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 Contents

Contributors to Volume 85 ix

The Von Hippel–Lindau Tumor Suppressor Complex and

Regulation of Hypoxia-Inducible Transcription
Ronald C. Conaway and Joan W. Conaway
I. The VHL Protein Regulates Transcription of Hypoxia-Inducible Genes 2
II. The VHL Tumor Suppressor Protein Is a Subunit of an E3 Ubiquitin Ligase That
Targets HIFα Subunits 3
III. Prolyl Hydroxylase: The Oxygen Sensor in Hypoxic Signaling? 6
IV. Conclusion 7
References 8

Retinoblastoma Tumor Suppressor and Genome Stability

Lei Zheng and Wen-Hwa Lee
I. Introduction 14
II. Rb and Chromosome Segregation 16
III. Rb and Chromosome Replication 26
IV. Rb and DNA Damage Response 28
V. Global Connections of Rb and Chromatin 31
VI. Haploinsufficiency of Rb in Maintaining Genome Stability 36
VII. Perspectives 38
References 40

The Abl Family Kinases: Mechanisms of Regulation

and Signaling
Ann Marie Pendergast
I. Introduction 51
II. The Abl Tyrosine Kinases: Structure and Conservation 52
III. Expression Patterns and Subcellular Localization 57
IV. Regulation of Abl Tyrosine Kinase Activity 59

v
vi Contents

V. Role of Abl Kinases in the Regulation of Cell Growth and Survival 74

VI. Role of Abl Kinases in Cellular Stress Responses 77
VII. Role of Abl Kinases in the Regulation of Cytoskeletal Dynamics 80
VIII. What Are the Substrates of Abl Kinases in Normal Cells? 88
IX. Conclusions and Future Directions 89
References 91

Cellular Immunity to the Her-2/neu Protooncogene

Rolf. Kiessling, W. Z. Wei, F. Herrmann, J. A. Lindencrona, A. Choudhury,
K. Kono, and B. Seliger
I. Introduction 102
II. The HER Receptor Family and Its Ligands 103
III. HER-2 and Cellular Transformation 105
IV. HER-2 Overexpression in Tumors of Different Histology 106
V. Validity of Methods Employed for the Assessment of HER-2/neu Status 108
VI. HER-2 as a Shared Tumor-Associated Antigen 109
VII. New Treatment Modalities for HER-2-Overexpressing Tumor Cells 114
VIII. HER-2/neu Expression and Immune Escape 121
IX. Clinical Trials Based on HER-2-Specific Tumor Vaccines 126
X. Concluding Remarks and Future Directions 131
References 133

A New Challenge for Successful Immunotherapy by Tumors

That Are Resistant to Apoptosis: Two Complementary Signals
to Overcome Cross-Resistance
Chuen-Pei Ng and Benjamin Bonavida
I.Introduction 146
II.Apoptosis as Cytotoxic Mechanisms of T Lymphocytes 149
III.The Road Map of Apoptosis: All Roads Lead to Caspases 150
IV. Cross-Talking between the Two Apoptotic Pathways and Cross-Resistance 153
V. Inhibition of Apoptosis as a Mechanism of Cross-Resistance 154
VI. Sensitization of Resistant Tumor Cells to Cytotoxic Lymphocytes/
Factors-Mediated Apoptosis 156
VII. Conclusions 164
References 167

Cell Volume and Ion Changes during Apoptotic Cell Death

Mireia Gómez-Angelats and John A. Cidlowski
I. The Universality of Apoptotic Volume Decrease 176
II. The Cell Volume “Constancy” 177
III. The Role of Cell Death-Induced Ion Movements across the Plasma Membrane 179
IV. Signal Transduction Mechanisms and Apoptotic Volume Decrease 191
Contents vii

V. Concluding Remarks 194

References 194

Mitochondria and Apoptosis: New Therapeutic Targets

David M. Hockenbery, Christopher D. Giedt, Jason W. O’Neill,
Michael K. Manion, and Deborah E. Banker
I. Introduction 203
II. Review of Metabolic Changes in Cancer Cells 204
III. Targeting Abnormal Metabolism in Cancer Cells 206
IV. Selective Distribution of Compounds in Cancer Mitochondria 207
V. Mitochondrial Pathways in Apoptosis 209
VI. Permeability Transition Pores in Apoptosis 210
VII. Apoptotic Regulators in Mitochondria 220
VIII. Conclusions 230
References 230

Index 243
This Page Intentionally Left Blank
 Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Deborah E. Banker, Divisions of Clinical Research and Human Biology, Fred

Hutchinson Cancer Research Center, Seattle, Washington 98109 (203)
Benjamin Bonavida, Department of Microbiology, Immunology, and Mole-
cular Genetics, Jonsson Comprehensive Cancer Center, UCLA School of
Medicine, Los Angeles, California 90095 (145)
A. Choudhury, Department of Oncology, Immune and Gene Therapy Labo-
ratory, Karolinska Institutet, CCK, 17176 Stockholm, Sweden (101)
John A. Cidlowski, Laboratory of Signal Transduction, National Institute
of Enviromental Health Sciences, National Institutes of Health, Research
Triangle Park, North Carolina 27709 (175)
Joan W. Conaway, The Stowers Institute for Medical Research, Kansas City,
Missouri 64110; Department of Biochemistry and Molecular Biology, Uni-
versity of Kansas Medical Center, Kansas City, Kansas 66160; Department
of Biochemistry and Molecular Biology, University of Oklahoma Health
Sciences Center, Oklahoma City, Oklahoma 73190 (1)
Ronald C. Conaway, The Stowers Institute for Medical Research, Kansas
City, Missouri 64110; Department of Biochemistry and Molecular Biology,
University of Kansas Medical Center, Kansas City, Kansas 66160 (1)
Christopher D. Giedt, Divisions of Clinical Research and Human Biology,
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
(203)
Mireia Gómez-Angelats, Laboratory of Signal Transduction, National
Institute of Enviromental Health Sciences, National Institutes of Health,
Research Triangle Park, North Carolina 27709 (175)
F. Herrmann, Department of Internal Medicine, The Johannes Gutenberg-
University, III. 55101 Mainz, Germany (101)
David M. Hockenbery, Divisions of Clinical Research and Human Biol-
ogy, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
(203)
Rolf Kiessling, Department of Oncology, Immune and Gene Therapy Labo-
ratory, Karolinska Institutet, CCK, 17176 Stockholm, Sweden (101)

ix
x Contributors

K. Kono, Department of Surgery, Yamanashi Medical University, Yamanashi-

Ken, 409-38, Japan (101)
Wen-Hwa Lee, Department of Molecular Medicine/Institute of Biotech-
nology, University of Texas Health Science Center at San Antonio, San
Antonio, Texas 78245 (13)
J. A. Lindencrona, Department of Oncology, Immune and Gene Therapy
Laboratory, Karolinska Institutet, CCK, 17176 Stockholm, Sweden (101)
Michael K. Manion, Divisions of Clinical Research and Human Biology, Fred
Hutchinson Cancer Research Center, Seattle, Washington 98109 (203)
Chuen-Pei Ng, Department of Microbiology, Immunology, and Molecu-
lar Genetics, Jonsson Comprehensive Cancer Center, UCLA School of
Medicine, Los Angeles, California 90095 (145)
Jason W. O’Neill, Divisions of Clinical Research and Human Biology, Fred
Hutchinson Cancer Research Center, Seattle, Washington 98109 (203)
Ann Marie Pendergast, Department of Pharmacology and Cancer Biology,
Duke University Medical Center, Durham, North Carolina 27710 (51)
B. Seliger, Department of Internal Medicine, The Johannes Gutenberg-
University, III. 55101 Mainz, Germany (101)
W. Z. Wei, Karmanos Cancer Institute, Wayne State University, Detroit,
Michigan 48201 (101)
Lei Zheng, Department of Molecular Medicine/Institute of Biotechnology,
University of Texas Health Science Center at San Antonio, San Antonio,
Texas 78245 (13)∗

∗ Current affiliation: Laboratory of Biochemistry and Molecular Biology, The Rockefeller

University, New York, New York 10021

 The Von Hippel–Lindau Tumor
Suppressor Complex and Regulation
of Hypoxia-Inducible Transcription
1,2 1,2,3,
Ronald C. Conaway and Joan W. Conaway *
1
The Stowers Institute for Medical Research, Kansas City, Missouri 64110
2
Department of Biochemistry and Molecular Biology, University of Kansas
Medical Center, Kansas City, Kansas 66160
3
Department of Biochemistry and Molecular Biology, University of Oklahoma
Health Sciences Center, Oklahoma City, Oklahoma 73190

I. The VHL Protein Regulates Transcription of Hypoxia-Inducible Genes

II. The VHL Tumor Suppressor Protein Is a Subunit of an E3 Ubiquitin Ligase
That Targets HIFα Subunits
A. The Elongin BC Complex
B. Cul2
C. Rbx1
D. The VHL Protein Is the Substrate Recognition Subunit of the
VHL Ubiquitin Ligase
III. Prolyl Hydroxylase: The Oxygen Sensor in Hypoxic Signaling?
IV. Conclusion
References

The von Hippel–Lindau (VHL) tumor suppressor gene on chromosome

3p25.5 was isolated in 1993 by a combined genetic analysis of VHL disease
kindreds and positional cloning (Latif et al., 1993). VHL disease is an autoso-
mal dominant familial cancer syndrome that predisposes affected individuals
to a variety of highly vascularized tumors, including clear cell renal carci-
nomas, cerebellar hemangiomas and hemangioblastomas, retinal angiomas,
and pheochromocytomas. Notably, the VHL gene is also mutated in the ma-
jority of sporadic clear cell renal carcinomas (Gnarra et al., 1994) and in
sporadic hemangioblastomas (Kanno et al., 1994).
Initial investigations revealed that the VHL gene is expressed in most tis-
sues and cell types and, further, that the human VHL gene encodes two
functionally indistinguishable isoforms, a full-length 213-amino protein and
a 160-amino acid protein generated by translation initiation at an internal
ATG (Iliopoulos et al., 1998; Schoenfeld et al., 1998). Although few clues
∗ Corresponding author: Tel: 816-926-4091; Fax: 816-926-2091; e-mail: jlc@stowers-

institute.org

Advances in CANCER RESEARCH Copyright 2002, Elsevier Science (USA).

0065-230X/02 $35.00 1 All rights reserved.
2 Conaway and Conaway

to the function of the VHL protein were provided by direct inspection of its
amino acid sequence, over the past few years findings from biochemical and
molecular genetic studies have shed considerable light on the VHL protein
and implicated it in ubiquitin-dependent proteolysis. Below, we discuss these
recent findings, which are providing critical insights into the role of the VHL
protein in normal cell function and in disease.

I. THE VHL PROTEIN REGULATES TRANSCRIPTION

OF HYPOXIA-INDUCIBLE GENES

Although it is presently not known exactly why loss of the VHL gene re-
sults in tumorigenesis, one of the first clues to the function of the VHL protein
came with the discovery that clear cell renal carcinoma cells lacking a func-
tional VHL gene misregulate and constitutively express hypoxia-inducible
genes like glucose transporter-1 (GLUT1), vascular endothelial growth fac-
tor (VEGF), and transforming growth factor α (TGFα) (Gnarra et al., 1996;
Iliopoulos et al., 1996; Siemeister et al., 1996; Knebelmann et al., 1998;
de Paulsen et al., 2001). Evidence implicating the VHL protein in regula-
tion of transcription of hypoxia-inducible genes (Maxwell et al., 1999) was
subsequently obtained in studies of VHL (−/−) clear cell renal carcinoma
cells.
The expression of hypoxia-inducible genes is repressed in normal cells
grown in a plentiful supply of oxygen, but is strongly induced in cells de-
prived of oxygen. The transcription of hypoxia-inducible genes is controlled
by hypoxia-inducible transcription factors (HIFs). HIFs activate transcrip-
tion of hypoxia-inducible genes in response to decreases in cellular oxygen
levels through interactions with hypoxia response elements (HREs) in their
promoter-regulatory regions. HIFs are heterodimers composed of one of the
basic helix-loop-helix (bHLH) PAS (PER-ARNT-SIM) domain HIFα pro-
teins and the aryl hydrocarbon receptor nuclear translocator ARNT (Wang
et al., 1995; Li et al., 1996; Semenza, 2000b).
HIFs are regulated by oxygen-dependent ubiquitylation and proteasomal
degradation of their HIFα subunits (Huang et al., 1996; Salceda and Caro,
1997; Huang et al., 1998). Whereas the ARNT subunit of HIFs is present
in cells at constitutively high levels, the HIFα subunits are continuously
synthesized but rapidly ubiquitylated and destroyed by the proteasome in
normal cells grown in a plentiful supply of oxygen. In contrast, in normal
cells deprived of oxygen, ubiquitylation of HIFα subunits is attenuated, and
they are free to dimerize with ARNT, enter the nucleus, and activate their
hypoxic transcriptional program. HIF1α subunits include two short, con-
served C-terminal regions referred to as the oxygen-dependent degradation
Von Hippel–Lindau Tumor Suppressor Complex 3

domains (ODDs), which are essential for their oxygen-dependent ubiquity-

lation and destruction and which are independently capable of promoting
oxygen-dependent degradation of heterologous ODD-containing fusion pro-
teins (Huang et al., 1998; Ohh et al., 2000; Cockman et al., 2000; Tanimoto
et al., 2000; Masson et al., 2001).
Evidence that the VHL protein regulates expression of hypoxia-inducible
genes at least in part through regulation of HIFα ubiquitylation came from
the work of Ratcliffe, Maxwell, and coworkers (Maxwell et al., 1999), who
observed that HIFα protein levels are substantially elevated in clear cell renal
carcinoma cells lacking a functional VHL gene because of a defect in their
ubiquitylation. The increased HIFα protein levels in VHL (−/−) cells was
found to correlate well with the observed increases in expression of hypoxia-
inducible genes, and reintroduction of a wild-type VHL gene into these cells
was sufficient to restore their proper oxygen-dependent ubiquitylation and
destruction of HIFα, as well as to restore proper regulation of expression of
hypoxia-inducible genes.

II. THE VHL TUMOR SUPPRESSOR PROTEIN

IS A SUBUNIT OF AN E3 UBIQUITIN LIGASE
THAT TARGETS HIFα SUBUNITS

Although the finding that VHL (−/−) renal carcinoma cells fail to ubiqui-
tylate HIFα subunits suggested that the VHL protein functions at some stage
upstream of HIFα in hypoxic signaling, a variety of evidence from biochem-
ical studies provided the first clues to the mechanism of VHL action in this
process, by demonstrating that the VHL protein is the substrate recognition
subunit of a multiprotein E3 ubiquitin ligase with remarkable structural sim-
ilarity to SCF ubiquitin ligases (Patton et al., 1998; Deshaies, 1999). Initial
efforts to identify VHL-interacting proteins led to the discovery that the VHL
protein associates stably in vitro and in cells with the heterodimeric Elongin
BC complex (Duan et al., 1995; Kibel et al., 1995; Kishida et al., 1995) and
with Cullin family member Cul2 (Pause et al., 1997, 1999; Lonergan et al.,
1998). Purification of an endogenous VHL-containing complex identified an
additional VHL-associated protein, Rbx1 (Kamura et al., 1999).

A. The Elongin BC Complex

The Elongin BC complex, which was first identified through its interac-
tion with RNA polymerase II elongation factor Elongin A (Aso et al.,
1995), is composed of the 118-amino acid Elongin B and 112-amino acid
4 Conaway and Conaway

Elongin C proteins. Elongin B is composed of an 84-amino acid N-terminal

ubiquitin-like domain and a 34-amino acid, proline- and leucine-rich
C-terminal tail. Elongin C shares significant sequence similarity with the
SCF ubiquitin ligase subunit Skp1. The Elongin BC complex was found to
interact with the VHL and Elongin A proteins through an approximately
12-amino acid sequence motif referred to as the BC-box, with consensus
[(A,P,S,T)LXXXCXXX(A,I,L,V)] (Kibel et al., 1995; Aso et al., 1996; Ohh
et al., 1999). Solution of the crystal structure of a VHL-Elongin BC com-
plex by Pavletich and coworkers (Stebbins et al., 1999) revealed that binding
of Elongin BC to the BC-box is governed by interaction of the highly con-
served leucine at position 2 in the N-terminus of the BC-box motif with a
hydrophobic pocket created by residues in the C-terminal half of Elongin C.
Elongin B binds to a short N-terminal Elongin C region and does not appear
to interact directly with the BC-box. The importance of the VHL–Elongin
BC interaction in both VHL tumor suppressor function and VHL regulation
of hypoxia-inducible genes is highlighted by the observation that a fraction
of VHL mutations found in VHL disease and in sporadic clear cell renal
carcinomas disrupt the VHL–Elongin BC interaction and result in defects in
HIFα ubiquitylation (Ohh et al., 2000; Cockman et al., 2000).

B. Cul2

Cul2 is a member of the Cullin protein family, which includes the SCF
ubiquitin ligase subunit Cul1 (also referred to as Cdc53 in S. cerevisiae). To
date, the mammalian Cullin protein family includes at least seven members
designated Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5 (Kipreos et al., 1996),
and the cyclosome subunit APC2 (Yu et al., 1998). In addition to the well-
characterized role of Cul1/Cdc53 as an integral subunit of SCF ubiquitin
ligases (Deshaies, 1999), biochemical and genetic evidence suggests that
Cullins play a wide role in cellular regulation in such diverse processes as
cell cycle regulation, signal transduction, and transcriptional regulation. The
founding Cullin family member Cul5 (also referred to as the vasopressin-
activated, calcium-mobilizing-1 [VACM-1] protein) was initially identified
as a cytoplasmic arginine vasopressin receptor (Burnatowska-Hledin et al.,
1995). Although VACM-1 has been shown to function in signal transduc-
tion through its abilities to mobilize calcium, to stimulate production of
D-myo-inositol 1,4,5-trisphosphate, and to inhibit production of cAMP
(Burnatowska-Hledin et al., 2000), its mechanism of action in these pro-
cesses is not known. Mutations of the C. elegans Cul1 and Cul2 genes result
in cell cycle defects. C. elegans Cul1 mutants exhibit hyperplasia of many cell
types, suggesting that it is required for transition of C. elegans cells from G1
to G0 or from G1 to the apoptotic pathway during development (Kipreos
Von Hippel–Lindau Tumor Suppressor Complex 5

et al., 1996). C. elegans Cul2 is required for normal mitotic chromosome

condensation and for transition of proliferating C. elegans cells from G1 to S
(Feng et al., 1999). Mice lacking either the Cul1 or Cul3 genes exhibit early
embryonic cell cycle defects correlating with misregulation of the cellular lev-
els of cyclin E (Singer et al., 1999; Dealy et al., 1999), and a Cul1-containing
ubiquitin ligase containing the F-box protein Fbw7 has been shown to target
cyclin E (Koepp et al., 2001). In addition, as discussed in more detail below,
mammalian Cul2 functions as a component of the VHL ubiquitin ligase in
regulation of hypoxia-inducible transcription.

C. Rbx1

Direct biochemical purification of the endogenous VHL protein from rat

liver revealed that as much as 90% of the VHL protein in cells is present
in a multiprotein complex containing, in addition to Elongin BC and Cul2,
a novel RING finger protein designated Rbx1 (Kamura et al., 1999) [also
referred to as ROC1 (Tan et al., 1999; Ohta et al., 1999) or Hrt1 (Seol et al.,
1999; Blondel et al., 2000)]. The discovery that Rbx1 is not only a subunit
of the VHL complex, but also an essential subunit of SCF ubiquitin ligases
(Kamura et al., 1999; Tan et al., 1999; Ohta et al., 1999; Seol et al., 1999;
Skowyra et al., 1999), further strengthened the striking structural similarity
between the VHL complex and SCF ubiquitin ligases.
Dissection of the mechanism of action of SCF ubquitin ligases has shown
that the Cul1 and Rbx1 proteins interact to form a Cul1/ Rbx1 module
that plays an essential role in ubiquitylation of target proteins by recruiting
and activating the E2 ubiquitin conjugating enzyme Cdc34 (Deshaies et al.,
1999; Seol et al., 1999; Skowyra et al., 1999). To date, all known Cullin
family members have been shown to be capable of assembling with Rbx1 to
reconstitute Cullin / Rbx1 modules that can activate E2 ubiquitin conjugating
enzymes, suggesting that all Cullin proteins will function at least in part as
subunits of ubiquitin ligases (Deshaies et al., 1999; Tan et al., 1999; Ohta
et al., 1999).

D. The VHL Protein Is the Substrate Recognition

Subunit of the VHL Ubiquitin Ligase

Together with the observation that VHL (−/−) clear cell renal carcinoma
cells do not properly ubiquitylate and destroy HIFα subunits under normoxic
conditions, recognition of the striking structural similarities between the
VHL complex and SCF ubiquitin ligases made it tempting to speculate that
the VHL complex might function as a ubiquitin ligase (Lisztwan et al., 1999;
6 Conaway and Conaway

Iwai et al., 1999) that targets HIFα subunits. Indeed, reconstitution and
characterization of the 5-subunit VHL complex revealed that it interacts
specifically with and promotes ubiquitylation of HIFα in vitro by the E2
ubiquitin conjugating enzyme hUbc5a (Kamura et al., 2000).
In addition to Cul1/Cdc53, Rbx1, and the Elongin C-like protein Skp1,
SCF ubiquitin ligases contain one of a large number of F-box proteins that
are responsible for recognizing, binding specifically to, and recruiting target
proteins to the SCF for ubiquitylation (Patton et al., 1998; Deshaies et al.,
1999; Bai et al., 1996). Supporting a similar role for the VHL protein in
function of the VHL ubiquitin ligase, VHL is capable of binding directly to
HIFα subunits, and a significant fraction of VHL mutations found in VHL
disease and in sporadic clear cell renal carcinomas abolish both the VHL–
HIFα interaction and HIFα ubiquitylation in vitro (Maxwell et al., 1999;
Ohh et al., 2000; Cockman et al., 2000; Tanimoto et al., 2000).

III. PROLYL HYDROXYLASE: THE OXYGEN SENSOR

IN HYPOXIC SIGNALING?

The finding that the VHL tumor suppressor protein is the substrate recog-
nition subunit of an E3 ubiquitin ligase that targets HIFα for rapid ubiq-
uitylation and destruction in cells grown in plentiful oxygen provided a
plausible explanation for the observed constitutive expression of hypoxi-
cally inducible genes in VHL (−/−) cells. This observation provided little
information, however, on the mechanism by which HIFα ubiquitylation is
regulated by cellular oxygen levels. Evidence from several recent studies has
shed light on the mechanism underlying hypoxic signal transduction and
has demonstrated that an oxygen-dependent posttranslational modification
of HIFα plays a crucial role in this process (Jaakkola et al., 2001; Ivan et al.,
2001; Yu et al., 2001).
It was known that treatment of normal cells with CoCl2 or the iron chela-
tor desferrioxamine mimicked the hypoxic response and resulted in stabi-
lization of HIFα and activation of the hypoxic transcriptional program (Zhu
and Bunn, 2001; Semenza, 1999). In addition, several laboratories observed
that the VHL–HIFα interaction is disrupted in cells grown under hypoxic
conditions or in the presence of CoCl2 or desferrioxamine (Cockman et al.,
2000; Ivan et al., 2001; Epstein et al., 2001), raising the possibility that the
VHL–HIFα interaction is governed by an oxygen- and iron-dependent post-
translation modification of either VHL or HIFα. Further investigation led
to the intriguing discovery that the VHL–HIFα interaction depends strongly
on an oxygen- and iron-dependent hydroxylation of specific proline residues
in the HIFα ODD by a CoCl2-sensitive prolyl hydroxylase (Masson et al.,
Von Hippel–Lindau Tumor Suppressor Complex 7

2001; Jaakkola et al., 2001; Ivan et al., 2001; Yu et al., 2001). Characteriza-
tion of the enzymatic properties of the HIFα prolyl hydroxylase in crude cell
lysates revealed that it belonged to the family of 2-oxoglutarate-dependent
dioxygenases, but was distinct from the only known members of this en-
zyme family, the prolyl hydroxylases responsible for modifying collagen in
the lumen of the endoplasmic reticulum (Jaakkola et al., 2001). Efforts to
identify the HIFα prolyl hydroxylase were greatly aided by Aravind and
Koonin (Aravind and Koonin, 2001), who had independently exploited a
bioinformatics approach to identify members of the AlkB, Leprecan, and
Egl-9 protein families as potential novel prolyl hydroxylases. A direct test of
the abilities of representative members of each of these three protein fam-
ilies to modify HIFα led to the discovery that Egl-9 proteins are, indeed,
HIFα prolyl hydroxylases that modify HIFα and promote the VHL–HIFα
interaction (Epstein et al., 2001; Bruick and McKnight, 2001).
Despite significant progress dissecting the role of the VHL complex and
HIFα proline hydroxylation in hypoxic gene regulation, the mechanisms
underlying hypoxic signaling are still unclear. Because Egl-9 proteins use
molecular oxygen as the oxygen donor for hydroxylation of HIFα in a reac-
tion with kinetics directly proportional to the concentration of oxygen, it has
been proposed that Egl-9 could serve as the primary oxygen sensor in hypoxic
gene regulation (Epstein et al., 2001; Bruick and McKnight, 2001). Direct
evidence in support of this model, however, has not been presented, and ad-
ditional evidence from a variety of studies suggests that signaling pathways
involving reactive oxygen species, protein nitrosylation, or protein phosphor-
ylation may also contribute to hypoxic regulation of HIFα (Semenza, 1999,
2000a). Finally, whether the Egl-9 dioxygenases function alone to modify
HIFα or whether they exist in cells in association with other proteins that
participate in regulation of HIFα is not known and awaits purification and
characterization of the endogenous Egl-9 proteins.

IV. CONCLUSION

The discovery that the VHL protein is an integral component of a novel

type of E3 ubiquitin ligase that negatively regulates expression of hypoxia-
inducible genes like TGFα and VEGF has provided key insights into the
molecular basis of VHL disease. In light of evidence that TGFα functions
as an autocrine growth factor for VHL (−/−) clear cell renal carcinoma
cells (dePaulsen et al., 2001), it is possible that overexpression of TGFα
by VHL (−/−) cells could account for the deregulated cell growth of VHL
tumors. In addition, constitutive expression of VEGF by VHL (−/−) cells
could underlie the highly vascularized nature of VHL tumors. Notably,
8 Conaway and Conaway

VHL (−/−) clear cell renal carcinoma cells exhibit, in addition to misreg-
ulation of expression of hypoxia-inducible genes, a remarkably pleiotropic
collection of additional phenotypes including (i) defects in assembly of an ex-
tracellular fibronectin matrix (Ohh et al., 1998), (ii) failure to properly induce
the cdk inhibitors p21 and p27 under a variety of conditions (Pause et al.,
1998; Schoenfeld et al., 2000), and (iii) defects in endoplasmic reticulum-
associated degradation (ERAD) of misfolded proteins (Gorospe et al., 1999).
Whether VHL disease and all these diverse phenotypes can be entirely ac-
counted for by defects in the VHL ubiquitin ligase remains to be determined.

ACKNOWLEDGMENT

Work in the authors’ laboratory is supported by NIH Grant R37 GM41628.

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