8885d_c12_466 2/20/04 1:27 PM Page 466 mac76 mac76:385_reb:

466 Chapter 12 Biosignaling

CH3 12.9 Regulation of the Cell Cycle

N by Protein Kinases
CH3 OH
C C CH3 One of the most dramatic roles for protein phosphory-
lation is the regulation of the eukaryotic cell cycle. Dur-
ing embryonic growth and later development, cell divi-
sion occurs in virtually every tissue. In the adult
O organism most tissues become quiescent. A cell’s “deci-
RU486 sion” to divide or not is of crucial importance to the or-
(mifepristone) ganism. When the regulatory mechanisms that limit cell
division are defective and cells undergo unregulated
Another steroid analog, the drug RU486, is used to ter- division, the result is catastrophic—cancer. Proper cell
minate early (preimplantation) pregnancies. An antag- division requires a precisely ordered sequence of bio-
onist of the hormone progesterone, RU486 binds to the chemical events that assures every daughter cell a full
progesterone receptor and blocks hormone actions es- complement of the molecules required for life. Investi-
sential to implantation of the fertilized ovum in the gations into the control of cell division in diverse eu-
uterus. ■ karyotic cells have revealed universal regulatory mech-
The classic mechanism for steroid hormone action anisms. Protein kinases and protein phosphorylation are
through nuclear receptors does not explain certain ef- central to the timing mechanism that determines entry
fects of steroids that are too fast to be the result of al- into cell division and ensures orderly passage through
tered protein synthesis. For example, the estrogen- these events.
mediated dilation of blood vessels is known to be
independent of gene transcription or protein synthesis, The Cell Cycle Has Four Stages
as is the steroid-induced decrease in cellular [cAMP].
Cell division in eukaryotes occurs in four well-defined
Another transduction mechanism is probably responsi-
stages (Fig. 12–41). In the S (synthesis) phase, the
ble for some of these effects. A plasma membrane pro-
DNA is replicated to produce copies for both daughter
tein predicted to have seven transmembrane helical seg-
ments binds progesterone with very high affinity and
mediates the inhibition of adenylyl cyclase by that hor-
mone, accounting for the decrease in [cAMP]. A second
nonclassical mechanism involves the rapid activation of M Phase
the MAPK cascade by progesterone, acting through the Mitosis (nuclear
soluble progesterone receptor. This is the same recep- G2 Phase division) and G0 Phase
No DNA cytokinesis Terminally
tor that, in the nucleus, causes the much slower changes synthesis. (cell division) differentiated
in gene expression that constitute the classic mecha- RNA and yield two cells withdraw
nism of progesterone action. How the MAPK cascade is protein daughter cells. from cell cycle
synthesis indefinitely.
activated is not yet clear. continue.
G0
M
G2 1h Reentry point
SUMMARY 12.8 Regulation of Transcription 3–4 h A cell returning
from G0
by Steroid Hormones enters at early
G1 phase.
■ Steroid hormones enter cells and bind to G1
specific receptor proteins. S
6–12 h

■ The hormone-receptor complex binds specific 6–8 h G1 Phase

RNA and protein
regions of DNA, the hormone response synthesis. No DNA
elements, and regulates the expression of synthesis.
nearby genes by interacting with transcription S Phase
Restriction point
factors. DNA synthesis
A cell that passes this
doubles the
■ Two other, faster-acting mechanisms produce amount of DNA point is committed
in the cell. RNA to pass into S phase.
some of the effects of steroids. Progesterone
and protein also
triggers a rapid drop in [cAMP], mediated by a synthesized.
plasma membrane receptor, and binding of
progesterone to the classic soluble steroid FIGURE 12–41 Eukaryotic cell cycle. The durations (in hours) of the
receptor activates a MAPK cascade. four stages vary, but those shown are typical.
8885d_c12_467 2/23/04 9:12 AM Page 467 mac76 mac76:

12.9 Regulation of the Cell Cycle by Protein Kinases 467

cells. In the G2 phase (G indicates the gap between Levels of Cyclin-Dependent Protein Kinases Oscillate
divisions), new proteins are synthesized and the cell
approximately doubles in size. In the M phase (mitosis), The timing of the cell cycle is controlled by a family of
the maternal nuclear envelope breaks down, matching protein kinases with activities that change in response
chromosomes are pulled to opposite poles of the cell, to cellular signals. By phosphorylating specific proteins
each set of daughter chromosomes is surrounded by a at precisely timed intervals, these protein kinases or-
newly formed nuclear envelope, and cytokinesis pinches chestrate the metabolic activities of the cell to produce
the cell in half, producing two daughter cells. In em- orderly cell division. The kinases are heterodimers with
bryonic or rapidly proliferating tissue, each daughter a regulatory subunit, cyclin, and a catalytic subunit,
cell divides again, but only after a waiting period (G1). cyclin-dependent protein kinase (CDK). In the ab-
In cultured animal cells the entire process takes about sence of cyclin, the catalytic subunit is virtually inac-
24 hours. tive. When cyclin binds, the catalytic site opens up, a
After passing through mitosis and into G1, a cell ei- residue essential to catalysis becomes accessible (Fig.
ther continues through another division or ceases to di- 12–42), and the activity of the catalytic subunit in-
vide, entering a quiescent phase (G0) that may last creases 10,000-fold. Animal cells have at least ten dif-
hours, days, or the lifetime of the cell. When a cell in ferent cyclins (designated A, B, and so forth) and at
G0 begins to divide again, it reenters the division cycle least eight cyclin-dependent kinases (CDK1 through
through the G1 phase. Differentiated cells such as he- CDK8), which act in various combinations at specific
patocytes or adipocytes have acquired their specialized points in the cell cycle. Plants also use a family of CDKs
function and form; they remain in the G0 phase. to regulate their cell division.

FIGURE 12–42 Activation of cyclin-dependent protein kinases (CDKs) by cyclin

and phosphorylation. CDKs, a family of related enzymes, are active only when
associated with cyclins, another protein family. The crystal structure of CDK2 with
and without cyclin reveals the basis for this activation. (a) Without cyclin (PDB
ID 1HCK), CDK2 folds so that one segment, the T loop (red), obstructs the
binding site for protein substrates and thus inhibits protein kinase activity. The
binding site for ATP (blue) is also near the T loop. (b) When cyclin binds (PDB ID
1FIN), it forces conformational changes that move the T loop away from the
active site and reorient an amino-terminal helix (green), bringing a residue critical
to catalysis (Glu51) into the active site. (c) Phosphorylation of a Thr residue (dark
orange space-filling structure) in the T loop produces a negatively charged residue
that is stabilized by interaction with three Arg residues (red ball-and-stick struc-
(a)
tures), holding CDK in its active conformation (PDB ID 1JST).

(b) (c)
8885d_c12_468 2/20/04 1:28 PM Page 468 mac76 mac76:385_reb:

468 Chapter 12 Biosignaling

G1 S G2 M G1 In a population of animal cells undergoing synchro-

nous division, some CDK activities show striking oscil-
Cyclin B–CDK1 lations (Fig. 12–43). These oscillations are the result of
four mechanisms for regulating CDK activity: phosphor-
Kinase activity

ylation or dephosphorylation of the CDK, controlled

Cyclin A–CDK2
degradation of the cyclin subunit, periodic synthesis of
Cyclin E–CDK2
CDKs and cyclins, and the action of specific CDK-
inhibiting proteins.

Regulation of CDKs by Phosphorylation The activity of a

CDK is strikingly affected by phosphorylation and de-
Time phosphorylation of two critical residues in the protein
(Fig. 12–44a). Phosphorylation of Tyr15 near the amino
FIGURE 12–43 Variations in the activities of specific CDKs during
the cell cycle in animals. Cyclin E–CDK2 activity peaks near the G1
terminus renders CDK2 inactive; the P –Tyr residue is
phase–S phase boundary, when the active enzyme triggers synthesis
in the ATP-binding site of the kinase, and the negatively
of enzymes required for DNA synthesis (see Fig. 12–46). Cyclin charged phosphate group blocks the entry of ATP. A
A–CDK2 activity rises during the S and G2 phases, then drops sharply specific phosphatase dephosphorylates this P –Tyr
in the M phase, as cyclin B–CDK1 peaks. residue, permitting the binding of ATP. Phosphorylation

(a)
3
Cyclin-CDK complex forms, but phosphorylation
2 on Tyr 15 blocks ATP-binding site; still inactive.
Cyclin
synthesis P Tyr 4
leads to its CDK Phosphorylation of Thr160 in
accumulation. T loop and removal of Tyr15
Cyclin phosphoryl group activates
cyclin-CDK manyfold.
Cyclin
P Tyr Thr P
5
CDK phosphorylates
phosphatase, which
1 activates more CDK.
No cyclin
present; Pi
Phosphatase Phosphatase
CDK is
inactive. P P
Thr
CDK CDK

DBRP DBRP
P 7
6
CDK phosphorylates DBRP triggers
DBRP, activating it. addition of ubiquitin
molecules to cyclin
by ubiquitin ligase.
8
Cyclin is
degraded CDK U
by proteasome,
Cyclin
leaving CDK
inactive. U U U U

(b)

FIGURE 12–44 Regulation of CDK by phosphorylation and prote- manyfold. (b) The active cyclin-CDK complex triggers its own inac-
olysis. (a) The cyclin-dependent protein kinase activated at the time tivation by phosphorylation of DBRP (destruction box recognizing
of mitosis (the M phase CDK) has a “T loop” that can fold into the protein). DBRP and ubiquitin ligase then attach several molecules of
substrate-binding site. When Thr160 in the T loop is phosphorylated, ubiquitin (U) to cyclin, targeting it for destruction by proteasomes,
the loop moves out of the substrate-binding site, activating the CDK proteolytic enzyme complexes.
8885d_c12_469 2/20/04 1:28 PM Page 469 mac76 mac76:385_reb:

12.9 Regulation of the Cell Cycle by Protein Kinases 469

of Thr160 in the “T loop” of CDK, catalyzed by the CDK- Growth factors,

cytokines
activating kinase, forces the T loop out of the substrate-
binding cleft, permitting substrate binding and catalytic MAPK
activity. cascade
One circumstance that triggers this control mecha-
nism is the presence of single-strand breaks in DNA, Phosphorylation of
which leads to arrest of the cell cycle in G2. A specific Jun and Fos in nucleus
protein kinase (called Rad3 in yeast), which is activated
by single-strand breaks, triggers a cascade leading to the
inactivation of the phosphatase that dephosphorylates transcriptional
Tyr15 of CDK. The CDK remains inactive and the cell is regulation
arrested in G2. The cell will not divide until the DNA is
Cyclins, Transcription
repaired and the effects of the cascade are reversed. CDKs factor E2F
Controlled Degradation of Cyclin Highly specific and pre- transcriptional
cisely timed proteolytic breakdown of mitotic cyclins regulation

regulates CDK activity throughout the cell cycle. Enzymes for

Progress through mitosis requires first the activation DNA synthesis
then the destruction of cyclins A and B, which activate
the catalytic subunit of the M-phase CDK. These cyclins
contain near their amino terminus the sequence
Arg–Thr–Ala–Leu–Gly–Asp–Ile–Gly–Asn, the “destruc-
tion box,” which targets them for degradation. (This us- Passage from
G1 to S phase
age of “box” derives from the common practice, in dia-
gramming the sequence of a nucleic acid or protein, of FIGURE 12–45 Regulation of cell division by growth factors. The
enclosing within a box a short sequence of nucleotide path from growth factors to cell division leads through the enzyme
or amino acid residues with some specific function. It cascade that activates MAPK; phosphorylation of the nuclear tran-
does not imply any three-dimensional structure.) The scription factors Jun and Fos; and the activity of the transcription fac-
protein DBRP (destruction box recognizing protein) tor E2F, which promotes synthesis of several enzymes essential for
recognizes this sequence and initiates the process of cy- DNA synthesis.
clin degradation by bringing together the cyclin and an-
other protein, ubiquitin. Cyclin and activated ubiqui-
tin are covalently joined by the enzyme ubiquitin ligase the nucleus to activate transcription of their genes. Syn-
(Fig. 12–44b). Several more ubiquitin molecules are thesis of E2F is in turn regulated by extracellular sig-
then appended, providing the signal for a proteolytic en- nals such as growth factors and cytokines (inducers
zyme complex, or proteasome, to degrade cyclin. of cell division), compounds found to be essential for
What controls the timing of cyclin breakdown? A the division of mammalian cells in culture. These growth
feedback loop occurs in the overall process shown in factors induce the synthesis of specific nuclear tran-
Figure 12–44. Increased CDK activity activates cyclin scription factors essential to the production of the
proteolysis. Newly synthesized cyclin associates with enzymes of DNA synthesis. Growth factors trigger phos-
and activates CDK, which phosphorylates and activates phorylation of the nuclear proteins Jun and Fos, tran-
DBRP. Active DBRP then causes proteolysis of cyclin. scription factors that promote the synthesis of a variety
Lowered [cyclin] causes a decline in CDK activity, and of gene products, including cyclins, CDKs, and E2F. In
the activity of DBRP also drops through slow, constant turn, E2F controls production of several enzymes es-
dephosphorylation and inactivation by a DBRP phos- sential for the synthesis of deoxynucleotides and DNA,
phatase. The cyclin level is ultimately restored by syn- enabling cells to enter the S phase (Fig. 12–45).
thesis of new cyclin molecules.
Inhibition of CDKs Finally, specific protein inhibitors
The role of ubiquitin and proteasomes is not limited
bind to and inactivate specific CDKs. One such protein
to the regulation of cyclin; as we shall see in Chapter 27,
is p21, which we discuss below.
both also take part in the turnover of cellular proteins,
a process fundamental to cellular housekeeping.
These four control mechanisms modulate the ac-
Regulated Synthesis of CDKs and Cyclins The third mech- tivity of specific CDKs that, in turn, control whether a
anism for changing CDK activity is regulation of the rate cell will divide, differentiate, become permanently qui-
of synthesis of cyclin or CDK or both. For example, cy- escent, or begin a new cycle of division after a period
clin D, cyclin E, CDK2, and CDK4 are synthesized only of quiescence. The details of cell cycle regulation, such
when a specific transcription factor, E2F, is present in as the number of different cyclins and kinases and the
8885d_c12_470 2/20/04 1:28 PM Page 470 mac76 mac76:385_reb:

470 Chapter 12 Biosignaling

combinations in which they act, differ from species to

DNA
species, but the basic mechanism has been conserved damage
in the evolution of all eukaryotic cells.
Active p53
CDKs Regulate Cell Division by Phosphorylating transcriptional
regulation
Critical Proteins ↑[p21]
p21
We have examined how cells maintain close control of Active
CDK activity, but how does the activity of CDK control
the cell cycle? The list of target proteins that CDKs are
CDK2 CDK2
known to act upon continues to grow, and much remains p21
to be learned. But we can see a general pattern behind Cyclin E Cyclin E
CDK regulation by inspecting the effect of CDKs on the
Inactive P
structures of laminin and myosin and on the activity of
pRb pRb
retinoblastoma protein.
The structure of the nuclear envelope is maintained transcriptional
pRb
in part by highly organized meshworks of intermediate regulation

filaments composed of the protein laminin. Breakdown E2F E2F Enzymes

for DNA
of the nuclear envelope before segregation of the sister Inactive synthesis
chromatids in mitosis is partly due to the phosphoryla- Active
tion of laminin by a CDK, which causes laminin filaments
Passage
to depolymerize. from
A second kinase target is the ATP-driven actin- G1 to S
myosin contractile machinery that pinches a dividing Cell division Cell division
cell into two equal parts during cytokinesis. After the blocked by p53 occurs normally
division, CDK phosphorylates a small regulatory subunit
of myosin, causing dissociation of myosin from actin fil- FIGURE 12–46 Regulation of passage from G1 to S by phosphory-
aments and inactivating the contractile machinery. Sub- lation of pRb. When the retinoblastoma protein, pRb, is phosphory-
sequent dephosphorylation allows reassembly of the lated, it cannot bind and inactivate EF2, a transcription factor that pro-
motes synthesis of enzymes essential to DNA synthesis. If the
contractile apparatus for the next round of cytokinesis.
regulatory protein p53 is activated by ATM and ATR, protein kinases
A third and very important CDK substrate is the
that detect damaged DNA, it stimulates the synthesis of p21, which
retinoblastoma protein, pRb; when DNA damage is
can bind to and inhibit cyclin E–CDK2 and thus prevent phosphory-
detected, this protein participates in a mechanism that
lation of pRb. Unphosphorylated pRb binds and inactivates E2F, block-
arrests cell division in G1 (Fig. 12–46). Named for the
ing passage from G1 to S until the DNA has been repaired.
retinal tumor cell line in which it was discovered, pRb
functions in most, perhaps all, cell types to regulate cell
division in response to a variety of stimuli. Unphosphor- phase, thereby avoiding the potentially disastrous trans-
ylated pRb binds the transcription factor E2F; while fer of a defective genome to one or both daughter cells.
bound to pRb, E2F cannot promote transcription of a
group of genes necessary for DNA synthesis (the genes SUMMARY 12.9 Regulation of the Cell Cycle
for DNA polymerase
, ribonucleotide reductase, and
other proteins; Chapter 25). In this state, the cell cycle by Protein Kinases
cannot proceed from the G1 to the S phase, the step
■ Progression through the cell cycle is regulated
that commits a cell to mitosis and cell division. The pRb-
E2F blocking mechanism is relieved when pRb is phos- by the cyclin-dependent protein kinases
phorylated by cyclin E–CDK2, which occurs in response (CDKs), which act at specific points in the
to a signal for cell division to proceed. cycle, phosphorylating key proteins and
When the protein kinases ATM and ATR detect dam- modulating their activities. The catalytic
age to DNA, such as a single-strand break, they activate subunit of CDKs is inactive unless associated
p53 to serve as a transcription factor that stimulates the with the regulatory cyclin subunit.
synthesis of the protein p21 (Fig. 12–46). This protein ■ The activity of a cyclin-CDK complex changes
inhibits the protein kinase activity of cyclin E–CDK2. In during the cell cycle through differential
the presence of p21, pRb remains unphosphorylated and synthesis of CDKs, specific degradation of
bound to E2F, blocking the activity of this transcription cyclin, phosphorylation and dephosphorylation
factor, and the cell cycle is arrested in G1. This gives of critical residues in CDKs, and binding of
the cell time to repair its DNA before entering the S inhibitory proteins to specific cyclin-CDKs.