymes in the immediate vicinity of the B cell
receptor (BCR). This figure is based on information taken from Singh et al. 2005.

there are 13 MAPK kinase kinases (MAPKKKs) respons- p38 pathway

ible for feeding information into the JNK pathway. One The p38 pathway is one of the major signalling cassettes of
way of trying to cope with this complexity is to exam- the mitogen-activated protein kinase (MAPK) signalling
ine specific examples such as the activation of JNK by the pathway. It functions in the control of apoptosis and the
interleukin-1 receptor (Module 2: Figure JNK signalling). release of cytokines by macrophages and neutrophils. The
The JNK pathway can also be activated through G pro- pathway takes its name from the family of p38 kinases,
tein-coupled receptors (GPCRs) using G proteins such as which are the MAPKs that interact with the final effectors
G12/13 . Just how G proteins feed into the cascade is unclear, (Module 2: Figure MAPK signalling).
but it seems that they activate the GTP-binding proteins The p38 pathway can be activated either by different
such as Rac and Cdc42. Alternatively, the arrestins that receptor mechanisms or by various environmental stresses
associate with G protein-coupled receptors (GPCRs) dur- such as osmotic, redox or radiation stress. For example, one
ing the process of receptor desensitization may function of the targets of the p38 pathway that is activated by UV
as a scaffold to bring together components of the JNK irradiation is one of the Cdc25 enzymes that control cell
pathway such as MKP7 and JNK3. cycle progression. Phosphorylation of Ser-323 on Cdc25B
The MAPK signalling system operates a negative- results in the binding of 14–3-3 protein, which then pre-
feedback loop in that some of the genes activated by the vents this enzyme from initiating entry into mitosis.
JNK pathway code for signalling components such as the The Toll receptor signalling pathway provides an ex-
scaffold protein JNK-interacting protein 1 (JIP1), which ample of how the p38 pathway is activated by a Toll re-
will bind JNK and thus limits its action. ceptor for lipopolysaccharide (LPS) (Module 2: Figure Toll
This JNK pathway contributes to the control of a large receptor signalling).
number of cellular processes:

• Phosphorylation of the transcription factor p53

(Module 4: Figure p53 domains). Mitogen-activated protein kinase (MAPK)
• The JNK pathway has been implicated in the signalling properties
mitogen-activated protein kinase (MAPK) signalling in The different mitogen-activated protein kinase (MAPK)
cardiac hypertrophy (Module 12: Figure hypertrophy signalling pathways have a number of important properties
signalling mechanisms). that greatly enhance their signalling efficiency.

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Module 2: Figure MAPK signalling

ERK pathway JNK pathway p38 pathway

Growth factors Osmotic stress FasL, Osmotic stress,
Stimulus redox stress, cytokines radiation
radiation

Transducers
Grb2 SOS Rac Cdc42 Rac Cdc42
Ras Rho

MAPKKK Raf-1, A-Raf, B-Raf MEKK1-4, MLK1-3, TAK1, TAK1

ASK1/3, TPL-2

MAPKK MEK1/2 MEK4/7 MEK3

MAPK ERK1/2 JNK1-3 p38αβγδ

Effectors Ets, Elk1, SAP1 c-Jun, Jun-B, Jun-D, ATF2, Ets, MEF2,
cPLA2 ATF2, Elk1 SAP1, MAPKAP2

Cellular Proliferation Proliferation, apoptosis, Apoptosis, cytokine

responses embryonic development release, cell cycle arrest

Organization of the main mitogen-activated protein kinase (MAPK) signalling pathways.

The generic pathway on the left summarizes the sequential organization of the mitogen-activated protein kinase (MAPK) signalling system. External
stimuli act through a variety of transducers to stimulate the first element of the signalling pathway, which is one of the 14 different MAPK kinase
kinases (MAPKKKs). This MAPKKK then phosphorylates the next element, which is one of the seven MAPK kinases (MAPKKs). This MAPKK then
phosphorylates one of the 12 MAPKs, the names of which define the different signalling pathways. The three main pathways are the ERK pathway
(Module 2: Figure ERK signalling), the JNK pathway (Module 2: Figure JNK signalling) and the p38 pathway. The components that make up these
different signalling pathways are summarized in the MAPK signalling toolkit (Module 2: Table MAPK signalling toolkit).

Module 2: Figure ERK signalling

Growth factors

GPCR PTKR

Grb2 SOS Ras

PLC Gq P
SHC
β γ Src P Raf-1
2+ P
KSR1
Ca
Pyk2 P MEK1/2
+
cPLA2
+ PKC P
+ ERK1/2
DAG P

P
cPLA2 +
ERK1/2
PL
Arachidonic
acid P
ERK1/2
P MKP-1

Negative
ETS SAP1 ELK1 feedback
MKP1 loop

Proliferation

The organization and topology of the extracellular-signal-regulated kinase (ERK) pathway.

The extracellular-signal-regulated kinase (ERK) pathway, which can be made up from different components (Module 2: Figure MAPK signalling), is
represented here by Raf-1, mitogen-activated protein kinase (MAPK)/ERK kinase 1/2 (MEK1/2) and ERK1/2, which can be activated by either protein
tyrosine kinase-linked receptors (PTKRs) or by G protein-coupled receptors (GPCRs). (See the text for further details.)

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Module 2: Figure JNK signalling

Interleukin-1

GPCR

RAC/
TRAF6 TAK1 Cdc42 Gα
12/13
P MEKK1 β γ
JIP1
P MKK7 PI3K-γ

P
JNK
P
Cytochrome c
JNK
Caspases
Negative feedback
loop
APOPTOSIS P ?
JNK
P

C-Jun ATF2 ELK1

JIP1

Proliferation,
embryonic development

The organization and topology of the c-Jun N-terminal kinase (JNK) pathway.
The c-Jun N-terminal kinase (JNK) pathway can be activated in many ways, including via different receptor mechanisms and by various environmental
stresses such as osmotic, redox and radiation stress. These different inputs are transduced by separate mechanisms that all feed into the JNK
signalling cascade. With regard to receptor activation, the JNK pathway can be activated by various cytokines such as interleukin-1 as illustrated
here. The interleukin-1 receptor (IL-1R) is composed of two receptor-binding domains that interact with interleukin-1 and a non-binding accessory
protein. Once activated by interleukin-1, the IL-1R recruits the adaptor protein tumour-necrosis-factor-receptor-associated factor 6 (TRAF6), which then
recruits the mitogen-activated protein kinase kinase kinase (MAPKKK) called transforming growth factor β-activated kinase 1 (TAK1) responsible for
initiating the phosphorylation cascade by phosphorylating MAPK/extracellular-signal-regulated kinase (ERK) kinase kinase 1 (MEKK1). The MEKK1
then phosphorylates the dual-specificity protein kinase MAPK kinase 7 (MKK7) responsible for phosphorylating the tyrosine and threonine residues on
JNK. This activation cascade occurs in the vicinity of the plasma membrane, where it is organized by the scaffolding protein JNK-interacting protein 1
(JIP1). Once JNK is phosphorylated, it leaves the multimolecular activation complex and then diffuses into the nucleus, where it activates transcription
factors responsible for controlling processes such as proliferation, apoptosis and embryonic development.

Temporal aspects of mitogen-activated protein kinase signalling hierarchies). Examples of such scaffolds are the
(MAPK) signalling scaffolding proteins kinase suppressor of Ras 1 (KSR1)
The outcome of these pathways is very dependent on tem- (Module 2: Figure ERK signalling) and JNK-interacting
poral aspects, particularly signal duration. For example, protein 1 (JIP1) (Module 2: Figure JNK signalling).
prolonged stimulation is necessary to induce cell prolifer- Other determinants of fidelity are the docking sites
ation. that enable the different mitogen-activated protein kinases
(MAPKs) to bind to their specific downstream effectors.

Fidelity
Another important property is the fidelity of these sig- Phenotypic remodelling of the mitogen-activated
nalling pathways. The three major signalling pathways protein kinase (MAPK) signalling pathway
share many common features: they often share signalling The mitogen-activated protein kinase (MAPK) signalling
components, they are often activated by similar inputs [es- pathway operates autoregulatory loops in that the dif-
pecially in the case of the c-Jun N-terminal kinase (JNK) ferent signalling pathways can regulate the expression
and p38 pathways], and they can also regulate similar cel- of their own signalling components. For example, the
lular processes. A question therefore arises as to how the extracellular-signal-regulated kinase (ERK) pathway can
fidelity of these signalling pathways is achieved in order regulate the expression of MAPK phosphatase 1 (MKP1)
to reduce cross-talk and to ensure that they carry out (Module 2: Figure ERK signalling), whereas the c-Jun N-
their particular functions. It seems that much of this fi- terminal kinase (JNK) pathway induces the expression of
delity is achieved by using molecular scaffolds to hold JNK-interacting protein 1 (JIP1) (Module 2: Figure JNK
together all the components of each signalling pathway in signalling). This induction of MKP1 and JIP1 effectively
a multimolecular complex. In this way, information can set up negative-feedback loops that limit the activity of the
be passed from one component to the next without inter- MAPK signalling pathway, and is an example of signalsome
ference from other signalling pathways (Module 6: Figure stability.

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r77

Phenotypic remodelling of the MAPK signalsome may Module 2: Table NF-κB signalling toolkit
result in the abnormal cell proliferation observed in The nuclear factor κB (NF-κB) signalling toolkit
polycystic kidney disease (Module 12: Figure polycystins Nuclear factor κB (NF-κB) Comments
transcription system
and polycystic kidney disease). NF-κB/Rel family of
transcription factors
p50 (NF-κB1) p50 has a p105 precursor protein
p52 (NF-κB2) p52 has a p100 precursor protein
Nuclear factor κB (NF-κB) signalling p65 (NF-κB3, also known
pathway as RelA)
The transcription factor nuclear factor κB (NF-κB) is RelB
c-Rel
activated by a large number of external stimuli such as Inhibitor of NF-κB (IκB) family
the tumour necrosis factors (TNFs), interleukin-1 (IL-1) IκBα Bind to p65 and c-Rel
and the pathogen-associated molecular patterns (PAMPs), IκBβ
IκBγ
which are responsible for controlling processes such as IκBε
inflammation, cell proliferation and apoptosis. NF-κB be- IκBζ Binds preferentially to p50
longs to the group of transcription factors that lie latent in Bcl3 B-cell lymphoma 3 binds to
p50/p50 and p53/p53
the cytoplasm and then translocate into the nucleus upon homodimers.
activation (mechanism 2 in Module 4: Figure transcription Inhibitor of NF-κB (IκB)
factor activation). This diversity of downstream effector kinases (IKKs)
IKKα
processes indicates that there must be separate signalling IKKβ
pathways, and this is evident from the nuclear factor κB NEMO (IKKγ) A regulatory subunit responsible
(NF-κB) signalling toolkit, which contains multiple iso- for the interaction with
upstream kinases on the
forms both of the transcription factors (the NF-κB/Rel receptor complex (Module 2:
family) and of the activation components. This complex- Figure NF-κB activation)
ity is carried through to the nuclear factor κB (NF-κB) Associated proteins
Cdc37
signalling pathway, where there are many variations on Hsp90
the basic theme of NF-κB activation (i.e. Mechanism 2 in
Module 4: Figure transcription factor activation).
There are two remarkable aspects of the NF-κB sig- of transcription factors normally function as heterodimers,
nalling pathway. Firstly, it can control a very large number and the p50/p65 complex was the first to be discovered.
of genes that are often activated as large cohorts in spe- Some of the family members can form homodimers, such
cific cells by different stimuli. Secondly, it is used by a as p50/p50 and p52/p52, and these act as repressors of
number of different signalling systems with subtle vari- NF-κB-sensitive genes.
ations in the mechanism and the components that are used
to convey information into the nucleus. The tumour nec- Inhibitor of nuclear factor κB (NF-κB) (IκB)
rosis factor α (TNFα) signalling pathway and the Toll The inhibitor of nuclear factor κB (NF-κB) (IκB) family
receptor signalling pathway will be described to illustrate are characterized by an ankyrin repeat domain (Module
the main features of the NF-κB signalling pathway. The 2: Figure NF-κB, IκB and IKK structure), which func-
receptor activator of nuclear factor κB (NF-κB) ligand tions in its interaction with NF-κB to form the inactive
(RANKL), which is a transmembrane protein that belongs complex that resides in the cytoplasm. The IκB inhibits
to the TNF family of cytokines, activates the RANKL re- transcription by masking the nuclear localization signal
ceptor (RANK) that uses the NF-κB signalling pathway (NLS) on NF-κB, which thus prevent it from entering the
to control osteoclastogenesis (Module 8: Figure osteoclast nucleus. The NF-κB/IκB complex remains inactive within
differentiation). the cytoplasm until the IκB is removed following activa-
tion of the nuclear factor κB (NF-κB) signalling pathway.
Nuclear factor κB (NF-κB) signalling toolkit Much of the specificity within the NF-κB signalling path-
The NF-κB signalling toolkit is composed of four main way depends on these IκB isoforms being able to bind to
classes of signalling components (Module 2: Table NF-κB different dimers of the NF-κB/Rel family.
signalling toolkit).
Inhibitor of nuclear factor κB (IκB) kinases (IKKs)
Nuclear factor κB (NF-κB)/Rel family The inhibitor of nuclear factor κB (IκB) kinases (IKKs)
The nuclear factor κB (NF-κB)/Rel family consists of function as heterodimers and are responsible for phos-
five members (Module 2: Table NF-κB signalling toolkit). phorylating IκB to mark it for subsequent degradation
There is some confusion concerning the nomenclature of by the proteasome. The kinase domain is located in the
this family. The notations shown at the beginning of the N-terminus, whereas the C-terminus has leucine zipper
table (p50, p52, p65, RelB and c-Rel) will be used here. and helix–loop–helix domains (Module 2: Figure NF-κB,
All members of the family share an N-terminal Rel ho- IκB and IKK structure). These two protein-association
mology domain (RHD), which binds to DNA (Module domains enable the enzyme to associate with a large
2: Figure NF-κB, IκB and IKK structure). The RHD is multisubunit complex in the cytoplasm. Another import-
also used to associate NF-κB with the inhibitor of nuclear ant component of this complex is nuclear factor κB (NF-
factor κB (NF-κB) (IκB) proteins. The NF-κB/Rel family κB) essential modulator (NEMO) (IKKγ), which is a

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Module 2: Figure NF-κB, IκB and IKK structure

NF-κB/Rel family
p65 RHD

c-Rel RHD

RelB RHD

p50 RHD

p52 RHD

Ankyrin repeat
IκB family domain

IκBα
IκBβ
IκBγ

IκBε

Bcl3

IKK family
IKKα Ser kinase ZIP HLH

IKKβ Ser kinase ZIP HLH

NEMO CCD CCD ZIP

Zn finger

The structure of components of the nuclear factor κB (NF-κB) signalling toolkit.

The nuclear factor κB (NF-κB)/Rel family all share a Rel homology region (RHD). The mauve box in the N-terminal region of RelB is a putative leucine
zipper region. The p50 and p52 isoforms also have ankyrin repeat domains that resemble those found in the inhibitor of NF-κB (IκB) family. The IκB
kinase (IKK) family has both kinase (IKKα and IKKβ) and regulatory components [NF-κB essential modulator (NEMO)]. ZIP, leucine zipper; HLH,
helix–loop–helix domain; CCD, coiled-coil domain. Redrawn from Handbook of Cell Signalling, Vol. 3 (R.A. Bradshaw and E.A. Dennis, eds), Westwick,
J.K., Schwamborn, K. and Mercurio, F., NFκB: a key integrator of cell signalling, pp. 107–114. Copyright (2003), with permission from Elsevier; see
Westwick et al. 2003.

regulatory/structural subunit responsible for the inter- can activate other signalling pathways (Module 1: Figure
action with upstream kinases on the receptor as part of cytokines). In this section, attention will focus on the NF-
the tumour necrosis factor α (TNFα) signalling pathway κB signalling pathway, which is used by the tumour nec-
(Module 2: Figure NF-κB activation). rosis factor α (TNFα). It is considered to be one of the
The tumour suppressor CYLD binds to NEMO and ‘classical’ mechanisms as illustrated by the following se-
inhibits the subsequent phosphorylation of IκB as part of quence of events (Module 2: Figure NF-κB activation):
the TNFα signalling pathway.
1. Occupation of the TNF receptor (TNF-R) by TNF
Tumour necrosis factor α (TNFα) signalling induces receptor oligomerization to form a complex
pathway that attracts the adaptors TRADD and the TNFR-
The basic operation of the nuclear factor κB (NF-κB)/Rel associated factor 2 (TRAF2), which is an adaptor
family of transcription factors is that they are activated that belongs to the TNF-receptor-associated factor
in the cytoplasm and then translocate into the nucleus to (TRAF) family.
activate transcription (mechanism 2 in Module 4: Figure 2. TRAF2 is a RING domain E3 ubiquitin ligase that as-
transcription factor activation). There are a number of vari- sociates with the heterotrimeric ubiquitin-conjugating
ations in the way that this translocation process is initiated, (E2) complex that contains Ubc13 and Uev1A. This
depending on the nature of the incoming signals and the is a K63 ubiquitinating complex that adds ubiquitin
receptors that are being activated. In addition to the activ- chains to TRAF2 and this then helps to recruit the
ation of NF-κB, these non-enzyme-containing receptors receptor-interacting protein 1 (RIP1), which is also

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r79

ubiquitinated. These ubiquitin chains provide an im- (Module 11: Figure formation and action of PAMPs).
portant scaffolding role in the assembly of additional These PAMPs act on Toll-like receptors (TLRs) and the
elements of the signal transduction pathway. IL-1 receptor (IL-1R) and these form a TLR/IL-1R fam-
3. The developing receptor complex attracts additional ily that act through a similar Toll receptor signalling path-
components such as the transforming growth factor β way (Module 1: Figure cytokines). The primary function
activated kinase-1 (TAK1), the TAK1-binding (TAB) of this signalling pathway is to stimulate the transcrip-
proteins 2 and 3 (TAB2/3) and the multisubunit cyto- tional processes that result in the expression of a wide
plasmic complex containing the inhibitor of NF-κB range of inflammatory cytokines and immunoregulators
(IκB) kinase (IKK) α/IKKβ dimer and the regulatory (Module 2: Figure Toll receptor signalling). As such, the
NF-κB essential modifier (NEMO) subunit, which is Toll receptor signalling pathway has to transmit informa-
a central player in this translocation sequence. tion from the TLRs and the IL-1R on the cell surface to the
4. Once this complex is complete, the TAK1 phos- transcriptional factors such as nuclear factor κB (NF-κB)
phorylates and activates IKKβ. and activating protein 1 (AP-1) (c-Jun/Fos) that act within
5. The IKKβ then phosphorylates IκBα on two sites the nucleus. This information transfer system will be il-
(Ser-32 and Ser-36). lustrated by reference to the response of the receptor
6. The phosphorylated IκBα is then susceptible to ubi- TLR4 to lipopolysaccharide (LPS) (Module 11: Figure
quitination by the Skp1/cullin/F-box (SCF) ubiquitin formation and action of PAMPs). As for many other sig-
ligase. nalling pathways, information is transferred through both
7. The polyubiquitinated IκBα is then sent to the protea- protein–protein interactions and protein phosphorylation
some, where it is degraded, resulting in the liberation reactions. The ubiquitin signalling system also has an im-
of the NF-κB heterodimer p50/p65. portant role in orchestrating this Toll receptor signalling
8. The NF-κB is imported into the nucleus, where it pathway as shown in the following sequence (Module 2:
binds to the κB promoter elements to activate expres- Figure Toll receptor signalling):
sion of many different genes.
9. Transcription ceases when NF-κB is exported from 1. The TLR4 is a transmembrane protein that has
the nucleus. One of the genes activated by NF-κB is leucine-rich repeats in its ectodomain, while the
IκBα, which thus sets up a negative-feedback loop, cytoplasmic domain has a Toll/interleukin 1 (IL-
because it binds to the NF-κB that is exported from 1) receptor (TIR) domain. The lipopolysaccharide
the nucleus to reconstitute the inactive cytoplasmic (LPS) that initiates this signalling pathway binds first
complex. to an extracellular LPS-binding protein (LBP) and
10. The ubiquitin signalling pathway contributes to to CD14, which is a glycosylphosphatidylinositol-
the recovery of this signalling cascade after TNFα anchored membrane glycoprotein, and this complex
is withdrawn by removing the ubiquitin scaffolds carries the LPS to the Toll-like receptor 4 (TLR4).
that hold together the transducing complex. The 2. The TIR domain on the TLR4 receptor forms ho-
deubiquitinating enzymes A20 and CYLD are par- mophilic interaction with the TIR domain of the
ticularly active in removing the ubiquitin chains. The adaptor proteins TIR adaptor protein (TIRAP) and
reversible ubiquitination of signal transducing com- MyD88. The latter is specific for certain TLRs, such
ponents is thus an essential part of the processing of as TLR4, but not others. The other end of these ad-
information by this TNF signalling pathway. aptors have a death domain that draws in the IL-
1 receptor-associated kinases 1 and 4 (IRAK-1 and
Tumour necrosis factor (TNF)-receptor-associated IRAK-4), which undergoes an autophosporylation re-
factor (TRAF) family action that enables them to bind to another adaptor
The tumour necrosis factor (TNF)-receptor-associated called TRAF6, which belongs to the tumour necrosis
factor (TRAF) family has seven members, which func- factor (TNF)-receptor-associated factor (TRAF) fam-
tion in the signalling pathways of various TNF receptor ily that has a critical role to play in the next series of
superfamily and Toll/interleukin-1 receptor mechanisms. reactions.
TRAF2 functions as an adaptor for the tumour necrosis 3. The IRAK-1 and TRAF6 dissociate from the receptor
factor receptor (Module 2: Figure NF-κB activation). In and move in to the cytoplasm.
the case of osteoclasts, it is TRAF6 that couples the 4. The TRAF 6 is a RING domain E3 ubiquitin lig-
receptor activator of nuclear factor κB (NF-κB) ligand ase that associates with the heterotrimeric ubiquitin-
(RANKL) receptor (RANK) to downstream signalling conjugating (E2) complex that contains Ubc13 and
pathways (Module 8: Figure osteoclast differentiation). Uev1A. This is a K63 ubiquitinating complex that
results in the autoubiquitination of TRAF6. The
Toll receptor signalling pathway ubiquitinated TRAF6 then binds the transforming
The Toll receptor signalling pathway plays a central growth factor β activated kinase-1 (TAK1) and the
role in inflammatory responses (Module 11: Figure in- TAK1-binding (TAB) proteins 2 and 3 (TAB2/3). The
flammation). This pathway is activated by two types of multisubunit cytoplasmic complex containing the in-
stimuli: cytokines, represented by interleukin-1 (IL-1), hibitor of NF-κB (IκB) kinase (IKK) α/IKKβ dimer
and stimuli derived from pathogens that are known and the regulatory NF-κB essential modifier (NEMO)
as pathogen-associated molecular patterns (PAMPs) subunit are also drawn into the complex. The

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r80

Module 2: Figure NF-κB activation

TNFα TNFα TNFα

TNFR

DD

DD

DD
DD

DD
DD

DD
10
Ubc13/ Ubc13/
Uev1a Ubiquitin Uev1a A20 Ubiquitin

TRAF2
TRAF2

TRAF2
TRADD 2 CYLD
RIP1 RIP1
TAB2/3
IKKα NEMO TAB2/3
IKKβ P TAK1
TAK1
5 4 3
P P P P NEMO
6
Proteasome IκBα IκBα IκBα IKKα IKKβ
p50 p65 p50 p65 p50 p65
7

p50 p65 NF-κB 9 IκBα

Export
Import

8 IκBα
IL-6
p50 p65 CYLD
κB A20

The ‘classical’nuclear factor κB (NF-κB) signalling pathway activated by the tumour necrosis factor receptor (TNFR).
The p50 and p65 isoforms of the nuclear factor κB (NF-κB)/Rel family form the NF-κB dimer that is activated in the tumour necrosis factor α (TNFα)
signalling pathway. The activated TNF receptor (TNFR) recruits a signalling complex to the membrane (Steps 2–4), which contains the inhibitor of
NF-κB (IκB) kinase (IKK) α/IKKβ dimer that is responsible for phosphorylating the IκBα subunit that retains p50/p65 in the cytoplasm (Step 5). When
the IκBα is phosphorylated, it is ubiquitinated and degraded by the proteasome (Steps 6 and 7). The p50/p65 homodimer is imported into the nucleus
(Step 8), where it activates a large number of genes. One of these genes codes for IκBα, which sets up a negative-feedback loop by exporting
p50/p65 from the nucleus (Step 9). Adapted from Trends Biochem. Sci., Vol. 30, Viatour, P., Merville, M.-P., Bours, V. and Chariot, A., Phosphorylation
of NF-κB and IκB proteins: implications in cancer and inflammation, pp. 43–52. Copyright (2004), with permission from Elsevier; see Viatour et al.
2005.

Module 2: Figure Toll receptor signalling

LBP TLR4
1
Cd14

LPS

MyD88 P P Ubiquitin
IRAK-1 4

IRAK-1 Ubc13/
TIRAP Uev1a
P 3 A20
IRAK-4 TRAF6 10
P CYLD
2 TAB2/3
TRAF6 TAK1 Proteasome
NEMO
IKKα P P
IKKβ IκBα 8
6
P
5 P P
7
IκBα
JNK p38 IκBα
p50 p65 p50 p65
NF-κB

Import Inflammatory cytokines

9 TNF-α;IL-6; IL-1 β
P
Immunoregulators
JUN Fos p50 p65
TGF-β; prostaglandins
CRE κB PDE4a

The Toll receptor signalling pathway.

The lipopolysaccharide (LPS) that initiates this signalling pathway binds first to an extracellular LPS-binding protein (LBP) and to CD14, which is a
glycosylphosphatidylinositol-anchored membrane glycoprotein, and this complex carries the LPS to the Toll-like receptor 4 (TLR4). The activated TLR4
then recruits a signalling complex to relay information to both the p38 and nuclear factor κB (NF-κB) signalling pathways to induce the transcription
of a number of inflammatory cytokines and immunoregulators as described in the text.

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resulting activation of TAK1 is then responsible for 2. The TLR9, which responds to CpG DNA, and the
relaying information out to other components of the TLR7/8 that bind ssRNA both recruit the adaptor
signalling pathways. protein MyD88. The latter then acts through TRAF6,
5. The TAK1 activates both the c-Jun N-terminal kinase which is one of the tumour necrosis factor (TNF)-
(JNK) pathway and the p38 pathway (Module 2: Fig- receptor-associated factor (TRAF) family, to stimulate
ure MAPK signalling). The JNK and p38 function the IKKαβ. The latter then activates the transcrip-
to phosphorylate transcription factors such as AP- tion factor NF-κB through the Toll receptor signalling
1, which binds to the cyclic AMP response element pathway described in more detail earlier (Module 2:
(CRE) site. Figure Toll receptor signalling).
6. The other action of TAK1 is to phosphorylate the 3. The NF-κB is imported into the nucleus where it func-
IKKβ component of the inhibitor of NF-κB (IκB) tions to induce the transcription of both the inflam-
kinase (IKK) α/IKKβ dimer. matory cytokines and the genes for type I interferon
7. The phosphorylated IKKβ then acts to phosphorylate (IFN).
IκBα to liberate the NF-κB. 4. When activated by dsRNA, the TLR3 receptor in-
8. The phosphorylated IκBα is then susceptible to ubi- teracts with the TIR-domain-containing adaptor pro-
quitination by the Skp1/cullin/F-box (SCF) ubiquitin tein inducing IFN-β (TRIF). The latter then activ-
ligase. The polyubiquitinated IκBα is then sent to the ates the two IKK-related proteins TBK1 [TRAF-
proteasome, where it is degraded. family member-associated NF-κB activator (TANK)-
9. The NF-κB enters the nucleus, binds to the κB site binding protein] and IKKε, which is also known as
and activates the genes that code for the inflammatory inducible IKK (iIKK).
cytokines and immunoregulators that contribute to 5. The activated TBK1/IKKε complex then phos-
inflammatory responses (Module 11: Figure inflam- phorylates the interferon-regulatory factors (IFRs)
mation). IRF3 and IRF7. When phosphorylated, these two
10. An important aspect of the recovery of this signalling IRFs form homodimers that are then imported into
cascade after LPS is withdrawn, is the removal of the nucleus where they contribute to the transcription
the ubiquitin scaffolds that hold together the trans- of the type I interferons (IFN-α and IFN-β).
ducing complex. The deubiquitinating enzymes A20
and CYLD are particularly active in removing the ubi- Retinoic acid-inducible gene I (RIG-I)-like receptors
quitin chains. This ubiquitin signalling system thus (RLRs)
plays an essential role in the processing of informa- 6. Those viral PAMPs that are directed into the cyto-
tion by this Toll receptor signalling pathway. plasm interact with cytoplasmic receptors such as the
This signalling pathway is particularly evident on res- RIG-I-like receptors (RLRs) (see yellow box in the
ident macrophages (Module 11: Figure macrophage sig- middle of Module 2: Figure viral recognition).
nalling) and mast cells (Module 11: Figure mast cell sig- 7. The two main RLRs are retinoic acid-inducible gene I
nalling) that respond to invading pathogens. (RIG-I) itself and melanoma-associated gene 5
(MDA5). These two receptors have a characteristic
Virus recognition and antiviral responses DExD/H box helicase domain that is responsible
The recognition and initiation of antiviral responses, which for binding double-stranded RNA (dsRNA). RIG-I
is a part of the innate immune system, depend upon the ac- can also respond to 5’ triphosphate ssRNA. These
tivation of a number of signalling pathways such as the Toll two receptors also have a pair of N-terminal caspase
receptor signalling pathway. When viruses enter cells they recruitment domains (CARD), which are the func-
are broken down into fragments such as double-stranded tional transducing components that signal to down-
RNA (dsRNA), single-stranded RNA (ssRNA), 5 tri- stream elements. The LGP2 (laboratory of genetics
phosphate ssRNA or CpG DNA, which are located either and physiology-2), which also has a helicase domain,
in the cytoplasm or within the endosomal compartment can also bind dsRNA, but it lacks the CARD domains
(Module 2: Figure viral recognition). These fragments are and thus fails to transduce cellular signals but may act
examples of the pathogen-associated molecular patterns as a negative regulator of MDA5 and RIG-I.
(PAMPs) that are responsible for triggering a variety of in- 8. The CARD domains on MDA5 and RIG-I interact
flammatory responses (Module 11: Figure formation and with the IFN-β promoter stimulator-1 (IPS-1), which
action of PAMPs). The following sequence of reactions also contains an N-terminal CARD domain and is
describes how three main groups of receptors detect these attached to the outer mitochondrial membrane. IPS1
viral PAMPs to initiate a number of antiviral signalling is also known as MAVS (mitochondrial antiviral sig-
pathways (Module 2: Figure viral recognition): nalling), VISA (virus-inducing signalling adaptor) or
Toll-like receptors (TLRs) CARDIF (CARD adaptor inducing IFN-β). The IPS1
protein then stimulates the TBK1/IKKε complex that
1. Those viral PAMPs, which are directed into the then phosphorylates the interferon-regulatory factors
endosome, interact with Toll-like receptors (TLR3, (IFRs) IRF3 and IRF7 as described above (see step 5).
TLR7/8 and TLR9) that are located in the endosomal
membrane (see green box on the left of Module 2: Nucleotide oligomerization domain (NOD) protein-like
Figure viral recognition). receptors (NLRs)

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r82

Module 2: Figure viral recognition

Viruses

1 6
Endosome
TLRs NLRs
RLRs 7

5’Triphosphate ssRNA
dsRNA NALP3
CpG
ssRNA dsRNA dsRNA
DNA MDA5 P
TLR9 TLR3 P 9
LGP2 P
TLR7/8
ASC
RIG-I
MyD88 2 TRIF IPS1
Caspase-1
MyD88 4 8
Mitochondrion 10
TRAF6
IκBα
p50 p65 TBK1
NF-κB IKKε
IKKα IKKβ p50 p65 Pro-IL-1β
P P P P IL-1β
IκBα 3 IRF3 5 IRF7
Import

Type I IFN genes

TNF-α, IL-6
IL-1β P P IFN α/β
p50 p65 p50 p65 IRF3 IRF7
Inflammatory
κB cytokines κB

Viral recognition and antiviral responses.

When viruses enter cells they are degraded into short fragments of double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), 5’ triphosphate
ssRNA or CpG DNA. There are three main groups of sensors that detect these fragments: Toll-like receptors (TLRs, see green box), retinoic acid-
inducible gene I (RIG-I)-like receptors (RLRs, see yellow box) and nucleotide oligomerization domain (NOD)-like receptors (NLRs, see pink box).
These different receptors then activate different signalling systems to induce transcription of the inflammatory cytokines and interferon as described
in the text. The information used to construct this figure was taken from McCartney and Colonna (2009) and Takeuchi and Akira (2009).

9. The double-stranded RNA (dsRNA) in the cytoplasm metabolism occurs through different pathways that gen-
can also interact with the nucleotide oligomerization erate further signalling molecules such as diacylglycerol
domain (NOD) protein-like receptors (NLRs) (see (DAG) and lysophosphatidic acid (LPA).
pink box on the right of Module 2: Figure viral re-
cognition). One of the NLRs is the Nacht domain-, Phospholipase D (PLD) activation
Leucine-rich repeat-, and PYD-containing protein 3 Mammals have two phospholipase D (PLD) genes (PLD1
(NALP3), which is also known as cryopyrin that re- and PLD2), both of which have splice variants. Most atten-
sponds to dsRNA to activate caspase-1. The interac- tion has focused on PLD1 as a signal transducer because it
tion between NALP3 and caspase-1 is facilitated by an has a low basal activity that increases markedly in response
adaptor called apoptosis-associated speck-like protein to external stimuli. On the other hand, PLD2 has a high
containing a CARD (ASC). The interaction between basal activity and its role in signalling is uncertain. While
these three proteins takes place in a macromolecular most of the PLD2 is located on the plasma membrane,
complex known as the inflammasome. PLD1 is found predominantly on intracellular membranes
10. The activated caspase-1 contributes to the develop- (e.g. Golgi, endoplasmic reticulum and endosomes), but
ment of an inflammatory response by cleaving pro- has also been located at the plasma membrane, particu-
IL-1β to form the inflammatory cytokine interleukin larly at caveolae. The structure of the enzyme contains
1β (IL-1β). motifs responsible for its membrane location and catalytic
activity (Module 2: Figure PLD isoforms). The activity
of PLD1 increases following stimulation of both protein
Phospholipase D (PLD) signalling tyrosine kinase-linked receptors (PTKRs) and G protein-
pathway coupled receptors (GPCRs) (Module 2: Figure PLD sig-
The phospholipase D (PLD) signalling pathway functions nalling). One of the problems with trying to understand
by generating phosphatidic acid (PA), which acts to reg- the signalling function of this pathway is to determine
ulate a wide range of cellular processes. Phospholipase D just how these different receptors act to stimulate PLD1.
(PLD) activation depends upon a number of mechanisms, The fact that the activation of PLD1 seems to depend on
and these may vary depending on where the signalling the prior activation of other signalling pathways such as
mechanism is located within the cell. The primary mes- the diacylglycerol (DAG)/protein kinase C (PKC) sig-
senger produced by this signalling pathway is PA, and nalling cassette or the PtdIns 3-kinase signalling cassette
phosphatidic acid (PA) action is carried out through a (Module 2: Figure PLD signalling) suggests that the PLD
number of downstream effectors. Phosphatidic acid (PA) signalling pathway is not an autonomous signalling system,

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r83

Module 2: Figure PLD isoforms

Palmitate Palmitate
(Cys 240) (Cys 241)

HKD HKD
PLD1 PX PH I II III IV 1072

Palmitate Palmitate
(Cys 240) (Cys 241)

HKD HKD
PLD2 PX PH I II III IV 1034

The domain structure of phospholipase D (PLD).

The two isoforms of phospholipase D (PLD) have similar domain structures. Palmitoylation of Cys-240 and Cys-241 within the pleckstrin homology
(PH) domain play a critical role in localizing the enzyme to membranes. There are four conserved sequences (I–IV). Domains II and IV contain
HXKXXXXD, the so-called HKD motif, which come together to form the catalytic domain. The C-terminal region has a phox homology (PX) domain
and a PH domain responsible for the binding of PtdIns4,5P2 during activation of the enzyme (Module 2: Figure PLD signalling).

Module 2: Figure PLD signalling

Growth Chemotactic
factors Antigen Chemokines factors
LPA

PIP2 PLCγ DAG PLCβ PIP2

PI3K
PKCα
Ras LPA
PIP3 Ral-GDS
PA PLA2
hydrolase
Protein phosphatase 1
Arf Rho RalA
Sphingosine kinase
PA +
mTOR

Membrane budding and fusion

PIP PtdIns4P-5K PIP2 PLD1 PC
Plasma membrane OR
Endosomal membranes Membrane trafficking
Cytoskeletal organization
Inflammation
Phagocytosis
Proliferation
+ Secretion (Mast cells)
Respiratory burst

The phospholipase D (PLD) signalling pathway.

This signalling pathway depends upon the enzyme phospholipase D1 (PLD1), which associates primarily with intracellular membranes (e.g. Golgi and
endosomes), but is also found in the plasma membrane. The activation of PLD1 is linked to many receptors through a number of mechanisms that may
vary depending on the location and which of its many functions PLD is carrying out. One activator is protein kinase Cα (PKCα), through a mechanism
that is independent of its catalytic activity. Other major regulators are various small G proteins. Arf and Rho may be activated by the lipid messenger
PtdIns3,4,5-trisphosphate (PIP3 ) formed by the PtdIns 3-kinase signalling cassette, whereas Ral A may be activated via Ras and the GTP/GDP
exchange factor Ral-GDS. In addition, PLD1 has an absolute requirement for PtdIns4,5P2 (PIP2 ), which may be part of a positive-feedback loop
because the PtdIns4P 5-kinase (PtdIns4P-5K) is activated by phosphatidic acid (PA). The action of PA is terminated either by a PA phosphohydrolase,
which removes phosphate to leave behind diacylglycerol (DAG), or by a phospholipase A2 (PLA2 ) to produce lysophosphatidic acid (LPA).

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r84

but should be considered more as a downstream effector (SPHK). As illustrated by the figures in Module 2: Fig-
of these other signalling pathways. ure sphingolipid metabolism, the relative cellular levels of
these sphingolipids vary enormously. The precursor SM is
Phosphatidic acid (PA) action present at the highest level and this then declines during
The primary messenger of the phospholipase D (PLD) sig- the different metabolic transformations.
nalling pathway is the lipid phosphatidic acid (PA), which An important aspect of this sphingomyelin signalling
has a number of actions within the cell (Module 2: Figure pathway is the cellular location and function of these dif-
PLD signalling). The signalling function of PA is mainly ferent metabolic steps (Module 2: Figure sphingomyelin
directed towards the regulation of various enzymes such as signalling):
stimulation of the target of rapamycin (TOR) and sphin-
gosine kinase or inhibition of protein phosphatase 1. It
also plays an important role in regulating phagocytosis 1. Sphingomyelin synthesis is carried out first in the en-
(Module 4: Figure phagosome maturation). doplasmic reticulum (ER), where ceramide is formed,
In addition to such signalling functions, a local accumu- and is completed by the Golgi. The first step is carried
lation of PA may also alter the physical properties of the out by serine palmitoyl transferase (SPT) that com-
membrane by creating curvatures to facilitate the forma- bines serine and palmitate to form dihydrosphingosine
tion of vesicles for intracellular trafficking. (dhSph), which is then converted into dihydroceram-
ide (dhCer) by dihydroceramide synthase (CerS). Fi-
Phosphatidic acid (PA) metabolism nally, a desaturase converts dhCer into ceramide (Cer).
Two separate enzymes carry out the metabolism of phos- 2. The ceramide formed at the ER is then transported
phatidic acid (PA). It can be dephosphorylated to diacyl- to the Golgi by a ceramide transfer protein (CERT).
glycerol (DAG) by a PA phosphohydrolase or it can be A sphingomyelin synthase (SMS) located in the Golgi
partially deacylated by a phospholipase A2 (PLA2 ) to form converts ceramide into sphingomyelin (SM), which is
lysophosphatidic acid (LPA) (Module 2: Figure PLD sig- transferred to the membrane through a vesicle trans-
nalling). The LPA released from the cell is a potent agonist port mechanism.
on receptors of the endothelial differentiation gene (EDG) 3. The hydrolysis of SM in the plasma membrane is activ-
family (Module 2: Figure sphingomyelin signalling). ated by receptors sensitive to stimuli such as tumour
necrosis factor α (TNFα), interleukin 1, CD28 and
Fas. The coupling between receptors and the neutral
Sphingomyelin signalling pathway sphingomyelinases (SMases) has been worked out in
The sphingomyelin signalling pathway that has been im- some detail for the TNFα receptor. A factor associated
plicated in the control of a whole host of cellular pro- with neutral SMase (FAN) functions to couple the en-
cesses through the generation and function of ceramide zyme to the neutral SMase activation domain (NSD) of
and sphingosine 1-phosphate (S1P), which are the main the TNFα receptor. These components of the sphin-
messengers operating in this signalling pathway. The ac- gomyelinase signalling pathway are often highly con-
tion of sphingosine 1-phosphate (S1P) is complicated be- centrated in lipid rafts and caveolae, which represent
cause, in addition to acting internally, S1P is released from the site where some of the sphingomyelinases function
cells to function as an external ligand acting on cell-surface to hydrolyse sphingomyelin.
receptors. This sphingomyelinase signalling pathway also 4. The activation of neutral SMase at the plasma mem-
produces ceramide, another messenger that plays a signi- brane provides one of the major sources of ceramide
ficant part in processes such as cell proliferation, apoptosis (Cer), which can then be metabolized to other sig-
and the response of the cell to stress and injury. One of the nalling sphingolipids (Module 2: Figure sphingolipid
difficulties in understanding this pathway is its pleiotropic metabolism). A ceramidase (CDase) hydrolyses Cer to
effect on cells with responses that are often diametrically sphingosine (Sph), which can then be phosphorylated
opposed, such as proliferation and apoptosis. A clue to by sphingosine kinase (SPHK) to form sphingosine 1-
this complexity lies in the fact that the pathway can spawn phosphate (S1P). Since the latter is soluble, it leaves the
a number of messengers, and the action of these is very membrane and is free to diffuse both into and out of
dependent on the current state of the cell, especially with the cell (see later). The SPHK is activated by a number
regard to what other signalling pathways are active. of signalling pathways using messengers such as Ca2+ ,
DAG, cyclic AMP and ERK1/2 that are produced by
Generation and function of ceramide and signalling pathways activated by growth factors and
sphingosine 1-phosphate (S1P) hormones. Ceramide can also be phosphorylated by
The generation of ceramide and sphingosine 1-phosphate a ceramide kinase to form ceramide 1-phosphate, for
(S1P), which are the two main bioactive ‘messenger’ lip- which there is no apparent function.
ids of the shingomyelin signalling pathway, begins with 5. A variety of stress stimuli, such as ionizing radiation,
the hydrolysis of sphingomyelin (SM) by various sphin- UV irradiation and reactive oxygen species (ROS) act
gomyelinases (SMases) to form ceramide (Module 2: Fig- by stimulating the acidic sphingomyelinases (SMases)
ure sphingolipid metabolism). A ceramidase (CDase) then localized in lysosomes to hydrolyse sphingomyelin
cleaves off one of the fatty acid chains to form sphingosine (SM) to form ceramide (Cer) (Module 2: Figure sphin-
(Sph), which is then phosphorylated by sphingosine kinase gomyelin signalling).

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r85

Module 2: Figure sphingolipid metabolism

Sphingomyelin Ceramide Sphingosine Sphingosine-1-PO4

(SM) (Cer) (Sph) (S1P)

30,000 3,000 100 1

O C HO C O C HO C C OH C OH
H H
N C N C N C N C
H H
C C C C
SMase CDase SPHK O
O OH OH
O P O O P OH
O O
C
C
CH 3 N CH 3
CH 3

Sphingolipid metabolism.
The sphingomyelin signalling pathway depends on the conversion of sphingomyelin (SM) into a series of bioactive lipids capable of activating
various signalling mechanisms as described in Module 2: Figure sphingomyelin signalling. The numbers represent the relative cellular levels of these
sphingolipids. SMase, sphingomyelinase; CDase, ceramidase; SPHK, sphingosine kinase. Information for this Figure was taken from Hannun and
Obeid (2008).

Module 2: Figure sphingomyelin signalling

TNFα Growth factors N S1P LPA

Hormones P
EDG
ABCC1 receptors

Neutral CDase β/γ Gq

N SMase N N SPHK Ca 2+ PI 3-K
P FAN OH OH DAG 8 Ras
SM Cer
N Sph ERK1/2
3
Golgi 4 N S1P
P DAG InsP3
2 Phospho-
SPPs ethanolamine
6 S1P lyase
OH

Ca 2+
N

Cer
Ionizing PKC
CAPPs PKB
Desaturase radiation ROS
CAPK
JNK ERK1/2 CaMKII
5
ER 7 PKCζ
N

dhCer
Acidic Cathepsin D 9
SMase
CerS 1
N
P

Apoptosis Survival
N
N

dhSph
OH

Cell cycle Proliferation

SPT SM Cer arrest Anti-inflammatory
Lysosome Senescence responses
Serine + palmitate

The sphingomyelin signalling pathway.

A number of stimuli can activate the neutral or acidic sphingomyelinases (SMase) to hydrolyse sphingomyelin (SM) to ceramide (Cer), which is then
converted into sphingosine (Sph) by ceramidase (CDase). The sphingosine is converted into sphingosine 1-phosphate (S1P) by a sphingosine kinase
(SPHK), which is sensitive to other signalling pathways using messengers such as Ca2+ , diacylglycerol (DAG), cyclic AMP (cAMP) and extracellular-
signal-regulated kinase 1/2 (ERK1/2). Ceramide can activate a number of targets, and some of these can activate apoptosis. On the other hand, S1P
can promote survival and proliferation by passing out of the cell, where it functions as an external ligand to activate endothelial differentiation gene
(EDG) receptors.

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r86

6. S1P is metabolized via two separate pathways: it can This sphingomyelin signalling pathway has been implic-
be converted back into sphingosine by a sphingosine ated in a number of cellular processes:
1-phosphate (S1P) phosphatase (SPP) or it can be
cleaved by an S1P lyase to give palmitaldehyde and • S1P has been proposed to release the Ca2+ necessary to
phosphoethanolamine. The lyase has a 20-amino-acid activate the formation of PtdIns3P on endosomes during
transmembrane domain that positions the enzymes in phagosome maturation (Module 4: Figure phagosome
cellular membranes. maturation).
• S1P appears to contribute to the CD28 co-stimulatory
The sphingomyelinase signalling pathway is thus sens- pathway during T cell activation (Module 9: Figure T
itive to external signals at a number of discrete steps. The cell signalling map).
production of ceramide is accelerated by receptors sens- • S1P contributes to the communication between en-
itive to cytokines such as TNFα or to various stress sig- dothelial cells and pericytes during angiogenesis (see
nals such as ionizing radiation and reactive oxygen species Step 6 in Module 9: Figure angiogenesis signalling).
(ROS). The formation of S1P can also be enhanced by
growth factor and hormone receptors that elevate second Sphingosine 1-phosphate (S1P)
messengers such as Ca2+ , DAG, ERK1/2 or cyclic AMP The key enzyme in the formation of sphingosine 1-
that act by stimulating SPHK. In the absence of these sig- phosphate (S1P) is sphingosine kinase (SPHK). The mes-
nals, the main messenger will be ceramide, but this will senger function of S1P is complicated by the fact that it
switch over to S1P if the SPHK is activated. This inter- may have both intra- and extra-cellular actions.
relationship between ceramide and S1P has led to a cer- S1P can exert its extracellular action either in an
amide/S1P rheostat model, where a balance between these autocrine mode (activate the same cell from which it is
two messengers is thought to determine cell fate. In gen- released) (see Step 8 in Module 2: Figure sphingomyelin
eral, ceramide seems to tip the balance in favour of cell signalling) or a paracrine mode (diffuse away to activate
cycle arrest, senescence and apoptosis, whereas S1P pro- neighbouring cells). This extracellular action is mediated
motes survival and proliferation (Module 2: Figure sphin- by endothelial differentiation gene (EDG) receptors, so-
gomyelin signalling). The different outcomes controlled called because they were first described in human um-
by these two messengers may depend on their ability to bilical vein endothelial cells induced to differentiate by
activate separate signalling cascades. phorbol esters. There is a family of these EDG receptors
that are all G protein-coupled receptors (GPCRs) capable
7. The ceramide formed at the plasma membrane of activating most of the conventional signalling pathways
or in the lysosomes acts on a number of tar- (Module 1: Table G protein-coupled receptors). The EDG
gets such as ceramide-activated protein kinase 1, 3, 5, 6 and 8 receptors mediate the action of S1P, whereas
(CAPK), protein kinase Cζ (PKCζ), cathepsin D the EDG 2, 4 and 7 receptors appear to respond to lyso-
and ceramide-activated protein phosphatases (CAPP), phosphatidic acid (LPA), a related lysolipid. The EDG re-
such as PP1 and PP2A. Most of these ceramide- ceptors that function to induce a mobilization of internal
sensitive targets act to promote cell cycle arrest and Ca2+ act by stimulating the formation of inositol 1,4,5-
apoptosis either directly or indirectly. For example, trisphosphate (InsP3 ). One function of the EDG receptors
the cathepsin D converts Bid into tBid to promote ap- is to regulate cell motility and directional migration. An
optosis (see Step 3 in Module 11: Figure apoptosis). example of the latter is preosteoclast chemotaxis where S1P
On the other hand, the CAPPs such as PP2A can de- directs preosteoclasts form the bone marrow back into the
phosphorylate various components that are used by blood (Module 8: Figure preosteoclast homeostasis).
the EDG receptors to drive cell survival and prolifer- The intracellular action of S1P is not as well defined,
ation. For example the dephosphorylation of protein mainly because it is often difficult to separate an intra-
kinase B (PKB) not only prevents it from inhibiting cellular action from that induced by its extracellular ac-
apoptosis but it will also reduce its ability to enhance tion. An intracellular action of S1P has been proposed for
proliferation. phagosome maturation, where it releases the Ca2+ neces-
8. One of the actions of the soluble sphingosine 1-phos- sary to activate the formation of PtdIns3P on endosomes
phate (S1P) is to diffuse out of the cell passing through (Module 4: Figure phagosome maturation). One of the
the ABCC1 transporter, which is a family mem- problems with trying to establish such an intracellular ac-
ber of the ATP-binding cassette (ABC) transporters tion for S1P is that it could also mobilize internal Ca2+
(Module 3: Table ABC transporters). The extracellu- by acting through the EDG receptors to generate InsP3 .
lar S1P then activates EDG receptors, which belong to However, there are indications that S1P can act directly to
the family of G protein-coupled receptors (GPCRs) release Ca2+ from an internal store. Unlike InsP3 , how-
(Module 1: Table G protein-coupled receptors) that ever, release occurs without the appearance of elementary
are capable of relaying information down a number of events. One of the difficulties with this hypothesis is that
signalling pathways. the channel on the endoplasmic reticulum (ER) that is
9. These signalling pathways control cellular pro- opened by S1P remains to be identified. An earlier pro-
cesses, such as cell survival, proliferation and anti- posal that S1P might act through Scamper has not been
inflammatory responses, which are opposite to those substantiated. Another suggestion is that S1P functions as
controlled by ceramide. the Ca2+ influx factor (CIF) that is proposed to link store

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depletion to Ca2+ entry. Another proposed action of S1P is kinase (MAPK) cascade and could account for those cases
to activate the extracellular-signal-regulated kinase (ERK) where ceramide promotes both inflammation and proli-
cascade to promote proliferation. Overexpression of the feration.
SPHK can induce tumour formation. Therefore the pre-
cise role of S1P and its relationship to ER signalling require
further clarification. Sphingosine kinase (SPHK)
Sphingosine kinase (SPHK) appears as two isoforms
Ceramide (SPHK1 and SPHK2), which have different tissue distri-
This is an enigmatic messenger in that it has been linked butions. SPHK1 is found at high levels in the lung and
to both proliferation and apoptosis, depending very much spleen, whereas SPHK2 is expressed mainly in liver and
on the background activity of other messenger systems. heart. Both have five highly conserved domains, with the
In keeping with its different functions, ceramide can ac- ATP-binding site and catalytic site located in the C2 do-
tivate a number of signalling components such as the main.
ceramide-activated protein kinase (CAPK), protein kinase The enzyme is both membrane-bound and free in the
Cζ (PKCζ) and ceramide-activated protein phosphatases cytosol, and there is some uncertainty as to how it is
(CAPP), such as PP1 and PP2A. Some of these ceramide activated by cell-surface receptors. It seems likely that
targets enable it to interact with other signalling systems it responds to various downstream signals emanating
such as the c-Jun N-terminal kinase (JNK) and mitochon- from these receptors such as Ca2+ /calmodulin, diacylgly-
drial systems. With regard to the latter, ceramide may ac- cerol (DAG)/protein kinase C (PKC), cyclic AMP/protein
tivate mitochondrial reactive oxygen species (ROS) form- kinase A (PKA) or extracellular-signal-regulated kinase
ation through ceramide-activated protein kinase (CAPK). 1/2 (ERK1/2) (see Step 4 in Module 2: Figure sphin-
It is important to stress that apoptosis can occur without gomyelin signalling). Another activator is oxidized low-
the need for ceramide. However, there is overwhelm- density lipoprotein (LDL), which may play a role in
ing evidence that the formation of ceramide can strongly cell proliferation. Consistent with the activation by vari-
tip the balance in favour of apoptosis. It contributes to ous messenger pathways is the fact that SPHK con-
this activation of the cell death programme by activat- tains consensus sequences for Ca2+ /calmodulin binding
ing the intrinsic pathway at the level of the mitochon- and phosphorylation sites for PKA, casein kinase II and
dria (Module 11: Figure apoptosis). One way of inter- PKC.
acting with the mitochondria depends on its activation
of the JNK pathway, which is known to induce apop-
Sphingosine 1-phosphate (S1P) phosphatase (SPP)
tosis (Module 2: Figure JNK signalling). Ceramide can
Mammals have two sphingosine 1-phosphate (S1P) phos-
also activate cathepsin D, which converts Bid into tBid,
phatases (SPPs). Both SPP1 and SPP2 have eight to ten
which is one of the pro-apoptotic members of the Bcl-2
transmembrane domains that locate the enzymes within
superfamily.
the endoplasmic reticulum (ER). SPP-1 is located mainly
in the placenta and kidneys, whereas SPP2 is found in the
Sphingomyelinases (SMases)
brain, heart, colon, kidney, small intestine and lung. These
Sphingomyelinases (SMases) exist in different isoforms
enzymes convert S1P back into ceramide and can diminish
that can be distinguished by their pH optima: acidic, neut-
survival and promote apoptosis (see Step 6 in Module 2:
ral or alkaline. The last is found in bile and plays a role in di-
Figure sphingomyelin signalling).
gestion. Sphingomyelin signalling seems to depend on the
acidic and neutral isoforms (Module 2: Figure sphingomy-
elin signalling). The neutral SMase functions to generate Ceramide-activated protein phosphatase (CAPP)
ceramide in response to the activation of receptors sensit- Ceramide-activated protein phosphatase (CAPP) is a
ive to stimuli such as tumour necrosis factor α (TNFα) and member of the protein phosphatase 2A (PP2A) family of
interleukin 1 (see Step 3 in Module 2: Figure sphingomy- serine/threonine phosphatases that consist of three sub-
elin signalling). The acidic form was originally identified units: A and B are regulatory, whereas C is the catalytic
in lysosomes where it generates ceramide in response to subunit. In the case of CAPP, ceramide acts to stimulate
stress stimuli (see Step 5). There also are indications that phosphatase activity by binding to one of the regulatory
the acidic SMase may act on sphingomyelin located in the B subunits. The ability of ceramide to inhibit growth is
outer leaflet. The acidic SMase is a component of lipid rafts probably mediated through PP2A.
and caveolae, which are microdomains of the plasma mem-
brane containing high levels of the precursor sphingomy-
elin. It is this acidic isoform that is defective in patients Scamper
with Niemann-Pick disease. A sphingolipid Ca2+ -release-mediating protein of the en-
doplasmic reticulum (ER) (Scamper) was originally pro-
Ceramide-activated protein kinase (CAPK) posed to be the Ca2+ channel on the ER that responds
Ceramide-activated protein kinase (CAPK) is a to signals from the sphingomyelin signalling pathway,
membrane-bound proline-directed protein kinase but more recent studies have shown that it has a single
that acts by phosphorylating Raf-1, thereby enabling membrane-spanning segment that seems to remodel the
ceramide to plug into the mitogen-activated protein actin cytoskeleton.

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r88

Module 2: Figure JAK and STAT structure

0 200 400 600 800 1000 1200

JH7 JH6 JH5 JH4 JH3 JH2 JH1

Pseudokinase Kinase
JAKs domain domain
Receptor
binding region

STATs
2&6 SH3 SH2

1,3,4 SH3 SH2 TAD

5A & 5B
STAT DNA Y S
dimerization binding
domain domain

Domain structure of the JAKs and STATs.

There are four mammalian Janus kinases (JAKs): JAK1, JAK2, JAK3 and Tyk2. They all have a similar domain structure, which has seven JAK
homology (JH) domains. JH1 is the kinase domain, whereas JH2 is a pseudokinase domain. Some of the other JH regions seem to contribute to
the binding of JAKs to various cell-surface receptors, where they function to activate the signal transducers and activators of transcription (STATs).
There are seven mammalian STAT genes. STATs 2 and 6 have 850 amino acids, whereas the others are somewhat shorter. The tyrosine residue
near residue 700 is phosphorylated during the activation domain and functions as a binding site for the Src homology 2 (SH2) sites on other STATs
during the dimerization process (Module 2: Figure JAK/STAT function). The DNA-binding domain is located between the SH3 (Src homology 3) and
SH2 domains. The C-terminus has a transcriptional activation domain (TAD), which, on the shorter-length STATs, contains a serine residue that can
modulate transcriptional activity when phosphorylated.

Janus kinase (JAK)/signal transducer Janus kinase (JAK)/signal transducer and

and activator of transcription (STAT) activator of transcription (STAT) structure
signalling pathway The two major components of the signalling pathway
The Janus kinase (JAK)/signal transducer and activator are the Janus kinases (JAKs) and their substrates the sig-
of transcription (STAT) signalling pathway provides a nal transducers and activators of transcription (STATs)
mechanism for rapidly activating gene transcription in (Module 2: Figure JAK and STAT structure). Of the four
response to a large number (>35) of external ligands. mammalian JAKs, three are expressed fairly ubiquitously,
This signalling pathway is mainly activated by cytokines whereas JAK3 is restricted to natural killer (NK) cells
such as interferon, but is also used by receptor tyr- and thymocytes, with some expression in vascular smooth
osine kinases [epidermal growth factor receptor (EGFR), muscle cells and endothelium. The main structural com-
platelet-derived growth factor receptor (PDGFR)], non- ponent of the JAKs is the kinase domain that functions to
receptor tyrosine kinases and G protein-coupled receptors phosphorylate the STATs at a key tyrosine in the region of
(GPCRs). The Janus kinase (JAK)/signal transducer and residue 700 during the signal transducer and activator of
activator of transcription (STAT) structure reveals the ma- transcription (STAT) activation cascade. The STATs have
jor features of these two components and how they are a number of functional domains whose three-dimensional
linked during the signal transducer and activator of tran- structure reveals how the STAT dimers are formed and
scription (STAT) activation cascade. Cell-surface recept- how they bind to DNA (Module 2: Figure STAT1/DNA
ors act by phosphorylating the STATs, which are latent complex).
transcription factors. Once phosphorylated, these STATs Signal transducer and activator of transcription
leave the membrane and then dimerize before migrating (STAT) activation cascade
into the nucleus where they bind to specific DNA-binding The signal transducers and activators of transcription
elements to activate transcription. There is considerable (STATs) are a family of latent cytoplasmic receptors that
evidence for a Janus kinase (JAK)/signal transducer and are activated by a phosphorylation cascade that can be
activator of transcription (STAT) function in growth and induced by many different receptors (e.g. cytokine re-
development. ceptors, tyrosine kinase-linked receptors, non-receptor

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r89

Module 2: Figure STAT1/DNA complex

Organization of a STAT1/DNA complex.

The ribbon diagram shown in (A) illustrates how the two DNA-binding domains of the two subunits attach the dimer to the DNA helix. The linker domain
(orange) attaches to the SH2 (Src homology 2) domains (light blue) that hold the molecule together through the intermolecular SH2–phosphotyrosine
interaction. The molecular surface representation shown in (B) has the same orientation as in (A). The local electrostatic potential over the cell surface
is represented by the colouring, with blue representing positive and red negative potentials. (Reproduced from Cell, Vol. 93, Chen, X., Vinkemeier,
U., Zhao, Y., Jeruzalmi, D., Darnell, Jr, J.E. and Kuriyan, J., Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA, pp. 827–839.
Copyright (1998), with permission from Elsevier; see Chen et al. 1998.

tyrosine kinases and G protein-linked receptors). The ver- 4. Once the STATs have docked, they are then phos-
satility of this signalling mechanism is greatly enhanced by phorylated by the JAKs on the tyrosine residue loc-
the way the Janus kinases (JAKs) and STATs can be mixed ated in the C-terminal region (Module 2: Figure JAK
and matched to generate an enormous number of com- and STAT structure).
binations as illustrated by the different cytokine receptors 5. The phosphorylated STATs, which are dimerized
(Module 2: Figure JAK/STAT heterogeneity). through an intermolecular SH2–phosphotyrosine in-
A typical activation cascade for cytokine receptors, teraction, leave the receptor.
which illustrates the essential role of the JAKs in a se- 6. These dimers are imported into the nucleus using
quence of tyrosine phosphorylations that culminates in the importin-α (Imp-α) and attach to promoter regions
phosphorylation and activation of the STATs, is outlined through their DNA-binding domains (Module 2: Fig-
in the following sequence (Module 2: Figure JAK/STAT ure STAT1/DNA complex).
function): 7. STATs activate the transcription of a number of tar-
get genes. One group of these genes code for the
1. Agonists induce dimerization by binding to the extra- suppressor of cytokine signalling proteins (SOCS),
cellular domains of the receptor subunits. which thus function as part of a negative-feedback
2. The JAKs, which are associated with the cytoplasmic loop to limit the action of the signalling pathway
domain of these receptors, phosphorylate each other. (Module 2: Figure JAK/STAT function).
3. The activated JAKs then phosphorylate tyrosine 8. STAT activity is terminated by a nuclear protein tyr-
residues on the receptors to provide docking sites for osine phosphatase (N-PTP) that removes the tyrosyl
the Src homology 2 (SH2) domains on the STATs. phosphate groups. The inactive STAT is then exported

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r90

Module 2: Figure JAK/STAT heterogeneity

TYPE I CYTOKINES
JAK1 STAT5a, STAT5b
IL-2, IL-7,IL-9, IL-15 STAT3
JAK3

JAK1
IL-4 STAT6
JAK3

JAK1,JAK2
IL-13 STAT6
Tyk2

IL-3, IL-5, G-CSF, JAK2 STAT5a, STAT5b

GM-CSF

IL-6, IL-11, OSM JAK1,JAK2

STAT3
CNTF, LIF, CT-1 Tyk2

IL-12 JAK2, Tyk2 STAT4

Growth STAT5a, STAT5b

JAK2
hormone STAT3

Prolactin JAK2 STAT5a, STAT5b

Erythropoietin JAK2 STAT5a, STAT5b

Thrombopoietin JAK2 STAT5a, STAT5b

TYPE II CYTOKINES
IFNα, IFNβ JAK1, Tyk2 STAT1, STAT2

IFNγ JAK1, Tyk2 STAT1

IL-10 JAK1, Tyk2 STAT3

Heterogeneity of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) combinations used by different cytokine receptors.
The cytokines acting on cell-surface receptors (blue and mauve boxes) activate different combinations of Janus kinases (JAKs) and signal transducers
and activators of transcription (STATs). CNTF, ciliary neurotrophic factor; GCSF, granulocyte colony-stimulating factor; IFN, interferon; IL, interleukin;
LIF, leukaemia inhibitory factor; OSM, oncostatin M. The organization of some of the type I cytokine receptors is shown in Module 1: Figure type I
cytokine receptors.

from the nucleus by chromosome region maintenance (JAK)/signal transducer and activator of transcription
1 (CRM1), thus completing the cycle. (STAT) signalling pathway (Module 2: Figure JAK/STAT
function). The SOCS act by inhibiting the Janus kinases
For some of the other receptors, these tyrosine phos- (JAKs) and thus operate a negative-feedback loop to limit
phorylations are carried out by kinases other than the the action of cytokines.
JAKs. After the STATs are phosphorylated, they leave There is a marked increase in expression of the SOCS-3
the receptor and then dimerize before translocating into isoform in hypothalamic neurons following the action of
the nucleus. This activation cascade can occur quickly, leptin during the control of food intake and body weight
with the activated STATs appearing in the nucleus within (Module 7: control of food intake).
minutes. Transcriptional activity can be modulated by
phosphorylation of the serine residue in the transcrip-
tional activation domain (TAD) of STAT 1, 3, 4, 5A and 5B Janus kinase (JAK)/signal transducer and
(Module 2: Figure JAK and STAT structure). In the case of activator of transcription (STAT) function in
STAT 1 and STAT4, phosphorylation enhances transcrip- growth and development
tional activity, whereas the binding of STAT5a to DNA is The Janus kinase (JAK)/signal transducer and activator of
greatly prolonged. transcription (STAT) signalling pathway has a primary role
in the regulation of growth and development, particularly
Suppressor of cytokine signalling proteins of haematopoietic cells. For example, it functions in the
(SOCS) interleukin-2 signalling pathway responsible for driving
The suppressor of cytokine signalling proteins (SOCSs) the final signalling steps in lymphocyte activation (Mod-
are induced during the activation of the Janus kinase ule 9: Figure T cell signalling map).

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r91

Module 2: Figure JAK/STAT function

Agonist

P P P P P P P P

JAK
P P P P P P
1 2
4 _

3
SH2 domain
DNA-binding STAT 5 P P
domain
SOCS
CRM1 Export Import Importin

8 6
Importin
CRM1
N-PTP Target genes
SOCS
P P 7

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) activation cascade.
A schematic summary of the main sequence of events responsible for the activation of signal transducers and activators of transcription (STATs).
Agonist activation of cell-surface receptors induces dimerization of receptor subunits, resulting in the stimulation of resident Janus kinases (JAKs). The
JAKs then carry out a sequential series of three phosphorylation reactions to activate the STATs, which then enter the nucleus to induce transcription.
The binding between STATs and DNA is shown in Module 2: Figure STAT1/DNA complex.

A mutation in the γc component of cytokine recept- roles in the Smad signalling mechanism that functions to
ors results in X-linked severe combined immune defi- transfer information from activated receptors on the cell
ciency (X-SCID). Since JAK3 associates with γc , it seemed surface to gene targets in the nucleus. This signalling mech-
likely that X-SCID may result from an alteration in the anism has two main components. Firstly, there is a process
JAK/STAT signalling pathway. This was confirmed when of transforming growth factor β (TGF-β) receptor ac-
SCID was found in patients carrying mutations in JAK3. tivation, which is responsible for activating the Smads.
SCID resulted from a dramatic reduction in the number of Secondly, there is the Smad activation of transcription.
T cells, highlighting the important role for this signalling There are a number of mechanisms for the modulation of
pathway in lymphocyte development. Smad signalling. Transforming growth factor β (TGF-β)
JAKs have also been implicated in certain forms of ma- inhibition of cell proliferation is one of the major functions
lignant transformation such as Sezary’s syndrome, v-Abl- of the Smad signalling pathway. It plays an important role
transformed cells and in some leukaemias. in the differentiation of intestinal cells. Alterations in the
signalling pathways controlled by the TGF-β superfamily
have been associated with many cancers of epithelial and
lymphoid origins, which fail to respond to the normal anti-
Smad signalling pathway proliferative effects of TGF-β. The TGF-β receptor is one
This signalling pathway takes its name from the Smads, of the tumour suppressors that are switched off in many
which are a collection of intracellular signalling molecules cancers and particularly in colorectal cancer (CRC).
that act collectively to transfer information from cell- An increase in the expression of TGF-β1 may play a
surface receptors into the nucleus. As such, some of the critical role in the transition from stable hypertrophy to
Smads function as transcription factors, whereas oth- congestive heart failure (CHF).
ers either facilitate or inhibit this transcriptional activ-
ity. These Smads mediate the action of the transforming
growth factor β (TGF-β) superfamily, which are cytokines Smad signalling toolkit
that regulate many cellular functions such as proliferation, The Smad signalling pathway is made up from a large
apoptosis, extracellular matrix formation and angiogen- number of components (Module 2: Table Smad signalling
esis. In addition, they play a critical role in controlling toolkit). Specific cell types express different combinations
events during early development and cell differentiation. of these components so that there is considerable variab-
There is an extensive Smad signalling toolkit, and the many ility in the nature of Smad signalling pathways. However,
components can be mixed and matched to assemble a large the overall organization is fairly similar and is best ex-
variety of Smad signalling pathways. The domain struc- emplified by the action of transforming growth factor β
ture of the Smad family illustrates their multifunctional (TGF-β) itself. The different components of the toolkit

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r92

Module 2: Table Smad signalling toolkit Module 2 Table continued

Components of the Smad signalling toolkit. Component Comment
Component Comment Smad regulatory factors
Ligand traps SARA Smad anchor for receptor activation
LAP Latency-associated polypeptide Smurf1 Smad ubiquitin-regulatory factor 1.
that binds TGF-β Smirf1 also regulates the
Decorin Binds TGF-β degradation of Rho during
α2 -Macroglobulin Binds TGF-β neutrophil chemotaxis (Module
Noggin Binds to BMPs (Module 8: Figure 11: Figure neutrophil
epidermal stem cell) chemotactic signalling)
Follistatin Binds to activins and BMPs. It Smurf2 Smad ubiquitin-regulatory factor 2
inhibits activin action on
gonadotrophs (Module 10:
Figure gonadotroph regulation)
Chordin/SOG Binds to BMPs
DAN/Cerberus Binds to BMPs have been grouped together on the basis of their function
Ligands in the signalling pathway (Module 2: Table Smad signalling
TGF-β1 Transforming growth factor β
(Module 1: Figure enzyme-linked toolkit):
receptors)
TGF-β2
TGF-β3 Ligand traps
Activin One of its functions is to increase These are a large group of proteins that control access
the transcription of FSH in
gonadotrophs (Module 10: of the ligands to their cell-surface receptors. These ligand
Figure gonadotroph regulation) traps can have an important inhibitory action since they
Myostatin Growth and differentiation factor 8 can prevent the ligands from reaching their cell-surface
(GDF8) (see Module 8: Figure
satellite cell activation) receptors. For example, follistatin inhibits the action of
Nodal activin on gonadotrophs (Module 10: Figure gonadotroph
BMP Bone morphogenetic factor regulation).
functions in the control of
epidermal stem cells (Module 8:
Figure epidermal stem cell),
proliferation of SNO cells
Ligands
(Module 8: Figure HSC The Smad signalling pathway is activated by a number of
regulation) and differentiation of closely related ligands. The flagship ligand is transforming
white fat cells (Module 8: Figure
white fat cell differentiation)
growth factor β (TGF-β), but there are a number of other
Inhibin Inhibits activin action in ligands that also act through the Smads.
gonadotrophs (Module 10:
Figure gonadotroph regulation)
Accessory receptors Accessory receptors
Betaglycan Facilitates the binding of TGF-β to
the Type II receptors. Mediates
These are cell-surface receptors that function as co-
the action of inhibins in receptors in that they promote the binding of the ligands
gonadotrophs (Module 10: to their signalling receptors.
Figure gonadotroph regulation)
Cripto
Endoglin Signalling receptors
Signalling receptors
Type I receptors There are two groups of signalling receptors (Type I
ALK1 Activin receptor-like 1 and II). These receptors contain a single membrane-
ALK2 Activin receptor-like 2 spanning region that separates the extracellular ligand-
ALK3 Activin receptor-like 3
ALK4 Activin receptor-like 4 binding domain from the cytosolic region that contains
ALK5 Activin receptor-like 5 a serine/threonine kinase domain. The Type I receptor
ALK6 Activin receptor-like 6 also contains a glycine/serine-rich (GS) domain, which is
ALK7 Activin receptor-like 7
Type II receptors phosphorylated by the Type II receptor during the signal
ActRII transduction process. Ligands act to complex these recept-
ActRIIB ors so that the Type II can then activate the Type I, which
BMPRII
TβRII in turn activates the Smads.
Receptor-regulated Smads
(R-Smads)
Smad1 Smads
Smad2 The Smads are the intracellular transducers of the Smad
Smad3
Smad5 signalling pathway. There are three Smad types. Receptor-
Smad8 regulated Smads (R-Smads), which are activated by the
Co-mediator Smad signalling receptors, carry information into the nucleus. A
(Co-Smad)
Smad4 A germ-line mutation in Smad4 has single co-mediator Smad (Co-Smad) acts together with the
been linked to juvenile polyposis R-Smads. Inhibitory Smads (I-Smads) set up a negative-
syndromes (JPSs). feedback loop to limit the activity of the R-Smads. These
Inhibitory Smads (I-Smad)
Smad6 different activities are reflected in the domain structure of
Smad7 the Smad family.

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Module 2: Figure Smad domain structure

Smurf1/2 Type I receptor

binding phosphorylation sites

PY P P
MH1 MH2 --SSXS- R-Smads
NLS (Smad1,2,3,5,8)

MH1 MH2 Co-Smad

NLS NES (Smad4)
Smurf1/2
binding

PY
MH2 I-Smads
(Smad6,7)

MAPK phosphorylation sites

CaMKII phosphorylation sites
PKC phosphorylation sites

The domain structure of the three Smad family members.

The receptor-regulated Smads (R-Smads) have a MAD homology domain 1 (MH1) in the N-terminal region and an MH2 in the C-terminal region.
These two domains play a critical role in carrying out the protein–protein and protein–DNA interactions. A nuclear localization signal (NLS) is located
within MH1 and functions in the transport of the R-Smads into the nucleus. The SSXS motif at the C-terminus contains the two serine residues that
are phosphorylated by the Type I receptors during the process of signal transduction (Module 2: Figure TGF-βR activation).

Smad regulatory factors Smad signalling mechanism

There are a number of proteins that contribute to the Smad The Smad signalling mechanism can be divided into two
signalling pathway. parts. Firstly, there is the process of transforming growth
factor β (TGF-β) receptor activation, which concerns
the way in which ligands such as TGF-β interact with
Domain structure of the Smad family
the signalling receptors to trigger Smad activation (Mod-
The main structural feature of the Smads are the two MAD
ule 2: Figure TGF-βR activation). The critical aspects of
homology domains (MH1 and MH2) that function both
this activation process are the phosphorylation reactions
in the protein–protein and protein–DNA interactions that
that occur within the receptor complex. The Type II re-
occur during the process of signal transduction (Mod-
ceptors are constitutively active and phosphorylate the
ule 2: Figure Smad domain structure). The regulatory
Type I receptors. These activated Type I receptors then
Smads also have a C-terminal SSXS motif, which is crit-
act to phosphorylate the Smads.
ical for the transduction process because two of the serine
The second part is the Smad activation of transcrip-
residues are phosphorylated by the type I receptors as part
tion, during which the phosphorylated receptor-regulated
of the Smad signalling mechanism. There also are numer-
Smads (R-Smads), together with their partner Smad4,
ous other sites that are phosphorylated by various kinases
translocate into the nucleus to induce gene transcription
from other signalling pathways. The linker region between
(Module 2: Figure Smad signalling).
the MH1 and MH2 domains contains a phosphotyrosine
(PY) motif, which is an interaction site for the binding of
the Smad ubiquitin-regulatory factors 1 and 2 (Smurf1/2), Transforming growth factor β (TGF-β) receptor
which is an ubiquitin ligase that controls the selective pro- activation
teolysis of the Smads. The single co-mediator Smad (Co- Activation of the transforming growth factor β (TGF-β)
Smad, i.e. Smad4) resembles the R-Smads in some aspects. receptor depends upon a series of reactions as illustrated by
It also has MH1 and MH2 domains, but here the latter is the following steps shown in Module 2: Figure TGF-βR
split. In addition to the NLS, it also has a nuclear export activation.
signal (NES). Smad4 lacks the C-terminal phosphoryla-
tion motif, but it does contain a number of phosphoryla- 1. The ligand, in this case TGF-β, is often held in a latent
tion sites. The two inhibitory Smads (I-Smads) lack an ligand complex by being bound to one of the ligand trap
MH1 domain, but they have the Smurf1/2-binding PY proteins such as latency-associated polypeptide (LAP)
motif. or decorin. When it dissociates from the ligand trap, it

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Module 2: Figure TGF-βR activation

Ligand trap (e.g. LAP, decorin)

TGF-β
1

2 TGF-β

P P P P MH1

SARA
Betaglycan 3
-SXSS-- MH2
Type II
receptors Type I 4
receptors
SARA 4

Ser/Thr kinase domain 5

Glycine-serine-rich

SARA
P P MH1
domain (GS) MH1 MH2
-SXSS--
-SXSS-- MH2

Smad2
GENE TRANSCRIPTION
Smad3
(See Module 2: Figure Smad signalling)

Activation of the transforming growth factor β (TGF-β) receptor superfamily.

The transforming growth factor β (TGF-β) receptor superfamily uses the Smad signalling pathway to transmit information into the cell. The main
features of this pathway can be illustrated by considering the action of TGF-β, which activates the TGF-β receptor complex through a sequential
series of reactions as described in the text.

can be taken up by one of the accessory receptors such tein (BMP) acts through Smad1, Smad5 or Smad8. Once
as betaglycan. these Smads have been phosphorylated on their C-terminal
2. The TGF-β then associates with the two TGF-β re- SSXS motif (Module 2: Figure TGF-βR activation) they
ceptor components to assemble an agonist/receptor leave the receptor, where they combine with Smad4 to form
complex. In the absence of ligand, the Type I and II a dimer that then translocates into the nucleus (Module 2:
receptor components exist as homodimers which are Figure Smad signalling). Once the dimer enters the nuc-
then brought together by TGF-β. leus, it recognizes and binds to a specific DNA motif.
3. When the two receptor types have been complexed The transcriptional activity of the Smads is facilitated
by TGF-β, the serine/threonine kinase domain on the by associating with other site-specific transcription factors
Type II receptors phosphorylates the serine residues (TFs). Their activity is also modulated by coactivators and
on the glycine/serine-rich (GS) region of the Type I repressors. For example, Smad4 binds directly to the co-
receptors. activator p300, which has an important role in activat-
4. These phosphorylated GS regions on the Type I re- ing transcription. The pathway on the right, which car-
ceptors provide a docking site for the MAD homology ries out the action of receptors that bind ligands such as
domain 1 (MH1) domain of Smad2 or Smad3. This re- bone morphogenetic protein (BMP), use Smad1, 5 or 8 to
cruitment of the Smads to the membrane is facilitated transmit information into the nucleus. As for the pathway
by Smad anchor for receptor activation (SARA). Once described above, Smad4 is again used as a partner for the
attached to the receptor, the SSXS motif is brought into translocation process, and the transcriptional processes are
contact with the serine/threonine kinase domain, and also similar.
two of the serine residues are phosphorylated. The Smads stimulate transcription of the collagen type I
5. Once Smad2/3 have been phosphorylated, their affinity during the fibrogenesis induced in activated hepatic stellate
for both the receptor complex and for SARA is reduced, cells in the liver (Module 7: Figure hepatic stellate cell).
and the two proteins pass into the cytoplasm. The ac-
tivated Smads then translocate into the nucleus where Modulation of Smad signalling
they activate transcription (Module 2: Figure Smad sig- There are a number of ways that the Smad signalling path-
nalling). way can be modulated. One mechanism is by an increase
in the expression of inhibitory Smads (I-Smads), which
Smad activation of transcription target the cell-surface receptors for degradation. This pro-
The receptor-regulated Smads (R-Smads) function as tran- cess is mediated by the Smad ubiquitin-regulatory factors
scription factors responsible for activating a large num- (Smurfs), which are a family of ubiquitin E3 ligases that
ber of target genes. Once they have been activated by the bind to the PY motif on the linker region of I-Smads
cell-surface receptors, the activated Smads translocate into (Module 2: Figure Smad domain structure). The E3 ligase
the nucleus (Module 2: Figure Smad signalling). Some of functions in the ubiquitin-proteasome system (Module 1:
the ligands, such as transforming growth factor β (TGF- Figure ubiquitin-proteasome system).
β), activin and Nodal activate receptors that are coupled The mitogen-activated protein kinase (MAPK) sig-
to Smad2 and Smad3, whereas bone morphogenetic pro- nalling pathway can also modulate Smad signalling by

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Module 2: Figure Smad signalling

TGFβ/activin/Nodal BMPs/GDFs

Smad4 P
P

Smad2 Smad1
Smad3 Smad5
Smad8
P P

Coactivator/ Coactivator/
repressor P repressor P
TGFβ/activin BMP
TF TF
target genes target genes
SBE SBE

The Smad signalling pathway links cell-surface transforming growth factor β (TGF-β) receptors to gene transcription.
There are two main types of Smad signalling. In the one shown on the left, receptors activated by ligands such as transforming growth factor β (TGF-β),
activin and Nodal phosphorylate either Smad2 or Smad3. The phosphorylated Smads then heterodimerize with the co-mediator Smad (Co-Smad)
Smad4 to form a dimer that then translocates into the nucleus. Once in the nucleus, the Smad dimer then binds to a specific Smad-binding element
(SBE), which has a GTCT motif that is recognized by the MAD homology domain 1 (MH1).

phosphorylating sites located in the linker regions of Wnt signalling pathways

Smad1 and 2 (Module 2: Figure Smad domain structure). The Wnt signalling pathways play a critical role in the
This is an example of signalling cross-talk in that it provides control of cell proliferation, cell fate specification and dif-
a mechanism for the tyrosine kinase-linked receptors to ferentiation. These different pathways are activated by ex-
antagonize the action of the transforming growth factor β tracellular lipoprotein signalling molecules called Wnts re-
(TGF-β) superfamily. sponsible for transmitting information between cells over
relatively short distances. There are three main pathways:
Transforming growth factor β (TGF-β) inhibition the canonical and two non-canonical pathways (Module 2:
of cell proliferation Figure Wnt signalling pathways).
One of the major functions of transforming growth factor The primary function of the canonical Wnt/β-catenin
β (TGF-β) is to inhibit cell proliferation by altering ex- pathway is to activate gene transcription to control pro-
pression of some of the key regulators of the cell cycle cesses during both development and in the adult organism.
signalling pathway. One of its actions is to increase the The functions of the non-canonical Wnt pathways are still
transcription of the p15INK4B gene that codes for p15, somewhat of an enigma, since their precise functions have
which is one of the cyclin-dependent kinase (CDK) in- not been clearly identified, but there are clear indications
hibitors that inhibits cyclin D/cyclin-dependent kinase 4 that they may function in planar cell polarity (PCP). The
(CDK4) complex that is one of the early events of the Wnt/planar cell polarity (PCP) pathway, which has been
cell cycle signalling cascade (Module 9: Figure cell cycle characterized in Drosophila, appears to act through various
signalling mechanisms). Another important action is to GTP-binding proteins. A closely related Wnt/Ca2+ sig-
repress c-Myc, which is one of the major transcriptional nalling pathway, which has been described in vertebrates,
activators of genes that function in cell proliferation. has a number of similar signalling components but also has
TGF-β plays an important role in regulating the pro- additional features such as the hydrolysis of PtdIns4,5P2 to
liferation of mesangial cells (Module 7: Figure mesangial activate signalling through InsP3 /Ca2+ and diacylglycerol
cells). It also regulates the proliferation of stem cells such (DAG)/protein kinase C (PKC).
as the skeletal muscle satellite cells (Module 8: Figure Satel-
lite cell activation) and the epidermal stem cells (Module
8: Figure epidermal stem cell). Wnts
Smad2 and 4 are tumour suppressors. A germline muta- The term Wnt results from the fusion of the names of
tion in Smad4 has been linked to juvenile polyposis syn- two orthologous genes, the Drosophila segment polarity
dromes (JPSs). gene Wingless (Wg) and a mouse proto-oncogene Int-1.

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Module 2: Figure Wnt signalling pathways

Canonical Wnt/catenin pathway Wnt/planar cell polarity pathway Wnt/Ca 2+ signalling pathway

Wnt
? Wnt5A,11

CRD
CRD
CRD
Wnt Wnt

Fz Fz Fz2, Fz6

PtdIns4,5P2
PLC
Dsh Dsh Dsh
LRP5/6
DAAM1

InsP3 DAG
β-catenin Rac Rho
? Ca 2+ PKC
JNK ROK
CaMKII
NFAT

p300 Wnt
β-catenin genes
P
Myosin II
LEF-1 TCF

Summary of the signalling mechanisms used by the canonical and non-canonical Wnt signalling pathways.
All three signalling pathways are activated by frizzled (Fz) receptors that depend upon the dishevelled protein (Dsh) as part of the transduction
mechanism to transfer information into the cell. Most is known about the canonical Wnt/β-catenin pathway (Module 2: Figure Wnt canonical pathway)
with less information on the two non-canonical pathways. In the Wnt/planar cell polarity (PCP) pathway, Dsh transfers information to the small GTP-
binding proteins Rho and Rac. The Wnt/Ca2+ signalling pathway is connected to the dishevelled-associated activator of morphogenesis 1 (DAAM1),
which relays information to the Rho pathway. In addition, Dsh also relays information to phospholipase C (PLC) to produce InsP3 and diacylglycerol
(DAG).

The human genome has 19 Wnt genes, many of which the left-hand panel in Module 2: Figure Wnt canonical
appear to have distinct functions. They contain numer- pathway:
ous cysteine residues, one of which is palmitoylated, thus
making the Wnts somewhat insoluble, which may help to 1. Formation of β-catenin by protein synthesis occurs
explain why they are short-range ligands. A large num- continuously.
ber of Wnt-binding proteins, such as secreted frizzled-re- 2. β-Catenin is drawn into a β-catenin degradation com-
lated protein (SFRP) and Wnt inhibitory factor (WIF) also plex, which consists of the scaffolding protein axin,
restrict their sphere of influence by acting as Wnt which binds the adenomatous polyposis coli (APC)
buffers. tumour suppressor, the protein phosphatase PP2A,
casein kinase Iα (CKIα), glycogen synthase kinase-3β
(GSK-3β) and β-catenin. Within this multiprotein de-
Canonical Wnt/β-catenin pathway gradation complex, the GSK-3β phosphorylates β-
The defining feature of the canonical Wnt/β-catenin path- catenin and thus targets it for destruction by the pro-
way is the transcription factor β-catenin, which is respons- teasome. Before β-catenin can be phosphorylated by
ible for regulating the transcription of Wnt target genes GSK-3β it must first be primed by phosphorylating
(Module 2: Figure Wnt canonical pathway). However, this Ser-45 by casein kinase Iα (CKIα). PP2A, which con-
is not the sole function of β-catenin, which also functions sists of a scaffolding A subunit and a regulatory B sub-
as a scaffolding protein in cell adhesion by providing a link unit (Module 5: Figure PP2A holoenzyme), may inac-
between cadherin and the actin cytoskeleton. Here we con- tivate the complex by dephosporylating GSK-3β.
sider how β-catenin functions in Wnt signalling to regulate 3. The phosphorylated β-catenin is recognized by the F-
gene transcription. There are a number of transcription box/β-TrCP/ubiquitin ligase complex, which targets it
factor activation mechanisms, and β-catenin belongs to for destruction by the proteasome.
those that depend on cell-surface receptors that generate 4. In the absence of β-catenin, the monomeric high-
cytosolic signals to activate latent transcription factors in mobility group (HMG) DNA-binding proteins lymph-
the cytoplasm, which are then imported into the nucleus ocyte enhancer factor-1 (LEF-1) and T cell factor
(Mechanism 2 in Module 4: Figure transcription factor (TCF) inhibit the transcription of Wnt genes. An addi-
activation). In the case of β-catenin, its cytosolic level is tional component of this repressor complex is histone
kept low because it is constantly being degraded by the deacetylase (HDAC), which prevents chromatin re-
proteasome as shown in the sequence of events shown in modelling through histone deacetylation. Wnt acts to

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inhibit the destruction of β-catenin, which then accu- cells used for the hair follicle cycle (Module 8: Figure epi-
mulates in the cytoplasm and can enter the nucleus to dermal stem cell).
promote transcription as shown in the right-hand panel
in Module 2: Figure Wnt canonical pathway. Wnt inhibitory factor 1 (WIF-1)
5. Wnt initiates the signalling process by binding together The Wnt inhibitory factor 1 (WIF-1) is a soluble extracel-
two cell-surface proteins: it binds to the cysteine-rich lular factor that binds to Wnt and prevents it from inter-
domain (CRD) of the frizzled (Fz) receptor and it acting with the frizzled receptor (Module 2: Figure Wnt
also draws in the Fz co-receptor LRP5 and LRP6 canonical pathway).
(LRP5/6), which are members of the low-density lipo- Secreted frizzled-related protein (sFRP)
protein receptor-related protein family (LRP). The secreted frizzled-related protein (sFRP) is a soluble
6. The LRP5/6 co-receptor is then phosphorylated by inhibitor that binds to Wnt and prevents it from interacting
membrane-bound casein kinase Iγ (CKIγ) isoform, with the frizzled receptor (Module 2: Figure Wnt canonical
which adds phosphates to multiple sites that have pathway).
PPPSP motifs. Some of these motifs are also phos-
phorylated by a membrane-associated GSK-3β. These Dickkopf (Dkk)
phosphorylated motifs then provide binding sites for Dickkopf (Dkk) binds to Kremen and the lipoprotein
the attachment of the scaffolding protein axin. Another receptor-related protein (LRP) co-receptor LRP5/6 and
key event is the binding of Dishevelled (Dsh), which this causes the complex to internalize and will thus inac-
is another scaffolding protein containing various signal tivate Wnt signalling.
transduction domains (e.g. DIX, PDZ and DEP). Dsh
becomes hyperphosphorylated by an unknown mech- Sclerostin (SOST)
anism and this contributes to its role in inhibiting the Sclerostin (SOST) inhibits Wnt signalling by binding to
degradation complex. Axin and Dsh also bind to each the lipoprotein receptor-related protein (LRP) co-receptor
other through their DIX domains. As the multiprotein LRP5/6 (Module 2: Figure Wnt canonical pathway). SOST
complex associates with the membrane, the organiza- is produced by the osteoclasts to inhibit the proliferation
tion of the subunits is altered so that the activity of and differentiation of the osteoblasts (Module 8: Figure
GSK-3β is inhibited, thus reducing the degradation of bone cell differentiation).
β-catenin. Loss-of-function mutations of LRP5 have Van Buchem disease is caused by a mutation in SOST
been linked to osteoporosis pseudoglioma (OPPG). that results in excessive activation of the Wnt signalling
7. When GSK-3β is inhibited, the newly synthesized β- pathway.
catenin is stabilized and accumulates within the cyto-
plasm, from where it can enter the nucleus to activate Function of canonical Wnt signalling
transcription. The canonical Wnt pathway has an important role to play
8. β-Catenin binds to LEF-1 and TCF to reduce their in regulating processes such as development and prolifer-
repressor activity to initiate the transcription of the Wnt ation:
genes. β-Catenin replaces HDAC with p300, which • It functions in dorsoventral specification, as has been
facilitates transcription by acetylating histones to re- shown for amphibia and zebrafish (Module 8: Figure
model chromatin. dorsoventral specification).
9. Activation of transcription of the Wnt target genes res- • It functions in the control of stem cell proliferation
ults in the activation of the developmental and prolifer- (Module 8: Figure stem cell function). For example, Wnt
ative effects that characterize the operation of the Wnt signalling stimulates the proliferation of epidermal stem
signalling pathway. cells during the hair follicle cycle (Module 8: Figure epi-
In summary, the Wnt signalling pathway acts by switch- dermal stem cell).
ing off the GSK-3β-dependent degradation pathway, thus • It controls the processes of osteoblastogenesis, which
enabling β-catenin to accumulate in the cytosol and to is responsible for the development of the bone-forming
enter the nucleus to activate transcription of the Wnt tar- osteoblasts (Module 8: Figure bone cell differentiation).
get genes. • It controls the differentiation of intestinal cells and al-
terations in its signalling components are a major cause
of colorectal cancer (CRC) (Module 12: Figure colon
Inhibitors of Wnt signalling cancer).
There are various extracellular molecules that can inhibit • The canonical Wnt pathway inhibits the differentiation
Wnt signalling. Some of these, such as the Wnt inhibit- of white fat cells (Module 8: Figure white fat cell dif-
ory factor 1 (WIF-1) and secreted frizzled-related protein ferentiation) and the differentiation of brown fat cells
(sFRP), bind to Wnt and thus prevent it from activating (Module 8: Figure brown fat cell differentiation).
the Fz receptor. There is another group, such as Dickkopf
(Dkk) and sclerostin (SOST) that interfere with the activ- The adenomatous polyposis coli (APC) protein, which
ity of the lipoprotein receptor-related protein (LRP) co- contributes to the cytoplasmic complex that degrades β-
receptor LRP5/6. These inhibitors play an important role catenin in the cytoplasm, is a potent tumour suppressor
in preventing the proliferation of stem cells until they are that is frequently mutated in cancer cells and partic-
required for tissue repair as occurs for the epidermal stem ularly in those that develop within the intestine such

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Module 2: Figure Wnt canonical pathway

LRP5/6 LRP5/6 WIF1 Wnt 5 WIF1

CRD
SOST Dkk1 Wnt SFRP
CRD SFRP Wnt
Fz LRP5/6 Fz
Kremen

CKIγ CKIγ P
P Dsh P
P P

GSK3β
Dsh

CKIα
Axin
6
Proteasome
P P P APC
P β-catenin B P
APC PP
3 A 2A

B
Axin Amino

A
β-catenin
acids

PP2A
Amino β-catenin protein GSK3β
acids synthesis and 2 7 β-catenin
CKIα β-catenin accumulates and
degradation cycle Protein
synthesis β-catenin translocates into
β-catenin β-catenin
Protein 1 the nucleus
degradation β-catenin
synthesis β-catenin complex

8 9
HDAC Wnt
4 p300
β-catenin genes
TCF
LEF-1
Ac Ac Ac Ac Ac TCF
LEF-1
Deacetylation Acetylation

The canonical Wnt/β-catenin signalling pathway.

The primary function of this signalling pathway is to regulate the activity of β-catenin, which controls transcription of the Wnt genes. The left-hand
panel illustrates the resting condition where the cytosolic level of β-catenin is kept low by its continuous degradation. In response to the arrival of Wnt
(as shown on the right), this degradation is inhibited and the level of β-catenin rises enabling it to induce the transcription of the Wnt genes.

as colorectal cancer (CRC). Germline mutations of Wnt/Ca2+ signalling pathway

APC are responsible for familial adenomatous polyposis The Wnt/Ca2+ signalling system (Module 2: Figure Wnt
(FAP). Mutation in the LRP5 gene, which binds to the signalling pathways) has been implicated in a number of
Wnt antagonist Dickkopf1 (DKK1), causes osteoporosis planar cell polarity (PCP) processes in vertebrates. There
pseudoglioma (OPPG) syndrome. are a number of similarities between this signalling path-
way and the PCP pathway in Drosophila. They both can
activate Rho and contraction of the actin/myosin system.
Wnt/planar cell polarity (PCP) pathway
The main difference is that the vertebrate system has an ad-
The Wnt/planar cell polarity (PCP) pathway has been
ditional signalling component in that it can activate Ca2+
characterized in insects, where it functions to establish
signalling. The activation of this Wnt/Ca2+ signalling path-
planar cell polarity (PCP) during development (Mod-
way depends upon the frizzled receptor plugging into the
ule 2: Figure Wnt signalling pathways). Just how this
inositol 1,4,5-trisphosphate (InsP3 )/Ca2+ signalling cas-
pathway functions is still unclear, but there are indica-
sette (Module 2: Figure InsP3 and DAG formation). There
tions that it relays information through the Rac signalling
are reports that the Ca2+ signalling events associated with
mechanisms and the Rho signalling mechanism, both of
this pathway may act to inhibit the canonical Wnt/catenin
which function in the remodelling of actin. The Rho path-
pathway during dorsal/ventral axis specification (Module
way acts through the Rho kinase (ROK) to activate con-
8: Figure dorsoventral specification).
traction of the actin/myosin system (Module 2: Figure
Mutations of the Fz4 receptor, which activates this
Rho signalling). These effects on actin remodelling and
Wnt/Ca2+ pathway, have been linked to familial exudative
contraction may play an important role in planar cell
vitreoretinopathy (FEVR).
polarity.
In vertebrates, there are similar PCP processes such as
neural tube closure, the orientation of hair cell stereociliary Casein kinase I (CKI)
bundles in the ear, mammalian hair follicle orientation and The casein kinase I (CKI) family has seven members
convergent extension (CE), during which there are large- (CKIα, α2, δ, ε, γ1, γ2 and γ3), which can have very
scale movements of cells that occur during gastrulation. different functions in cells.
Components of the insect planar cell polarity pathway
have also been described in vertebrates, where it includes Casein kinase Iα and α2
signalling through Ca2+ and has thus been referred to as CKIα functions as a priming kinase for glycogen syn-
the Wnt/Ca2+ signalling pathway. thase kinase-3β (GSK-3β) during the operation of the

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r99

Wnt signalling pathway (Module 2: Figure Wnt canonical Hedgehog signalling toolkit
pathway). The Hedgehog signalling pathway has a number of com-
ponents (Module 2: Table Hedgehog signalling toolkit)
Casein kinase Iδ whose names resemble those given to similar components
Casein kinase Iδ (CKIδ), which is 97% homologous with in Drosophila, where this signalling system was origin-
casein kinase Iε (CKIε), contributes to the operation of ally identified and characterized. While many of the mam-
the circadian clock by phosphorylating PER1 and PER2 malian components have similar functions to those found
to control the nuclear entry and stability of these clock pro- in insects, it is clear that there are important differences.
teins (Module 6: Figure circadian clock molecular mech- Since there are mammalian components that are not found
anism). in insects, the mechanism of Hedgehog activation of tran-
scription has to be worked out separately for vertebrates.
Casein kinase Iε
CKIε participates in circadian rhythmicity by phos- Hedgehog activation of transcription
phorylating the PER and CRY proteins to regulate their Hedgehog mediates its effects by activating gene tran-
nuclear entry and stability during the operation of the scription. Hedgehog uses mechanism 2 of the different
PER regulatory loop (Module 6: Figure circadian clock transcription factor activation mechanisms found in cells
molecular mechanism). Mutation of CKIε, which results (Module 4: Figure transcription factor activation). There
in a decrease in the ability of this kinase to phosphorylate are three Hedgehog transcription factors (GLI 1–3), which
the PER proteins of the circadian clock, is responsible for are held in an inactive state within the cytoplasm in rest-
familial advanced sleep phase syndrome (FASPS). ing cells (Module 2: Figure Hedgehog signalling path-
way). This inactive state is maintained by the Hedge-
Casein kinase Iγ1, γ2 and γ3 hog receptor patched (PTC), which inhibits the seven-
CKIγ is an unusual member of the family in that it has membrane-spanning protein smoothened (SMO) that acts
a palmitoylation site at its C-terminus and this fatty acid as the Hedgehog transducer. In the absence of a signal from
anchor attaches it to the plasma membrane. This membrane SMO, the GLI transcription factors are maintained in a lat-
location is critical for one of its main functions, which is ent state by interacting with a large number of cytoplasmic
to phosphorylate the frizzled lipoprotein receptor-related factors (Module 2: Table Hedgehog signalling toolkit). The
protein (LRP) co-receptor LRP5/6 during activation of the precise function of all these factors is still being worked
Wnt signalling pathway (Module 2: Figure Wnt canonical out. Hedgehog arriving at the cell surface induces a train of
pathway). events that activate these transcription factors so that they
translocate into the nucleus to induce gene transcription
Casein kinase II (CK2) (Module 2: Figure Hedgehog signalling pathway). First of
Casein kinase II (CK2) is a serine/threonine protein kinase. all, Hedgehog binds to its receptor PTC and this removes
It functions as a heterotetramer consisting of two 44 kDa the inhibitory effect of PTC on SMO. The latter is then
catalytic α-subunits and two regulatory β-subunits. It has able to activate GLI by removing it from the inhibitory
a unique ability to use GTP as well as ATP as a phosphate constraints of the cytoplasmic factors so that it is now free
donor. CK2 is also known as phosvitin kinase, glycogen to translocate into the nucleus to activate transcription.
synthase kinase 5, troponin T kinase and casein kinase G, Some of the genes that are activated are components of the
which reflects the fact that CK2 can phosphorylate many Hedgehog signalling pathway and thus set up both posit-
different substrates and thus contributes to many control ive and negative feedback loops. Many of the other genes
mechanisms. This multifunctional kinase has been implic- contribute to Hedgehog signalling functions.
ated in many cellular processes and seems to be particu-
larly active in controlling cell proliferation and has also Hedgehog signalling functions
been implicated in cell transformation and tumorigenesis. The Hedgehog signalling pathway functions both dur-
Many of its actions depend on its ability to phosphorylate ing development and during adult life. Its function dur-
transcription factors such as Myc, p53, Rb and activating ing development is wide-ranging in that it can control
protein 1 (AP-1). Although CK2 is constitutively active, cell proliferation, cell determination and pattern forma-
it can also be activated by certain growth factors (insulin, tion of the developing embryo and the final processes of
IGF-I and EGF). cell differentiation. Hedgehog signalling seems to play an
important role in controlling the development of organs
such as the skin, brain, digestive tract, pancreas and pro-
Hedgehog signalling pathway state. Mutations of components of this signalling pathway
The Hedgehog signalling pathway in mammals closely re- result in congenital malformations such as holoprosen-
sembles that originally discovered and characterized in cephaly, which is a cranial defect that occurs when the
Drosophila. The mammalian Hedgehog signalling toolkit midline structures of the brain and face fail to separate.
has many of the components found in insects. In compar- The most dramatic phenotype of such malformation is
ison with the latter, however, much less is known about cyclopia where only one eye is formed.
the mechanism of Hedgehog activation of transcription in The function of Hedgehog continues in the adult or-
mammals. There are multiple Hedgehog signalling func- ganism, where it plays a major role in the formation and
tions that operate during both development and adult life. maintenance of the stem cell population. Since Hedgehog

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Module 2: Table Hedgehog signalling toolkit

The Hedgehog signalling toolkit.
Component Comment
Hedgehog (Hh) ligands
Shh Sonic Hedgehog
Ihh Indian Hedgehog
Dhh Desert Hedgehog
Hedgehog-binding proteins
HIP Hh-interacting protein
Megalin A member of the low-density lipoprotein (LDL)-receptor-related family
Hedgehog receptors
PTC1 Patched 1
PTC2 Patched 2
Transducer
SMO Smoothened; a seven-membrane-spanning protein
Cytoplasmic regulators
KIF7 Functions to repress Sh responses
FU The fused serine/threonine protein kinase
SUFU Suppressor of fused; a negative regulator of GLI transcription factors
MIM Missing in metastasis; an actin-binding protein that potentiates transcription
Iguana A zinc-finger protein that promotes localization of GLI1
IFT88 Ciliary protein that regulates GLI function
IFT172 Ciliary protein that regulates GLI function
FKBP8 Antagonizes Shh action
SIL Cytoplasmic protein that acts downstream of PTC
Rab23 This regulator of vesicular traffic is a negative regulator of Hh signalling
Transcription factors
The multifunctional GLI transcription factors function to
either activate or repress transcription
GLI1 A zinc-finger transcriptional activator
GLI2 A zinc-finger transcriptional activator/repressor
GLI3 A zinc-finger transcriptional repressor
The function of some of these toolkit components are illustrated in Module 2: Figure Hedgehog signalling pathway.

Module 2: Figure Hedgehog signalling pathway

Hh

Hh
HIP
PTC SMO SMO Hh PTC

KIF7
SU X
SU GLI1 SU GLI1
KIF7 KIF7 SUFU
SUFU GLI1
GLI1

Positive Negative
Iguana + MIM feedback feedback
+

GLI1, MIM

PTC, HIP

GLI1 Wnt, BMP

Activation of gene transcription by the Hedgehog signalling pathway.

Under resting conditions, the patched (PTC) receptor for Hedgehog (Hh) inhibits the activity of the seven-membrane-spanning receptor smoothened
(SMO). In this inhibited state, SMO is not able to act on the complex of cytoplasmic factors that collectively regulate the transcription factor GLI1.
When Hh is present, it binds to PTC, and this removes the inhibitory effect of the latter on SMO, which is now capable of activating transcription
by releasing GLI from its associated cytoplasmic factors. GLI1 then translocates into the nucleus, where it activates a variety of genes that fall into
three main groups. One group consists of components of the signalling pathway, such as GLI1 and missing in metastasis (MIM), which set up a
positive-feedback loop. The second group consists of genes coding for PTC and Hh-interacting protein (HIP), which thus set up a negative-feedback
loop. The HIP protein acts on the outside to bind Hh and thus reduce its activity. The last group of genes code for proteins not involved in Hedgehog
signalling, but some do function in other signalling pathways such as Wnt (Module 2: Figure Wnt canonical pathway) and the bone morphogenetic
proteins (BMPs) (Module 2: Figure Smad signalling).

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Module 2 : Figure Notch signalling

Endosome
Delta
Jagged

1 2
S2 Notch 3
γ-secretase
S3 Proteasome
6 Ankyrin
repeats ADAM 4
NICD
PEST
UbUbUbUb
P

5 8 9
NICD SEL-10
CDK-8
P
Acetylation M
p300 MA Notch target genes HES1
SKIP HES2
CSL 7
HES7
HEY1
Gene repression 10 6 Gene activation HEY2
HEYL
ESR
Deacetylation HDAC
SMRT
CSL
SHARP
X NRARP

Notch signalling pathway.

One of the stimuli for the Notch signalling pathway is Jagged, which is located on the surface of the communicating cell. The receptor for Jagged is
Notch, which is located on the surface of the receiving cell. When Jagged interacts with Notch (Step 1), it triggers a series of steps that result in the
proteolytic release of the Notch intracellular domain (NICD), which then enters the nucleus to activate transcription of Notch target genes. See the text
for details of this sequence of events.

signalling plays such a major role in regulating cell prolif- fate (self-renewal) while the daughter cell (progenitor cell)
eration, it is not surprising that alterations in this signalling adopts a different state that will drive it towards prolifera-
pathway have been detected in many cancers such as skin tion and differentiation into a specific cell type (Module 8:
cancer and some brain cancers. Figure stem cell function). As these progenitor cells adopt
For example, patched (PTC) is a tumour suppressor that their new cell fate, they use the Notch signalling pathway
is inactivated in basal cell carcinomas (BCCs), medullo- to feed information back to suppress their neighbour from
blastomas, gliomas in the brain and prostate cancers. adopting a similar cell fate. This is a short-range informa-
Mutations of the PTC gene are responsible for Gorlin’s tion transfer mechanism that depends upon direct contact
syndrome, which is also known as nevoid basal cell car- between the cells, which is a hallmark of this signalling
cinoma syndrome (NBCC). The Hedgehog signalling pathway. For example, the stimulus Jagged is an integral
pathway may also contribute to the growth of tumours membrane protein located on the surface of communic-
by enhancing the activity of stromal cells that provide the ating cells, whereas the Notch receptor that responds to
tumour cell microenvironment that supports cancer cell Jagged is embedded in the surface of the receiving cell
survival and growth. Cancer cells release hedgehog that (Module 2: Figure Notch signalling).
then uses a paracrine mechanism to stimulate neighbouring The main feature of the transduction mechanism is de-
stromal cells such as the blood vessels, fibroblasts, immune ceptively simple. When Jagged binds to Notch, the Notch
cells and epithelial cells. These stromal cells assist cancer intracellular domain (NICD) is released into the cytoplasm
cell growth by providing both an extracellular matrix and and then diffuses into the nucleus where it induces the
essential growth factors such as insulin-like growth factor transcription of multiple Notch target genes. Despite its
(IGF) and Wnt. simplicity, this signalling system has an extensive number
of interacting components (Module 2: Table Notch sig-
nalling components). Some of these function during the
Notch signalling pathway signal transduction process itself, whereas others play a
The Notch signalling pathway is a highly conserved sig- role in the modulation of Notch signalling, which regu-
nalling system that functions in both development and lates the expression of the stimuli (Delta and Jagged) and
adulthood. Many of its functions relate to cell-fate de- the Notch receptor at the cell surface.
termination and this is particularly the case in its control The operation of the Notch signalling pathway depends
of binary cell-fate decisions in stem cells. During the assy- upon the following steps (Module 2: Figure Notch sig-
metrical divisions of stem cells, one cell retains its stem cell nalling):

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Module 2: Table Notch signalling components

Notch signalling toolkit.
Components Comments
Notch stimuli These Notch stimuli contain an N-terminal DSL (Delta, Serrate and LAG-2)
domain that interacts with the Notch receptor (Module 2: Figure Notch
signalling)
Delta 1
Delta 3
Delta 4 Responsible for lateral inhibition during angiogenesis (Module 9: Figure
angiogenesis signalling)
Delta-like-4 (DLL4)
Jagged 1
Jagged 2
Receptors
Notch 1
Notch 2
Notch 3
Notch 4
Notch modulators
Numb A major inhibitor of Notch. It facilitates the endocytic removal of Notch from the
plasma membrane (Module 2: Figure Notch modulation)

Crumbs May function to inhibit γ-secretase activity thereby reducing the activation
of Notch
Proteases
ADAM-10 Also known as Kuzbanian or MADM. Functions in neural and cardiac
development
ADAM-17 Also known as tumour necrosis factor-α (TACE)
Furin A protein convertase that cleaves site 1 (S1) during Notch receptor maturation
γ-secretase An enzyme complex containing presenilin, nicastrin, PEN2 and APH1
Glycosyltransferases
Fringe family A family of glycosyltransferase in the Golgi that glycosylates Notch to alter its
binding affinities to Delta and Jagged
Lunatic fringe
Radical fringe
Manic fringe
Transcriptional regulators
CSL
CSL [CBF-1, Su(H), Lag-1] represses Notch target genes (Module 2: Figure
Notch signalling)

MAM Mastermind
SKIP Ski-interacting protein interacts with CSL and the ankyrin repeats of NICD
Notch target genes
HES1 Hairy and enhancer of split 1 (HES1) represses transcription of tissue specific
genes responsible for differentiation
HES2
HES7
HEY1
HEY2
HEYL
ESR
Ubiquitin ligases
Itch A Hect domain E3 ligase that functions in Notch trafficking (Module 2: Figure
Notch modulation)
Deltex A Ring finger E3 ligase that functions in Notch trafficking (Module 2: Figure
Notch modulation)
Mind bomb (Mib) Ring finger E3 ubiquitin ligases that initiate the trafficking of Delta and Jagged
(Module 2: Figure Notch modulation)
Neutralized (Neur) Ring finger E3 ubiquitin ligases that initiate the trafficking of Delta and Jagged
(Module 2: Figure Notch modulation)
SEL10 A nuclear ubiquitin ligase that interacts with the phosphorylated form of NICD
(see Step 8 in Module 2: Figure Notch signalling)

1. Delta or Jagged located on one cell interacts with drawn into the endosome, they pull on Notch, which
the Notch receptor on a neighbouring cell. The N- undergoes a conformational change to expose site-2
terminal DSL (Delta, Serrate and LAG-2) domain (S2).
(yellow bar) on Delta/Jagged binds to the EGF- 3. S2 is cleaved by ADAM proteases such as ADAM-10
repeats 11 and 12 (red bar) on Notch. By itself, this and ADAM-17 (Module 1: Figure ADAMs proteases).
interaction with Notch seems to have little effect and The external domain is shed leaving behind a short
only initiates the signal transduction sequence as a res- transmembrane region and the intracellular domain,
ult of Delta/Jagged physically pulling on Notch. which then becomes a substrate for γ-secretase.
2. The endocytosis of Delta/Jagged seems to be a crit- 4. γ-Secretase is an enzyme complex made up of
ical step for receptor activation. As Delta/Jagged are presenilin, nicastrin, PEN2 and APH1 that cleaves

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the S3 site to release the Notch intracellular domain itor Numb has been observed in breast cancer. Mutations
(NICD). The activity of γ-secretase may be inhibited in Notch have been linked to T-cell acute lymphoblastic
by Crumbs, which is an apical polarity protein. leukaemia.
5. NICD released from the membrane diffuses into the
nucleus and binds to CSL (CBF-1, Suppressor of Hair- Modulation of Notch signalling
less, Lag-1). The primary mechanism for modulating Notch signalling
6. CSL normally acts to repress Notch target genes. is to adjust the mechanisms responsible for regulating both
Repression is enhanced by CSL providing a frame- ligand (Delta and Jagged) and receptor (Notch) maturation
work to recruit co-repressors such as SMRT, SHARP, (Module 2: Figure Notch modulation). In both cases, the
SKIP and CIR. This repression complex also recruits ligands and the receptor on the cell surface are taken up
histone deacetylase (HDAC) that deacetylates chro- into the endosomes and are then either recycled back to the
matin further shutting down transcriptional activity. cell surface or are degraded. The balance between recycling
When NICD binds to CSL, the repression complex is and degradation thus determines their level of membrane
disassembled and this paves the way for the assembly expression and thus sets the sensitivity of the Notch sig-
of an activation complex. An important part of the nalling pathway. The ubiquitin-proteasome system plays
complex is the co-activator Mastermind (MAM) and a critical role in regulating the trafficking of these two
the histone acetyl transferase p300. The latter is re- signalling components.
sponsible for protein acetylation of histones to open The trafficking of Delta and Jagged follows the fol-
up the chromatin to facilitate gene transcription. lowing series of events (Module 2: Figure Notch mod-
7. Gene activation results in an increase in the transcrip- ulation):
tion of Notch target genes. Some of these such as the
hairy and enhancer of split (HES) family encode tran- 1. The cytoplasmic domain of Delta and Jagged are ubi-
scriptional repressors responsible for suppressing the quitinylated by Ring finger E3 ubiquitin ligases such as
expression of tissue specific proteins, which accounts Mind bomb (Mib) and Neutralized (Neur).
for the ability of the Notch signalling pathway to in- 2. Endocytosis of these ubiquitylated proteins is facil-
hibit differentiation. itated by the protein epsin that binds to ubiquitin-
8. The inactivation of target gene transcription begins containing cargo proteins. In addition, epsin binds
when NICD is phosphorylated in the N-terminal to both clathrin and to phosphatidylinositol 4,5-
PEST domain by kinases such as cyclin-dependent bisphosphate (PtdIns4,5P2 ), which functions in the
kinase-8 (CDK-8). PtdIns4,5P2 regulation of membrane trafficking and en-
9. The phosphorylated NICD becomes a substrate for docytosis.
nuclear ubiquitin ligases such as SEL-10 and is then 3. The endosome then has two paths: it can move down
exported to the cytoplasm where it is degraded by the the lysosomal path resulting in degradation of Delta and
proteasome. Jagged. Alternatively, it can recycle back to the plasma
10. Once NICD is removed from the activation com- membrane where the ligands are then able to activate
plex and degraded, the different co-activators fall Notch.
away and are replaced by the repressors that result Different players function in the regulation of Notch traf-
in gene repression. Gene repression also depends on ficking as shown in the following steps (Module 2: Figure
the recruitment of histone deacetylase (HDAC) that Notch modulation):
deacetylates chromatin further shutting down tran-
scriptional activity. 4. Entry of Notch into the endosome is dependent on the
protein Numb, which is a major inhibitor of Notch
Notch signalling controls a number of developmental signalling. The Notch receptor interacts with Numb,
events and continues to have a role during adulthood par- which also binds to α-adaptin. The latter is a component
ticular in maintaining the stem cell population: of the adaptor protein-2 (AP2) complex responsible for
endocytosis. A Numb-associated kinase (NAK) is also
• Proliferation of satellite cells in skeletal muscle is en-
a component of this complex. The Notch receptors in
hanced by Notch signalling (Module 8: Figure Satellite
the endosome have two fates.
cell activation).
5. Some of the Notch receptors are ubiquitinated by
• The self renewal of haematopoietic stem cells (HSCs)
ubiquitin ligases such as Itch (a Hect domain E3 lig-
is facilitated by Notch signalling that acts by inhibiting
ase) and Deltex (a Ring finger E3 ligase).
differentiation (Module 8: Figure HSC regulation).
6. Ubiquitinylated notch receptors are transferred to the
• Notch signalling is responsible for the process of lat-
multivesicular body (MVB) en route to degradation by
eral inhibition that inhibits tip cell formation during
the lysosome.
angiogenesis (see Step 2 in Module 9: Figure angiogen-
7. Some of the Notch receptors are recycled back to the
esis signalling).
plasma membrane.
Alterations in Notch signalling have been implicated in a 8. These Notch receptors on the cell surface are then able
number of cancers. For example, overexpression of Notch to interact with ligands such as Delta and Jagged to
has been identified in both ovarian and medullablastomas. initiate the Notch signalling pathway (Module 2: Figure
An up-regulation of Notch and a decrease in the inhib- Notch signalling).

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Module 2: Figure Notch modulation

Ub

Ub
Ligand maturation Degredation

Ub 3
2
Ub

Epsin Recycling
1
Ub
ur b
Ne Mi Ub PtdIns4,5P2
Ub

Delta
Jagged
8
Notch

NAK
Numb 4
α-adaptin Recycling

7
MVB
Receptor maturation
Endosome
Ub
Itch 6
5 Degredation
Deltex Ub
Ub

Modulation of Notch signalling.

Trafficking through the endosomal compartment plays an important role in regulating the maturation/activity of the Notch ligands (shown at the top)
and the Notch receptor (shown at the bottom). See text for further details. Mib, Mind bomb; MVB, multivesicular body; Neur, Neutralized; NAK,
Numb-associated kinase; Ub, ubiquitin.

Delta and this is an important aspect of the modulation of Notch

Delta is a transmembrane protein that functions as one signalling.
of the stimuli that activate the Notch receptor (Module
2: Table Notch signalling components). The extracellu-
lar domain is made up of EGF-like repeats and there is Notch
an N-terminal DSL (Delta, Serrate and LAG-2) domain Notch is a transmembrane protein that functions as the
that interacts with the Notch receptor (Module 2: Fig- receptor for the Notch signalling pathway by respond-
ure Notch signalling). In order for Delta to function as a ing to stimuli such as Delta and Jagged (Module 2: Figure
stimulus, it has to undergo a maturation process, which Notch signalling). The large extracellular domain of Notch
depends upon its passage through the endosome (Module is made up of a variable number (29–36) of EGF-like re-
2: Figure Notch modulation). This trafficking through the peats and a Lin21/Notch repeat region. EGF-repeats 11
endosomal compartment determines the amount of Delta and 12 (red bar) on Notch provides the binding site that in-
in the membrane and this is an important aspect of the teracts with the N-terminal DSL (Delta, Serrate and LAG-
modulation of Notch signalling. 2) domain on Delta and Jagged. The cytoplasmic domain
The expression of Delta-1 is increased on the surface has a series of six ankyrin repeats and a terminal PEST
of myofibres during muscle damage and this activates the domain.
Notch signalling pathway to stimulate the proliferation of Notch undergoes a number of post-translational modi-
satellite cells (Module 8: Figure Satellite cell activation). fications as it moves through the endoplasmic reticulum
(ER) and the Golgi. While in the ER, certain sites on the
EGF repeats are fucosylated by the chaperone O-fut. After
Jagged the addition of these fucose groups, further extension of
Jagged is a transmembrane protein that functions as a one the carbohydrate chains is carried out by the Fringe family
of the stimuli that activate the Notch receptor (Module (Module 2: Table Notch signalling components). The de-
2: Table Notch signalling components). The extracellular gree of glycosylation can markedly influence the affinity
domain is made up of a cysteine-rich domain, EGF-like of the Notch receptor for its ligands. The last modification,
repeats and there is an N-terminal DSL (Delta, Serrate and which occurs in the Golgi, is the cleavage of the molecule
LAG-2) domain that interacts with the Notch receptor. at site 1 (S1) by the protease; the heterodimeric receptor is
In order for Jagged to function as a stimulus, it has to then inserted into the membrane.
undergo a maturation process, which depends upon its Trafficking of Notch through the endosomal compart-
passage through the endosome (Module 2: Figure Notch ment determines the amount of receptor in the membrane
modulation). This trafficking through the endosomal com- and this is an important aspect of the modulation of Notch
partment determines the amount of Delta in the membrane signalling (Module 2: Figure Notch modulation).

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Endoplasmic reticulum (ER) stress Unfolded protein response (UPR)

signalling An accumulation of misfolded proteins induces an unfol-
The endoplasmic reticulum (ER) has sophisticated stress ded protein response (UPR), which switches off ongoing
signalling pathways that enable it to adapt to a whole protein synthesis and also activates various transcriptional
host of stress factors mainly concerned with the way in cascades that result in the up-regulation of many of the
which proteins are synthesized and packaged. Mainten- key chaperones in an attempt to improve the defective
ance of a constant level of Ca2+ within the lumen of the protein packaging machinery (Module 2: Figure ER stress
ER is essential for the post-translational processing, fold- signalling). Activating transcription factor 6 (ATF6) is one
ing and export of proteins. This protein processing is car- of the transcription factors activated by the UPR.
ried out by a number of Ca2+ -sensitive chaperones such as
78 kDa glucose-regulatory protein (GRP78) [also known Endoplasmic reticulum (ER) overload response
as immunoglobulin heavy-chain-binding protein (BiP)], (EOR)
GRP94 (endoplasmin) and calnexin. Any decline in the lu- An excessive build-up of proteins, as occurs during viral in-
minal level of Ca2+ results in the accumulation of misfol- fections, switches on an endoplasmic reticulum (ER) over-
ded proteins and the activation of the ER stress signalling load response (EOR) that acts through the nuclear factor
pathways (Module 2: Figure ER stress signalling): κB (NF-κB) signalling cascade to stimulate the release of
interferons and cytokines as part of an inflammatory re-
sponse.
1. Oligomerization and autophosphorylation of PKR
(protein kinase R)-like ER kinase (PERK) sets off a Activation of apoptosis
phosphorylation cascade that culminates in the phos- Endoplasmic reticulum (ER) stress signalling pathways
phorylation and inactivation of the translation euka- can also contribute to apoptosis (Module 11: Figure ap-
ryotic initiation factor eIF-2α, resulting in protein syn- optosis). For example, one of the UPR pathways depends
thesis being switched off. upon the release of the transcription factor activating
2. Oligomerization and autophosphorylation of IRE1 ini- transcription factor 6 (ATF6), which acts to switch on
tiates one of the transcriptional signalling pathways re- C/EBP (CCAAT/enhancer-binding protein)-homologous
sponsible for the up-regulation of the various chaper- protein 10 (CHOP). Another mechanism depends upon
ones. the ER directly activating a subset of caspases during
3. Another of the transcriptional pathways depends ER stress. A critical component is caspase 12, which is
upon the activation of the ER membrane-bound associated with the ER membrane and is released by
activating transcription factor 6 (ATF6), which is proteolytic cleavage following ER stress. Several mech-
released from the ER to enter the nucleus, where anisms have been proposed for this activation process.
it interacts with the ER stress-response element One suggestion is that the stress sensor molecule IRE1
of the C/EBP (CCAAT/enhancer-binding protein)- recruits tumour-necrosis-factor-receptor-associated factor
homologous protein 10 (CHOP) gene. (TRAF) which then binds to caspase 12, making it sens-
4. The various chaperones are then expressed within the itive to the Ca2+ -responsive cysteine protease m-calpain.
ER, where they participate in protein folding. Another suggestion is that the hydrolysis of caspase 12 is
5. One of the genes activated during the stress response mediated by caspase 7, which is recruited to the membrane
is CHOP, which acts as a transcription factor and can during ER stress. An interesting aspect of this mechanism
contribute to apoptosis. is that glucose-regulated protein 78 kDa (GRP78) appears
6. Caspase 12, which is associated with the ER membrane, to inhibit this activation process by forming a complex
is also activated and contributes to ER stress-induced with caspase 7 and caspase 12. Once caspase 12 is released
apoptosis. into the cytosol, it activates a specific cascade involving
7. An excessive accumulation of proteins within the ER caspase 9 and caspase 3 in a cytochrome c-independent
results in the activation of the transcription factor manner.
nuclear factor κB (NF-κB), which acts to increase the
production of interferons and cytokines, so contribut-
ing to an inflammatory response. Metabolic messengers
There are a number of cellular metabolites that function as
metabolic messengers to integrate the activities of cellular
These stress pathways are then responsible for switching metabolism and cell signalling (Module 2: Figure meta-
off ongoing protein synthesis, for up-regulating the pro- bolic messengers). In this context, a metabolic messenger
duction of new chaperones, for inducing apoptosis and for is defined fairly widely: it includes components that are
activating inflammatory responses. The degree to which either a part of, or are derived from, cellular metabolism.
these different responses are activated depends upon the Metabolism is regulated at many different levels. The most
nature of the stress. The fact that the ER can up-regulate direct control depends upon feedback processes where cer-
chaperone levels results in the phenomenon of tolerance, tain substrates or products function as positive or negative
whereby treatment of cells with low levels of stress stimuli regulators of the enzymes that synthesize or metabolize
can make cells much more tolerant to subsequent stressful them. They are not signalling mechanisms in the strict
stimuli. sense, but are based on relatively straightforward mass

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Module 2: Figure ER stress signalling

P e1F-2 Protein synthesis OFF

PERK P P Protein synthesis ON Endoplasmic reticulum

e1F-2
Protein synthesis
Protein folding
1 Dysfunctional Normal
chaperones chaperones
STRESS
Export
Oligomerization Misfolded 4
protein

2 3 Excess
6 protein
Caspase-12 7
IRE1 P P
NF-kB INFLAMMATORY
ATF-6 APOPTOSIS RESPONSES
Transcription
factors
5
PDI: GRP78:
GRP94; CHOP Interleukins
Calreticulin etc Cytokines

Endoplasmic reticulum (ER) stress signalling pathways.

An accumulation of misfolded proteins or an excessive accumulation of normal proteins activate a number of signalling pathways. Chaperones within
the endoplasmic reticulum (ER) lumen are responsible for folding newly synthesized proteins into their tertiary structures prior to their export to the
Golgi. A variety of stress factors, including a decline in the luminal level of Ca2+ , results in dysfunctional chaperones and an accumulation of misfolded
proteins that can activate a number of signalling pathways.

Module 2: Figure metabolic messengers

+
Ca 2+ K+ + Ca 2+
ATP
TRPM2 P2Y P2X

K ATP α βγ
Ca 2+ + PLC Ca 2+

DAG InsP3

ADPR ATP
Cyclic AMP
NAD signalling
NADP HCO pathway
3
ADP ribosyl H S
cyclase Metabolic
messengers
cADPR AMP
AMP
signalling
+ NADH
NAADP pathway
NAD

Calcium +
signalling Gene
pathway transcription

Interaction between metabolic messengers and cell signalling pathways.

A number of metabolic intermediates can interact with various cell signalling pathways. ATP plays a significant role through its ability to close
ATP-sensitive K+ channels as occurs in insulin-secreting β-cells (Module 7: Figure β-cell signalling). ATP that is released from the cell functions
to activate ATP-sensitive P2X channels (Module 3: Figure P2X receptor structure). ATP may also play an important role in regulating the activity of
ADP-ribosyl cyclase that produces the Ca2+ -mobilizing messengers cyclic ADP-ribose (cADPR) and nicotinic acid–adenine dinucleotide phosphate
(NAADP) (Module 2: Figure cADPR/NAADP function). AMP is an important activator of the AMP signalling pathway (Module 2: Figure AMPK control of
metabolism). The breakdown product of cADPR is ADP-ribose (ADPR), which is an activator of the transient receptor potential melastatin 2 (TRPM2)
channel. Bicarbonate (HCO3 − ) is an activator of soluble adenylyl cyclase and thus contributes to the cyclic AMP signalling pathway (Module 2: Figure
cyclic AMP signalling).

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Module 2: Figure metabolic signalling

Metabolic messengers
Stimuli ROS/RNS Substrates Oxygen

1 3
+
NAD / NADH
+
NADP / NADPH
AMP/ATP

METABOLISM
_
SIGNALLING 9 HCO 3 8
PATHWAYS
NAADP
cADPR

7
4
Energy
6
CELLULAR
2 RESPONSES 5

Function of metabolic hormones in integrating the operation of cell metabolism and cell signalling pathways.
The signalling cascade begins with the arrival of an agonist (1) that recruits specific signalling pathways to activate a cellular response (2). Metabolism
uses oxygen and substrates (3) to provide the energy (4) that not only powers the cellular responses (5), but also drives signalling pathways (6). The
signalling pathways can also regulate the supply of energy by direct effects on metabolism (7). Superimposed on these more direct interactions, there
are a variety of metabolic messengers that form a complex signalling network (8 and 9) that integrate the activity of both the signalling and metabolic
cascades.

action reactions that ensure that metabolism proceeds in an AMP signalling pathway
orderly and regulated manner. However, some of the meta-
bolic components can be considered to be metabolic mes- Adenosine monophosphate (AMP)
sengers because they activate or modulate clearly defined Cells have an AMP signalling pathway that is activated by
signalling pathways. There are a number of such metabolic an increase in the AMP/ATP ratio, which results in the
messengers: activation of an AMP-activated protein kinase (AMPK).
AMP thus functions as a second messenger, since it is re-
• Adenosine triphosphate (ATP) sponsible for activating the signalling pathway. The AMPK
• Adenosine monophosphate (AMP) that responds to AMP has been referred to as the “fuel
• Fatty acids gauge” in that it responds to a decrease in the level of
• Bicarbonate (HCO3 − ) ATP. This signalling cascade is sensitive to many stim-
• NAD+ signalling pathways uli, such as cell stress, oxidative damage, hypoxia and
glucose deprivation. Once activated, AMPK induces an
The AMP signalling pathway is of major importance up-regulation of ATP-generating systems while simultan-
with regard to metabolic signalling systems. Components eously down-regulating processes that consume energy.
of various redox signalling systems, such as the nitric ox- One of its important actions is to reduce protein synthesis
ide signalling pathway, are also related to such metabolic when energy levels are low by regulating the activity of
signalling pathways. the target of rapamycin (TOR) (Module 9: Figure target of
Signalling through metabolites is a highly integrated sys- rapamycin signalling). Another of its actions is to regulate
tem because some of the signalling pathways activated by the transcription of genes that function in the metabol-
the metabolic messengers can feed back to regulate meta- ism of glucose, fatty acids and cholesterol. The AMPK
bolism (Module 2: Figure metabolic signalling). An ex- signalling pathway may also play a role in stimulating mi-
ample of how a change in metabolism can affect a signalling tochondrial biosynthesis.
pathway has been described in cardiac myocytes, where the AMPK has also been implicated in cell growth control,
addition of pyruvate to increase metabolism has a marked where it functions to regulate the protein kinase TOR
effect on Ca2+ signalling (Module 2: Figure pyruvate and that controls protein synthesis (Module 9: Figure target of
Ca2+ signalling). rapamycin signalling).

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Module 2: Figure pyruvate and Ca2+ signalling

The effect of pyruvate on Ca2+ signalling in cardiac myocytes.

The traces represent the Ca2+ spikes that are generated when isolated cardiac myocytes are electrically stimulated at 0.5 Hz. Under control conditions
(0 ), there are constant amplitude transients resulting from the release from the internal store. The size of this store is indicated by the amplitude of
the much larger transient when caffeine (10 mM) is added. The next series of transients were taken 1, 2 and 8 min after addition of 10 mM pyruvate,
which caused an increase in both the resting level and the transient amplitudes. This increase in the amplitude of the Ca2+ transients may have
resulted from an increase in the content of the internal store, as indicated by the very large caffeine-induced transient apparent at the end of the 8-min
sequence. The effect of pyruvate was reversible because the signalling system was back to its control values 4 min after washing out the pyruvate
(W). A possible interpretation of this experiment is that pyruvate enhanced metabolism and the increase in ATP concentration may have enhanced the
formation of cyclic ADP-ribose (cADPR) to stimulate the pump that transfers Ca2+ into the internal store (Module 2: Figure cADPR/NAADP function).
Reproduced from Zima, A.V., Kockskämper, J., Meijia-Alvarez, R. and Blatter, L.A. (2003) Pyruvate modulates cardiac sarcoplasmic reticulum Ca2+
release in rats via mitochondria-dependent and -independent mechanisms. J. Physiol. 550:765–783, with permission from Blackwell Publishing; see
Zima et al. 2003.

AMP-activated protein kinase (AMPK) AMPK can inhibit the activity of various transcrip-
AMP-activated protein kinase (AMPK) plays a critical role tion factors such as the sterol regulatory element-binding
in regulating the usage of fuels such as glucose and fatty protein 1c (SREBp1c) and hepatocyte nuclear factor 4α
acids. However, it can have additional functions such as (HNF4α), which regulate lipogenic and glycolytic genes
the regulation of insulin secretion by pancreatic β-cells, respectively. AMPK phosphorylates TORC2 to prevent
and may also play a role in controlling satiety centres in it from acting as a cofactor to activate genes respons-
the hypothalamus. AMPK is a trimeric protein (Module ible for gluconeogenesis. Glycogen can inhibit the activity
2: Figure AMPK structure) made up of multiple isoforms of AMPK by interacting with the glycogen-binding do-
of a catalytic α subunit (α1 and α2) associated with β- main (GBD) of the β subunit (Module 2: Figure AMPK
subunits (β1 and β2) and γ-subunits (γ1–γ3). Cells express structure). Finally, AMPK plays an important role in cell
different combinations of these different isoforms. The growth control by reducing protein synthesis when energy
activity of AMPK is regulated by both the intracellular levels are low by acting through TOR (Module 9: Figure
level of AMP and by phosphorylation through an AMPK target of rapamycin signalling).
kinase known as LKB1. In summary, AMPK switches the cell away from energy-
AMPK functions as a pleiotropic regulator of cell meta- requiring processes towards energy conservation. It does
bolism (Module 2: Figure AMPK control of metabolism). this by exerting rapid effects on processes such as glucose
It stimulates the translocation of the glucose transporter entry and glycolysis, as well as longer-term effects, by reg-
(GLUT4) to the plasma membrane, where it facilitates the ulating the transcription of both glycolytic and lipogenic
entry of glucose in skeletal muscle and heart cells. AMPK hormones (Module 2: Figure AMPK control of metabol-
can influence the balance between glycolysis and gluco- ism). Many of its actions are carried out in muscle cells and
neogenesis by stimulating the formation of fructose 2,6- liver. With regard to the latter, it can phosphorylate trans-
bisphosphate (F-2,6-P2 ), which is a potent regulator of ducer of regulated cyclic AMP response element-binding
glycolysis through its ability to activate 6-phosphofructo- protein 2 (TORC2), which is then prevented from act-
1-kinase (PFK-1) and to inhibit fructose-1,6-bisphosphate ing as a cofactor for the cyclic AMP response element-
1-phosphatase. The formation of F-2,6-P2 is regulated by binding protein (CREB) transcription factor, which can
two separate signalling pathways. AMPK promotes glyco- activate genes responsible for gluconeogenesis (Module 7:
lysis by stimulating the phosphofructokinase (PFK-2) of Figure liver cell signalling). However, AMPK can also reg-
the bifunctional enzyme, which has both kinase and phos- ulate the activity of other cells such as insulin-secreting β1
phatase activities. Cyclic AMP acting through the fructose- cells (Module 7: Figure β-cell signalling) and may func-
2,6-bisphosphate 2-phosphatase component lowers the tion in O2 -sensing by the glomus cells in the carotid
level of F-2,6-P2 , which reduces glycolysis and promotes body (Module 10: Figure carotid body chemorecep-
gluconeogenesis. tion). The fact that AMPK plays such a central role in

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r109

regulating energy metabolism has important implications NAD signalling pathways

for diabetes. As the name implies, the NAD signalling pathways en-
Glycogen storage disease in humans is caused by a muta- compass signalling systems that depend on nicotinamide–
tion of the AMPK γ-subunit. adenine dinucleotide (NAD+ ) and nicotinamide–adenine
dinucleotide phosphate (NADP+ ). These two meta-
bolic cofactors exist in the cell as NAD+ /NADH and
LKB1 NADP+ /NADPH redox couples and have a number of
LKB1 is a serine/threonine protein kinase that functions to important functions both as messengers and as precursors
activate AMP-activated protein kinase (AMPK) by phos- of other metabolic messengers.
phorylating Thr-172 on the α-subunit (Module 2: Figure
AMPK structure). The activation of AMPK is very de-
NAD and NADP as metabolic messengers
pendent upon LKB1. Inactivation of the latter is respons-
• NAD+ functions to regulate a number of cellular pro-
ible for Peutz-Jeghers syndrome. As such, LKB1 is con-
cesses including energy metabolism, gene transcription,
sidered to be one of the tumour suppressors.
DNA repair and perhaps ageing as well.
• NADH activates the C-terminal binding protein
Adenosine triphosphate (ATP) (CtBP), which is a transcriptional corepressor that func-
ATP is an important metabolic messenger in that it has tions during growth and development.
a number of different actions both as an internal and • NADH and NADPH regulate the transcription factors
an external signal (Module 2: Figure metabolic messen- Clock/BMAL1 and NPAS2/BMAL1 that control gene
gers). With regard to the former, one of its main func- expression during the operation of the circadian clock.
tions is to regulate the activity of the ATP-sensitive K+ • NADPH appears to regulate the ADP-ribosyl cy-
(KATP ) channel, which is particularly important in reg- clase that is responsible for producing both cyclic
ulating the release of insulin (Module 7: Figure β-cell ADP-ribose (cADPR) and nicotinic acid–adenine di-
signalling). It also plays a role in regulating the activ- nucleotide phosphate (NAADP) (Module 2: Figure
ity of the ADP-ribosyl cyclase that generates the Ca2+ - cADPR/NAADP function).
mobilizing messengers cyclic ADP-ribose (cADPR) and
nicotinic acid–dinucleotide phosphate (NAADP) (Module NAD and NADP as precursors of metabolic
2: Figure cADPR/NAADP function). ATP is also released messengers
from the cell and can activate P2X receptors that activate • NAD+ is a precursor of cyclic ADP-ribose (cADPR)
Ca2+ entry or P2Y receptors that are coupled to phos- signalling pathway that functions in Ca2+ signalling.
pholipase C (PLC) to generate inositol 1,4,5-trisphosphate • Metabolism of cADPR generates ADPR, which regu-
(InsP3 ) and diacylglycerol (DAG) (Module 2: Figure meta- lates transient receptor potential melastatin 2 (TRPM2),
bolic messengers). a member of the transient receptor potential (TRP) ion
channel family that controls the entry of external Ca2+
(Module 2: Figure cADPR/NAADP function).
Bicarbonate (HCO3 − ) • NADP+ is a precursor of nicotinic acid–adenine dinuc-
The CO2 produced during cellular metabolism is rap- leotide phosphate (NAADP) signalling pathway that
idly converted into bicarbonate (HCO3 − ), which acts as a functions in Ca2+ signalling.
messenger to report the current state of metabolism. The • NADPH is the substrate used to generate reactive oxy-
HCO3 − activates soluble adenylyl cyclase (Module 2: Fig- gen species (ROS), which function as second messen-
ure cyclic AMP signalling) to generate cyclic AMP that has gers to regulate a number of cellular proteins (Module
many signalling functions, including an effect on cellular 2: Figure plasma membrane ROS formation).
metabolism through its activation of glycogen metabolism.

Sterol sensing and cholesterol biosynthesis

Fatty acids The level of cholesterol in cell membranes is regulated by
Fatty acids can be considered as metabolic messen- a signalling system that can sense the level of sterols and
gers because they contribute to a number of meta- then relay information to the nucleus to adjust the tran-
bolic control mechanisms. For example, they con- scription of the genes responsible for cholesterol biosyn-
trol gluconeogenesis in liver cells by activating the thesis. The signalling system is based on membrane-bound
peroxisome-proliferator-activated receptor α (PPARα) transcription factors: the sterol regulatory element-bind-
(Module 7: Figure liver cell signalling). Free fatty acids ing proteins (SREBPs), which are integral membrane pro-
are also responsible for activating the uncoupling pro- teins located in the endoplasmic reticulum (ER). They have
tein 1 (UCP1) that provides the proton leak during two membrane-spanning domains with the free ends pro-
noradrenaline-induced heat production by brown fat cell jecting into the cytosol. The N-terminal region is the lat-
mitochondria (Module 7: Figure brown fat cell). ent transcriptional regulator, which is cleaved by a sterol-
The positive-feedback loop that free fatty acids (FFAs) regulated system of proteases (Module 2: Figure sterol
exert on the process of lipogenesis (i.e. Step 7 in Module sensing). Once released into the cytosol, these transcrip-
7: Figure metabolic energy network) may exacerbate the tion factors dimerize through a basic loop–helix–leucine
onset of obesity by enhancing fat storage. zipper and enter the nucleus, where they bind to sterol

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Cell Signalling Biology Michael J. Berridge r Module 2 r Cell Signalling Pathways 2 r110

Module 2: Figure AMPK structure

β GBD α γ BD
α
I
γ
C

Enzyme activation by
AMP and protein phosphorylation
by an AMPK kinase (LKB1)

β GBD α γ BD
α
I
P
AM γ
+ P
C
LKB1

Protein +
Protein- P AMP

Structure and function of AMP-activated protein kinase (AMPK).

AMP-activated protein kinase (AMPK) is an αβγ heterotrimer. The β-subunit, which contains a C-terminal αγ-binding domain (αγBD), functions as
a scaffold to organize the other two subunits. The α-subunit has an N-terminal catalytic domain (C), which is kept quiescent at rest by binding to
an autoinhibitory domain (I). This α-subunit is activated by both AMP and by an AMPK kinase (AMPKK). The γ-subunit binds AMP and undergoes
a conformation change that is transmitted to the α-subunit, causing the enzyme to open up so that its catalytic site can begin to phosphorylate its
substrate proteins. Enzyme activity is also regulated by an AMPKK known as LKB1, which phosphorylates Thr-172. The β-subunit also contains a
glycogen-binding domain (GBD), which enables AMPK to associate with glycogen that serves to inhibit the enzyme.

Module 2: Figure AMPK control of metabolism

Glucose Glucagon

Glut4 Cyclic AMP

Exocytosis
G-6-P PKA

LKB1 AMP + Gluconeogenesis

+ G-6-P
Glycogen + +
_
+ PFK
- 2
AMPK ase
F-6-P
-P 2
TORC P + + F -2,6
PFK-1
F-2,6-P F-1,6-P2 ase
TSC1/2 2
_ _ + _
TORC _
SREBp1c HNF4α F-1,6-P2
++
TOR
Lipogenic and
glycolytic genes
Pyruvate

Protein
Nucleus synthesis Glycolysis

The pleiotropic action of AMP-activated protein kinase (AMPK) on cell metabolism.

An increase in the level of AMP during metabolic stress activates AMP-activated protein kinase (AMPK), which then has a number of actions, as
outlined in the text.

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Module 2: Figure sterol sensing

Sterol signalling at the endoplasmic reticulum (ER) membrane.

Sterols bind to the sterol regulatory element-binding proteins (SREBP) cleavage-activating protein (SCAP) resulting in the activation of site-1 protease
(S1P), which then cleaves the SREBPs at a point on the luminal loop. A site-2 protease (S2P) then hydrolyses the N-terminal bHLH (basic helix–
loop–helix) fragment to release the cytosolic portion, which then diffuses into the nucleus to initiate gene transcription. Reproduced from Brown, M.S.
and Goldstein, J.L. (1999) A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. Proc. Natl. Acad. Sci. U.S.A.
96:11041–11048. Copyright (1999) National Academy of Sciences, U.S.A.; see Brown and Goldstein 1999).

regulatory elements on the genes that code for the en- Tybulewicz, V.L.J. (2005) Vav-family proteins in T-cell signalling.
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Welch, H.C.E., Coadwell, W.J., Stephens, L.R. and Hawkins, P.T. (2003)
acids and triacylglycerols) and lipid-uptake mechanisms. Phosphoinositide 3-kinase-dependent activation of Rac. FEBS Lett.
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bind to SCAP to inhibit the proteolytic cleavage of the Cyclic AMP signalling pathway
SREBPs. The ER thus plays a central role both in sensing Cooper, D.M. (2003) Regulation and organization of adenylyl cyclases
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Nuclear factor κB (NF-κB) signalling pathway

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Hedgehog signalling pathway

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Sphingomyelin signalling pathway

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Sterol sensing and the regulation of cholesterol

Smad signalling pathway biosynthesis
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Index of cited figures and tables

Figures Page no.

Module 1: Figure cytokines 1.26
Module 1: Figure Eph receptor signalling 1.24
Module 1: Figure homologous desensitization 1.55
Module 1: Figure non-receptor tyrosine kinases 1.39
Module 1: Figure PDGFR activation 1.23
Module 1: Figure signal transmission mechanisms 1.37
Module 1: Figure stimuli for cyclic AMP signalling 1.5
Module 1: Figure stimuli for enzyme-linked receptors 1.20
Module 1: Figure type I cytokine receptors 1.27
Module 1: Figure tyrosine kinase-linked receptors 1.21
Module 1: Figure ubiquitin–proteasome system 1.49
Module 2: Figure adenylyl cyclase structure 2.4
Module 2: Figure AMPK control of metabolism 2.110
Module 2: Figure AMPK structure 2.110
Module 2: Figure basic Ca2+ signalling mechanism 2.17
Module 2: Figure Ca2+ modules 2.24
Module 2: Figure Ca2+ signalling dynamics 2.18
Module 2: Figure Ca2+ signalling toolkit 2.23
Module 2: Figure Ca2+ transient mechanisms 2.26
Module 2: Figure Ca2+ -induced Ca2+ release 2.25
Module 2: Figure cADPR action in heart cells 2.31
Module 2: Figure cADPR action in neurons 2.32
Module 2: Figure cADPR metabolism 2.29
Module 2: Figure cADPR/NAADP function 2.30
Module 2: Figure Cdc42 signalling 2.15
Module 2: Figure cell signalling pathways 2.2
Module 2: Figure cell-specific Ca2+ signalsomes 2.22
Module 2: Figure cyclic AMP signalling 2.5
Module 2: Figure eNOS activation 2.58
Module 2: Figure ER stress signalling 2.106
Module 2: Figure ERK signalling 2.75
Module 2: Figure G protein binary switching 2.8
Module 2: Figure GSH/GSSG couple 2.65
Module 2: Figure H2 O2 metabolism 2.66
Module 2: Figure Hedgehog signalling pathway 2.100
Module 2: Figure heterotrimeric G protein signalling 2.10
Module 2: Figure inositol phosphate metabolism 2.41
Module 2: Figure InsP3 and DAG formation 2.42
Module 2: Figure InsP3 /Ca2+ signalling functions 2.42
Module 2: Figure InsP3 /DAG recycling 2.43
Module 2: Figure insulin receptor 2.53
Module 2: Figure JAK and STAT structure 2.88
Module 2: Figure JAK/STAT function 2.91
Module 2: Figure JAK/STAT heterogeneity 2.90
Module 2: Figure JNK signalling 2.76
Module 2: Figure MAPK signalling 2.75
Module 2: Figure metabolic messengers 2.106
Module 2: Figure metabolic signalling 2.107
Module 2: Figure NAADP structure 2.32
Module 2: Figure NF-κB activation 2.80
Module 2: Figure NF-κB, IκB and IKK structure 2.78
Module 2: Figure NO and cyclic GMP signalling 2.56
Module 2: Figure NO synthase mechanism 2.57
Module 2: Figure Notch modulation 2.104
Module 2: Figure Notch signalling 2.101

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Figures Page no.

Module 2: Figure peroxiredoxin catalytic cycles 2.66

Module 2: Figure phosphoinositide metabolism 2.34
Module 2: Figure phosphoinositide signalling systems 2.36
Module 2: Figure PI3-K family 2.35
Module 2: Figure PKC structure and activation 2.47
Module 2: Figure plasma membrane ROS formation 2.64
Module 2: Figure PLC structure and function 2.44
Module 2: Figure PLD isoforms 2.83
Module 2: Figure PLD signalling 2.83
Module 2: Figure protein kinase A (PKA) 2.6
Module 2: Figure PtdIns 3-kinase signalling 2.51
Module 2: Figure PtdIns structure 2.34
Module 2: Figure PtdIns4,5P2 regulation of K+ channels 2.50
Module 2: Figure PtdIns4,5P2 regulation of TRP channels 2.50
Module 2: Figure PtdIns4,5P2 signalling 2.48
Module 2: Figure pyruvate and Ca2+ signalling 2.108
Module 2: Figure Rac signalling 2.12
Module 2: Figure Ras signalling 2.11
Module 2: Figure reactive oxygen species (ROS) 2.63
Module 2: Figure recovery of protein oxidation 2.69
Module 2: Figure reversible and irreversible ROS oxidations 2.67
Module 2: Figure Rho signalling 2.13
Module 2: Figure Rho-regulated kinases 2.16
Module 2: Figure ROS effects on Ca2+ signalling 2.72
Module 2: Figure ROS formation and action 2.68
Module 2: Figure ROS microdomains 2.74
Module 2: Figure sites of ROS formation 2.64
Module 2: Figure Smad domain structure 2.93
Module 2: Figure Smad signalling 2.95
Module 2: Figure sphingolipid metabolism 2.85
Module 2: Figure sphingomyelin signalling 2.85
Module 2: Figure STAT1/DNA complex 2.89
Module 2: Figure sterol sensing 2.111
Module 2: Figure summary of redox signalling 2.60
Module 2: Figure temporal aspects 2.28
Module 2: Figure TGF-βR activation 2.94
Module 2: Figure thimerosal-induced Ca2+ signalling 2.70
Module 2: Figure Toll receptor signalling 2.80
Module 2: Figure viral recognition 2.82
Module 2: Figure Wnt canonical pathway 2.98
Module 2: Figure Wnt signalling pathways 2.96
Module 2: Figure phosphoinositide metabolism 2.34
Module 2: Figure Rho-regulated kinases 2.16
Module 3: Figure AMPA receptor phosphorylation 3.17
Module 3: Figure Ca2+ entry mechanisms 3.12
Module 3: Figure CaV 1.1 L-type channel 3.6
Module 3: Figure CaV 1.2 L-type channel 3.7
Module 3: Figure CaV 2 channel family 3.9
Module 3: Figure CFTR channel 3.52
Module 3: Figure cyclic nucleotide-gated channels 3.20
Module 3: Figure InsP3 R activation 3.63
Module 3: Figure InsP3 R structure 3.62
Module 3: Figure P2X receptor structure 3.19
Module 3: Figure polycystin domain structure 3.33
Module 3: Figure ryanodine receptor structure 3.59
Module 3: Figure STIM-induced Ca2+ entry 3.26
Module 3: Figure TRP channel phylogeny 3.26
Module 4: Figure actin remodelling 4.54

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Figures Page no.

Module 4: Figure annexin structure 4.5

Module 4: Figure Ca2+ -induced membrane fusion 4.18
Module 4: Figure calcineurin 4.14
Module 4: Figure CREB activation 4.41
Module 4: Figure ETS activation 4.43
Module 4: Figure FOXO control mechanisms 4.42
Module 4: Figure NFAT activation 4.39
Module 4: Figure p53 domains 4.29
Module 4: Figure phagosome maturation 4.20
Module 4: Figure S100 phylogenetic tree 4.7
Module 4: Figure SRF and AP-1 4.46
Module 4: Figure transcription factor activation 4.23
Module 5: Figure Ca2+ uptake and extrusion 5.15
Module 5: Figure ER/mitochondrial shuttle 5.25
Module 5: Figure mitochondrial Ca2+ signalling 5.23
Module 5: Figure mitochondrial motility 5.28
Module 5: Figure phospholamban mode of action 5.21
Module 5: Figure PP2A holoenzyme 5.9
Module 6: Figure caveolae molecular organization 6.15
Module 6: Figure circadian clock molecular mechanism 6.42
Module 6: Figure integrin signalling 6.21
Module 6: Figure IRS domain structure 6.9
Module 6: Figure modular lipid-binding domains 6.5
Module 6: Figure signalling hierarchies 6.2
Module 6: Figure vinculin function 6.22
Module 7: Figure α-cell signalling 7.126
Module 7: Figure β-cell signalling 7.125
Module 7: Figure brown fat cell 7.129
Module 7: Figure collecting duct function 7.103
Module 7: Figure control of food intake 7.6
Module 7: Figure control of pancreatic secretion 7.113
Module 7: Figure endothelial cell contraction 7.22
Module 7: Figure glomerulosa cell signalling 7.120
Module 7: Figure glycogenolysis and gluconeogenesis 7.115
Module 7: Figure HCl secretion 7.91
Module 7: Figure hepatic stellate cell 7.118
Module 7: Figure intestinal Ca2+ reabsorption 7.77
Module 7: Figure intestinal secretion 7.95
Module 7: Figure kidney Ca2+ reabsorption 7.104
Module 7: Figure L cell 7.94
Module 7: Figure lipolysis and lipogenesis 7.128
Module 7: Figure liver cell signalling 7.116
Module 7: Figure mesangial cell 7.106
Module 7: Figure metabolic energy network 7.5
Module 7: Figure osteoclast podosome 7.81
Module 7: Figure skeletal muscle E-C coupling 7.25
Module 7: Figure smooth muscle cell cGMP signalling 7.43
Module 7: Figure smooth muscle cell E-C coupling 7.42
Module 8: Figure bone cell differentiation 8.38
Module 8: Figure brown fat cell differentiation 8.48
Module 8: Figure dorsoventral specification 8.18
Module 8: Figure epidermal stem cell 8.32
Module 8: Figure HSC regulation 8.35
Module 8: Figure mammalian fertilization 8.5
Module 8: Figure osteoclast differentiation 8.41
Module 8: Figure Satellite cell activation 8.29
Module 8: Figure signalsome expression 8.26

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Figures Page no.

Module 8: Figure stem cell function 8.27

Module 8: Figure white fat cell differentiation 8.47
Module 9: Figure angiogenesis signalling 9.37
Module 9: Figure cell cycle signalling mechanisms 9.7
Module 9: Figure cytokinesis 9.15
Module 9: Figure G1 checkpoint signalling 9.18
Module 9: Figure growth factor signalling 9.22
Module 9: Figure immunological synapse structure 9.28
Module 9: Figure T cell signalling map 9.31
Module 9: Figure target of rapamycin signalling 9.24
Module 9: Figure TCR signalling 9.32
Module 9: Figure VEGF-induced proliferation 9.38
Module 10: Figure Ca2+ -induced synaptic plasticity 10.36
Module 10: Figure carotid body chemoreception 10.80
Module 10: Figure gonadotroph regulation 10.55
Module 10: Figure lactotroph regulation 10.57
Module 10: Figure medium spiny neuron signalling 10.39
Module 10: Figure neuronal gene transcription 10.40
Module 10: Figure nociception 10.77
Module 10: Figure phototransduction overview 10.67
Module 10: Figure phototransduction 10.67
Module 10: Figure postsynaptic density 10.11
Module 11: Figure apoptosis 11.20
Module 11: Figure autophagy 11.19
Module 11: Figure FcεRI mast cell signalling 11.9
Module 11: Figure formation and action of PAMPs 11.11
Module 11: Figure inflammation 11.2
Module 11: Figure macrophage signalling 11.12
Module 11: Figure mast cell inhibitory signalling 11.10
Module 11: Figure mast cell signalling 11.7
Module 11: Figure neutrophil chemotactic signalling 11.15
Module 11: Figure neutrophil chemotaxis 11.14
Module 11: Figure platelet activation 11.6
Module 12: Figure amyloid cascade hypothesis 12.6
Module 12: Figure amyloid plaques and tangles 12.10
Module 12: Figure astrocyte-induced neuronal death 12.11
Module 12: Figure colon cancer 12.45
Module 12: Figure hypertrophy signalling mechanisms 12.22
Module 12: Figure polycystins and polycystic kidney disease 12.57

Tables Page no.

Module 1: Table G protein-coupled receptors 1.32

Module 2: Table adenylyl cyclases 2.4
Module 2: Table Ca2+ signalling toolkit 2.19
Module 2: Table Hedgehog signalling toolkit 2.100
Module 2: Table heterotrimeric G proteins 2.9
Module 2: Table MAPK signalling toolkit 2.73
Module 2: Table monomeric G protein toolkit 2.14
Module 2: Table NF-κB signalling toolkit 2.77
Module 2: Table Notch signalling components 2.102
Module 2: Table redox signalling components 2.62
Module 2: Table Smad signalling toolkit 2.92
Module 3: Table ABC transporters 3.51
Module 3: Table properties of Ca2+ -sensitive K+ channels 3.42
Module 3: Table receptor-operated channel toolkit 3.13
Module 3: Table VOC classification 3.2

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Tables Page no.

Module 3: Table voltage-dependent K+ channels 3.37

Module 5: Table Ca2+ pumping toolkit 5.17
Module 5: Table PDE family properties 5.11
Module 5: Table PP1 regulatory, targeting and inhibitory subunits and proteins 5.6

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2009 Portland Press Limited www.cellsignallingbiology.org