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The von Hippel–Lindau tumor suppressor protein: new insights into oxygen sensing and cancer
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The von Hippel±Lindau tumor suppressor protein: new insights into oxygen sensing and cancer William Kim and William G Kaelin Jr The von Hippel±Lindau tumor suppressor protein (pVHL) is the substrate-recognition module of an E3 ubiquitin ligase that targets the alpha subunits of hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. Recognition of HIF by pVHL is linked to enzymatic hydroxylation of conserved prolyl residues in the HIF alpha subunits by members of the EGLN family. Dysregulation of HIF-target genes such as vascular endothelial growth factor and transforming growth factor a has been implicated in the pathogenesis of renal cell carcinomas and of hemangioblastomas, both of which frequently lack pVHL function. Addresses Howard Hughes Medical Institute, Dana-Farber Cancer Institute and Brigham and Women's Hospital, 44 Binney St, Boston, Massachusetts 02115, USA e-mail: [email protected]
Current Opinion in Genetics & Development 2003, 13:55±60 This review comes from a themed issue on Oncogenes and cell proliferation Edited by Frank McCormick and Kevin Shannon 0959-437X/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-437X(02)00010-2 Abbreviations ARNT aryl hydrocarbon receptor nuclear translocator CCT cytosolic chaperonin containing TCP-1 CTAD C-terminal transactivation domain EGFR epidermal growth factor receptor EGLN egg laying nine FIH-1 factor inhibiting HIF-1 HIF hypoxia-inducible factor NTAD N-terminal transactivation domain ODD oxygen-dependent degradation domain PDGF platelet-derived growth factor pVHL von Hippel±Lindau protein TGF transforming growth factor VEGF vascular endothelial growth factor
Introduction
von Hippel±Lindau (VHL) disease is caused by germline mutation of the VHL tumor suppressor gene and is characterized by the development of multiple tumors, including those of the blood vessels (hemangioblastomas) of the retina and central nervous system, clear-cell carcinoma of the kidney, and adrenal-gland tumors (pheochromocytomas) [1]. Tumor development in this setting is linked to somatic inactivation of the remaining wild-type VHL allele. In keeping with the Knudson two-hit model www.current-opinion.com
[1], biallelic VHL inactivation (as a result of mutation or hypermethylation) is also common in sporadic hemangioblastomas and clear-cell carcinomas. Surprisingly, however, somatic VHL mutations are rare in sporadic pheochromocytomas despite the fact that certain germline VHL mutations cause them. Indeed, some germline VHL mutations (e.g. those found in Type 2C VHL disease) cause familial pheochromocytomas without any of the other stigmata that are typical of VHL disease [2,3]. The VHL gene is ubiquitously expressed and VHL orthologs have been identi®ed in mouse, Drosophila and Caenorhabditis elegans. VHL gene encodes two different protein products with apparent molecular weights of 30 kD and 19 kD. The smaller form is biologically active and results from translational initiation from an internal, in-frame methionine. Why cells produce two forms of the VHL protein is still not clear and, for simplicity, `pVHL' will be used below when referring to the two isoforms generically. This review will focus on recent studies that link tumor suppression by pVHL to its ability to downregulate the heterodimeric transcription factor hypoxia-inducible factor (HIF), which is the key regulator of hypoxia-inducible genes, as well as the unanticipated discovery that HIF is enzymatically hydroxylated on speci®c prolyl and asparagine residues in an oxygen-dependent manner. The former regulates pVHL-binding and the latter regulates coactivator recruitment.
pVHL regulates HIF
Earlier studies showed that pVHL-defective tumor cells overproduce hypoxia-inducible mRNAs, such as the vascular endothelial growth factor mRNA, whether or not there are high levels of oxygen present [1]. Many hypoxiainducible genes are under the control of HIF, which consists of an alpha subunit encoded by one of three genes (HIF1a, HIF2a, or HIF3a) and a beta subunit HIF1b (also called aryl hydrocarbon receptor nuclear translocator or ARNT). HIF alpha subunits are normally polyubiquitinated and degraded if oxygen is present. In cells lacking pVHL, this polyubiquitination does not take place, which leads to the inappropriate accumulation of HIF and its downstream targets. Subsequently it was shown that pVHL is a component of a ubiquitin ligase complex (or E3) that polyubiquitinates HIF alpha subunits in the presence of oxygen. The pVHL ubiquitin ligase complex contains additional proteins called elongin B, elongin C, Cullin 2, and Rbx1 (also called ROC1 or Hrt1), and resembles the SCF-like ubiquitin ligase comCurrent Opinion in Genetics & Development 2003, 13:55±60
56 Oncogenes and cell proliferation
plexes (Skp1/Cdc53/F-box protein) that were ®rst described in yeast. The binding of pVHL to elongin B and elongin C is facilitated by CCT (cytosolic chaperonin containing TCP-1), and protects pVHL from autoubiquitination [4±7]. As is true for SCF complexes, pVHL ubiquitin ligase activity requires ongoing neddylation of the cullin component [8±11]. Cullin neddylation is thought to in¯uence the recruitment of an E2 ubiquitin conjugating enzyme [12]. pVHL binds directly to HIF alpha subunits and thus serves as a substrate recognition module in a manner similar to that of an F-box protein in an SCF complex. pVHL contains two subdomains, alpha and beta, that are frequently affected by disease-associated VHL mutations. The beta domain binds to HIF whereas the alpha domain binds to the elongins, which recruit the rest of the complex. Where polyubiquitination of HIF occurs in the cell is still unclear. pVHL shuttles between the nucleus and the cytoplasm in a transcription-dependent manner. One recent report indicated that pVHL polyubiquitinates HIF in the nucleus and transports it to the cytoplasm for proteasomal degradation [13]; however, a HIF variant carrying a heterologous membrane-targeting motif was ef®ciently polyubiquitinated in cells [14].
Hydroxylation of HIFa
Under hypoxic conditions HIF alpha subunits accumulate, bind to ARNT, and activate the transcription of hypoxia-inducible genes bearing canonical HIF DNAbinding sites. This implies that the recognition of HIF by pVHL, and therefore the functional consequences of such recognition, is linked to whether or not oxygen is present. In early 2001, several groups reported that the binding of pVHL to HIFa was dependent upon the hydroxylation of conserved prolyl residues within a region of HIF called the oxygen-dependent degradation domain (ODD) [15±17,18±20] (Figure 1). This modi®cation is inherently linked to oxygen availability as the hydroxyl oxygen atom is derived from molecular oxygen. The crystal structure of pVHL and a hydroxylated HIF peptide when bound together has been elucidated and reveals that the HIF prolyl hydroxy group forms two critical hydrogen bonds with hydrophilic residues within the pVHL beta domain [21,22]. Prolyl hydroxylases belong to a larger family of irondependent, 2-oxoglutarate-dependent dioxygenases [23, 24,25]. The classic prolyl hydroxylases are located in the endoplasmic reticulum and modify collagen. In contrast, HIF is an intracellular protein and the HIF prolyl hydroxylation sites do not resemble the sites that are modi®ed in collagen. It is now clear that the HIF prolyl hydroxylases belong to the EGLN family, which in human cells comprise EGLN1, EGLN2, and EGLN3 [25,26,27±29]. Disruption of the single EGLN family member (EGL9) in C. elegans or Drosophila leads to the Current Opinion in Genetics & Development 2003, 13:55±60
stabilization of transcriptionally active HIF [27,28]. Inhibition of mammalian EGLN with small molecule antagonists likewise leads to HIF stabilization [28,29]. HIF is regulated at multiple levels in addition to the protein turnover level. In particular, HIF contains two modular transcriptional activation domains: the N-terminal transactivation domain (NTAD), which overlaps with the ODD, and the C-terminal transactivation domain (CTAD). Transcriptional activation by the CTAD as an isolated polypeptide (e.g. when it is fused to a heterologous DNA-binding domain) is induced by hypoxia without a concomitant change in CTAD's abundance [30]. A speci®c asparagine residue within the CTAD is hydroxylated by a protein called FIH-1 (factor inhibiting HIF-1) in the presence of oxygen, which in turn prevents the CTAD from recruiting the coactivator proteins p300 and CBP [31±33,34±36] (Figure 1). FIH-1, like the EGLN family members, is an iron-dependent and 2-oxoglutarate dependent dioxygenase. In summary, hydroxylation affects HIF turnover as well as HIF transcriptional activation capability. The former is linked to the recognition of HIF by the pVHL ubiquitin ligase complex and the latter to the regulated interaction of the CTAD with p300 and CBP coactivator proteins. On the other hand, the inactivation of pVHL, either genetically or with peptidic inhibitors [37], is suf®cient to activate HIF target genes in the presence of oxygen. Thus, the presumed failure of the CTAD to recruit p300 and CBP does not translate into failure to activate transcription under all conditions. It is possible, for example, that activation of HIF target genes following pVHL inactivation is caused by NTAD, which can function in the absence of the CTAD as shown by the analysis of naturally occurring HIF splice variants [38].
Additional functions of pVHL
pVHL is suspected of having other functions in addition to its role in regulating HIF. For example, pVHL has been implicated in the control of extracellular-matrix deposition and turnover, epithelial differentiation, and cell-cycle exit, although some of these functions may, in time, be linked to HIF. Recent studies provided evidence that pVHL can downregulate Cyclin D1, perhaps accounting for the earlier ®nding that pVHL-defective cells fail to exit the cell-cycle normally under certain experimental conditions [39±41]. pVHL also affects ®bronectin matrix assembly and the expression of both metalloproteinases and tissue inhibitors of metalloproteinases. Perhaps the best evidence for HIF-independent pVHL functions has come from the study of VHL genotype± phenotype correlations. VHL mutations linked to Type 2C VHL disease encode proteins that seemingly retain the ability to recognize and polyubiquitinate HIF [2,3]. Although a quantitative defect in HIF regulation has not www.current-opinion.com
The von Hippel±Lindau tumor suppressor protein Kim and Kaelin 57
Figure 1
Nedd8 Elongin C Elongin B
Elongin B α
pVHL
Nedd8
Cul2
Elongin C α
CCT
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Ubc12
Cul2
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Nedd8
Elongin B
Elongin C α
Oxygen EGLN FIH-1
HIFα
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Cul2
Rbx1
pVHL HIFα
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Ub Ub Ub Ub Ub Ub
CTAD
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CTAD
NTAD
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Ub Ubiquitin Current Opinion in Genetics & Development
Oxygen-dependent regulation of HIF. CCT facilitates the formation of the pVHL±elongin B±elongin C complex, which then binds to Cul2 and Rbx1 (also called ROC1 or Hrt1). Cul2 is neddylated by the Nedd8 conjugating enzyme Ubc12. HIFa contains two transactivation domains called the NTAD and CTAD. In the presence of oxygen, the NTAD and CTAD are hydroxylated at conserved proly and asparagine residues by an EGLN family member and FIH-1 respectively. For simplicity, only one of two potential prolyl hydroxylation sites in the NTAD is shown. Hydroxylation of the CTAD displaces the p300 and CBP coactivators whereas hydroxylation of the NTAD creates a binding site for pVHL, which then directs the polyubiquitination of HIF.
been excluded, this observation raises the possibility that pVHL interacts biochemically with additional targets. With regard to this, pVHL has been reported to interact with atypical protein kinase C isoforms, VHL-interacting deubiquitinating enzyme 1, and the plant homeodomain protein Jade-1 [42±50].
Therapeutic modulation of the pVHL pathway
The pVHL mutants that are linked to hemangioblastoma and renal cell carcinoma are invariably defective with respect to HIF polyubiquitination. Accordingly, these two tumor types are highly vascular and frequently overproduce growth factors that are either directly or indirectly under the control of HIFs such as VEGF, plateletderived growth factor B (PDGF B), and transforming growth factor a (TGFa). VEGF and PDGF B have been linked to angiogenesis whereas TGFa and its receptor www.current-opinion.com
EGFR have been implicated in the control of renal epithelial proliferation [1,51]. Two groups recently reported that HIFa variants that escaped pVHL control by virtue of mutations within their pVHL-binding motifs could override certain aspects of pVHL tumor suppressor function in vitro and in vivo [52,53]. This establishes that suppression of HIF is necessary for pVHL tumor suppressor activity. For these reasons, agents that are directed against HIFresponsive growth factors such as VEGF, PDGF B, and TGFa, and their cognate receptors, warrant investigation as potential treatments for hemangioblastomas and renal cell carcinomas. There is now anecdotal evidence that the VEGF inhibitor SU5416 can cause symptomatic improvement in VHL patients with hemangioblastomas without inducing frank tumor regression [54,55]. Symptomatic Current Opinion in Genetics & Development 2003, 13:55±60
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improvement in this setting might be at least partly attributable to the decreased peritumoral edema that results from blocking the vascular-permeability effects of VEGF. A recent Phase II trial of a neutralizing VEGF antibody (AvastinTM, as manufactured by Genentech; generic name Bevacizumab) for the treatment of recurrent renal cancer showed a modest, but statistically signi®cant, prolongation in time to progression [56]. Additional VEGF inhibitors, some of which also block PDGF signaling, are being developed [57]. Theoretically, such agents could be combined with agents that block TGFa or epidermal growth factor receptor. In theory, activation of HIF target genes might promote cell survival in the context of acute or chronic hypoxia. In this regard, it is worth noting that there is no evidence that HIF is suf®cient to induce tumorigenesis and some evidence that it is not [58,59]. In any event, acute administration of a HIF agonist to patients with conditions such as myocardial infarction or stroke would not be expected to cause tumors. The HIF prolyl hydroxylase appears susceptible to pharmacological attack and the same would be expected to be true of FIH-1 [28,29]. One compound, FG-0041 (manufactured by Fibrogen), was shown to preserve left ventricular function when administered after acute myocardial infarction in rats [60]. Although this study was motivated by the ability of FG-0041 to inhibit collagen prolyl hydroxylase (and hence ®brosis), it now appears that the bene®cial effects that were observed may have been caused by inhibition of EGLN and the resulting stabilization of transcriptionally active HIF in the perinfarct zone [29]. It will be of interest to characterize more fully the biological effects of small molecules that inhibit the VHL ubiquitin ligase [37] or the HIF hydroxylases.
Conclusions and future prospects
The study of pVHL and its interaction with HIF has led to the discovery of a critical component of the mammalian oxygen-sensing pathway and, more broadly, of a previously unappreciated role for protein hydroxylation in intracellular signaling. It will be of interest to determine whether all three EGLN family members hydroxylate HIF in vivo and whether any of these proteins have substrates in addition to, or perhaps instead of, HIF. A related issue is whether the interaction of pVHL with any other cellular protein is likewise governed by prolyl hydroxylation. The available evidence suggests that downregulation of HIF target genes is necessary for suppression of renal cell carcinoma and hemangioblastoma by pVHL. From a therapeutic viewpoint, it will be more important to establish whether suppression of HIF is also suf®cient for tumor suppression in these settings and, if so, to identify which HIF target genes are required for the development of these tumors in vivo. Similarly, it will be important to Current Opinion in Genetics & Development 2003, 13:55±60
determine whether HIF dysregulation is suf®cient to cause tumor growth or whether the oncogenic effects of HIF are dependent upon the loss of other pVHL functions, perhaps in conjunction with mutations in genes other than VHL. Finally, preliminary data suggest that drugs that activate, rather than inhibit, HIF might one day play a role in the management of diseases characterized by tissue ischemia such as myocardial infarction and stroke.
Acknowledgements
We thank the members of the Kaelin Laboratory for many helpful discussions. We apologise to colleagues whose work was not cited because of space limitations or our oversight. Please bring signi®cant errors or omissions to our attention.
References and recommended reading
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The von Hippel±Lindau tumor suppressor protein Kim and Kaelin 59
13. Groulx I, Lee S: Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic traf®cking of the von Hippel±Lindau tumor suppressor protein. Mol Cell Biol 2002, 22:5319-5336. 14. Berra E, Roux D, Richard D, Pouyssegur J: Hypoxia-inducible factor-1a (HIF-1a) escapes O2-driven proteasomal degradation irrespective of its subcellular localization: nucleus or cytoplasm. EMBO Rep 2001, 2:615-620. 15. Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara J, Lane W, Kaelin WG: HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 2001, 292:464-468. See annotation [17]. 16. Jaakkola P, Mole D, Tian Y, Wilson M, Gielbert J, Gaskell S, Kriegsheim A, Hebestreit H, Mukherji M, Scho®eld C et al.: Targeting of HIF-alpha to the von Hippel±Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001, 292:468-472. See annotation [17]. 17. Yu F, White S, Zhao Q, Lee F: HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc Natl Acad Sci USA 2001, 98:9630-9635. This paper, and [15,16], show that binding of pVHL to HIF is regulated by enzymatic hydroxylation of a conserved prolyl residue within the HIF oxygen-dependent degradation domain. As oxygen is required for hydroxylation, these papers provide a satisfying explanation for why HIF turnover is oxygen-dependent. Moreover, these studies provided the ®rst evidence for a role of protein hydroxylation in intracellular signaling. 18. Yu F, White S, Zhao Q, Lee F: Dynamic, site-speci®c interaction of hypoxia-inducible factor-1alpha with the von Hippel±Lindau tumor suppressor protein. Cancer Res 2001, 61:4136-4142. 19. Masson N, Willam C, Maxwell P, Pugh C, Ratcliffe P: Independent function of two destruction domains in hypoxia-inducible factor-a chains activated by prolyl hydroylation. EMBO 2001, 20:5197-5206. 20. Kaelin WG Jr: How oxygen makes its presence felt. Genes Dev 2002, 16:1441-1445. 21. Min JH, Yang H, Ivan M, Gertler F, Kaelin WG Jr, Pavletich NP: Structure of an HIF-1alpha-pVHL complex: hydroxyproline recognition in signaling. Science 2002, 296:1886-1889. See annotation [22]. 22. Hon WC, Wilson MI, Harlos K, Claridge TD, Scho®eld CJ, Pugh CW, Maxwell PH, Ratcliffe PJ, Stuart DI, Jones EY: Structural basis for the recognition of hydroxyproline in HIF-1alpha by pVHL. Nature 2002, 417:975-978. This paper, and [21], reveal why binding of pVHL to HIF depends upon prolyl hydroxylation. The prolyl hydroxy group forms critical hydrogen bonds with two hydrophilic pVHL side-chains located in the otherwise hydrophobic pVHL b domain. In the unbound state these two side chains are partially solvent-exposed. Binding to HIF leads to the removal of the solvent groups, which would destabilize the pVHL structure were it not for these two hydrogen bonds, which cannot form with unmodi®ed proline. 23. Scho®eld CJ, Zhang Z: Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes. Curr Opin Struct Biol 1999, 9:722-731. 24. Kivirikko KI, Myllyharju J: Prolyl 4-Hydroxylases and their protein disul®de isomerase subunit. Matrix Biol 1998, 16:357-368. 25. Aravind L, Koonin EV: The DNA-repair protein AlkB, EGL-9, and leprecan de®ne new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome Biol 2001, 2:reasearch0007.0001-0007.0008. This paper was the ®rst to predict that EGL-9 might encode a novel prolyl hydroxylase. Moreover, this paper identi®ed a number of other potential protein hydroxylases, which suggests that protein hydroxylation may be more common than is appreciated. 26. Taylor MS: Characterization and comparative analysis of the EGLN gene family. Gene 2001, 275:125-132. 27. Bruick R, McKnight S: A conserved family of prolyl-4 hydroxylases that modify HIF. Science 2001, 294:1337-1340. See annotation [28]. www.current-opinion.com
28. Epstein A, Gleadle J, McNeill L, Hewitson K, O'Rourke J, Mole D, Mukherji M, Metzen E, Wilson M, Dhanda A et al.: C. elegans EGL-9 and mammalian homologs de®ne a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001, 107:43-54. This paper and [27] showed that Egl9 is the HIF prolyl hydroxylase using bioinformatic and genetic approaches, followed by biochemical validation experiments. 29. Ivan M, Haberberger T, Gervasi D, Michelson K, Gunzler V, Kondo K, Yang H, Sorokina I, Conaway R, Conaway J et al.: Biochemical puri®cation and pharmacological inhibition of a mammalian HIF prolyl hydroxylase. Proc Natl Acad Sci USA 2002, 99:13459-13464. The authors of this paper identify a series of structurally diverse smallmolecule EGLN antagonists. One such compound, FG-0041, activated HIF and HIF target genes in cell-based assays and was shown earlier [60] to be protective in a rat model of myocardial ischemia. 30. Sang N, Fang J, Srinivas V, Leshchinsky I, Caro J: Carboxyl-terminal transactivation activity of hypoxia-inducible factor 1alpha is governed by a von Hippel±Lindau protein-independent, hydroxylation-regulated association with p300/CBP. Mol Cell Biol 2002, 22:2984-2992. 31. Lando D, Peet D, Whelan D, Gorman J, Whitelaw M: Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science 2002, 295:858-861. This paper is the ®rst to demonstrate that the transcriptional-activation function of the HIF CTAD is regulated by oxygen-dependent hydroxylation of a conserved asparagine residue. 32. Lando D, Peet D, Gorman J, Whelan D, Whitelaw M, Bruick R: FIH-1 is a an asparaginyl hydroxylase that regulates the transcriptional activity of hypoxia inducible factor. Genes Dev 2002, 16:1466-1471. See annotation [33]. 33. Hewitson KS, McNeill LA, Riordan MV, Tian YM, Bullock AN, Welford RW, Elkins JM, Oldham NJ, Bhattacharya S, Gleadle JM et al.: Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family. J Biol Chem 2002, 277:26351-26355. This paper and [32] show that FIH-1, the HIF-interacting protein identi®ed earlier by Mahon et al. [36], is the enzyme responsible for the asparaginyl hydroxylation of HIF. 34. Freedman S, Sun Z, Poy F, Kung A, Livingston D, Wagner G, Eck M: Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1alpha. Proc Natl Acad Sci USA 2002, 99:5367-5372. 35. Dames S, Martinez-Yamout M, De Guzman R, Dyson H, Wright P: From the cover: structural basis for Hif-1alpha /CBP recognition in the cellular hypoxic response. Proc Natl Acad Sci USA 2002, 99:5271-5276. 36. Mahon P, Hirota K, Semenza G: FIH-1: a novel protein that interacts with HIF-1a and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev 2001, 15:2675-2686. 37. Willam C, Masson N, Tian YM, Mahmood SA, Wilson MI, Bicknell R, Eckardt KU, Maxwell PH, Ratcliffe PJ, Pugh CW: Peptide blockade of HIFalpha degradation modulates cellular metabolism and angiogenesis. Proc Natl Acad Sci USA 2002, 99:10423-10428. 38. Gothie E, Richard D, Berra E, Pages G, Pouyssegur J: Identi®cation of alternative spliced variants of human hypoxia-inducible factor-1alpha. J Biol Chem 2000, 275:6922-6927. 39. Bindra RS, Vasselli JR, Stearman R, Linehan WM, Klausner RD: VHL-mediated hypoxia regulation of cyclin D1 in renal carcinoma cells. Cancer Res 2002, 62:3014-3019. 40. Zatyka M, da Silva NF, Clifford SC, Morris MR, Wiesener MS, Eckardt KU, Houlston RS, Richards FM, Latif F, Maher ER: Identi®cation of cyclin D1 and other novel targets for the von Hippel±Lindau tumor suppressor gene by expression array analysis and investigation of cyclin D1 genotype as a modi®er in von Hippel±Lindau disease. Cancer Res 2002, 62:3803-3811. 41. Davidowitz E, Schoenfeld A, Burk R: VHL induces renal cell differentiation and growth arrest through integration of cell-cell and cell-extracellular matrix signaling. Mol Cell Biol 2001, 21:865-874. Current Opinion in Genetics & Development 2003, 13:55±60
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42. Okuda H, Saitoh K, Hirai S, Iwai K, Takaki Y, Baba M, Minato N, Ohno S, Shuin T: The von Hippel±Lindau tumor suppressor protein mediates ubiquitination of activated atypical protein kinase C. J Biol Chem 2001, 276:43611-43617.
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43. Okuda H, Hirai S, Takaki Y, Kamada M, Baba M, Sakai N, Kishida T, Kaneko S, Yao M, Ohno S et al.: Direct interaction of the beta-domain of VHL tumor suppressor protein with the regulatory domain of atypical PKC isotypes. Biochem Biophys Res Commun 1999, 263:491-497.
53. Maranchie JK, Vasselli JR, Riss J, Bonifacino JS, Linehan WM, Klausner RD: The contribution of VHL substrate binding and HIF1a to the phenotype of VHL loss in renal cell carcinoma. Cancer Cell 2002, 1:247-255. This paper and [52] provide evidence that HIF variants that escape pVHL control can override aspects of pVHL tumor suppressor activity. In particular, [52] shows that a HIF2a variant bearing a proline-to-alanine mutation could promote renal carcinoma cell growth in vivo despite the presence of wild-type pVHL.
44. Zhou MI, Wang H, Ross JJ, Kuzmin I, Xu C, Cohen HT: The von Hippel±Lindau (VHL) tumor suppressor stabilizes novel PHD protein Jade-1. J Biol Chem 2002, 277:39887-39898. 45. Li Z, Wang D, Na X, Schoen SR, Messing EM, Wu G: Identi®cation of a deubiquitinating enzyme subfamily as substrates of the von Hippel±Lindau tumor suppressor. Biochem Biophys Res Commun 2002, 294:700-709. 46. Li Z, Na X, Wang D, Schoen SR, Messing EM, Wu G: Ubiquitination of a novel deubiquitinating enzyme requires direct binding to von Hippel±Lindau tumor suppressor protein. J Biol Chem 2002, 277:4656-4662. 47. Datta K, Sundberg C, Karumanchi SA, Mukhopadhyay D: The 104-123 amino acid sequence of the beta-domain of von Hippel±Lindau gene product is suf®cient to inhibit renal tumor growth and invasion. Cancer Res 2001, 61:1768-1775. 48. Datta K, Nambudripad R, Pal S, Zhou M, Cohen HT, Mukhopadhyay D: Inhibition of insulin-like growth factor-I-mediated cell signaling by the von Hippel±Lindau gene product in renal cancer. J Biol Chem 2000, 275:20700-20706. 49. Pal S, Claffey K, Dvorak H, Mukhopadhyay D: The von Hippel±Lindau gene product inhibits vascular permeability factor/vascular endothelial growth factor expression in renal cell carcinoma by blocking protein kinase C pathways. J Biol Chem 1997, 272:27509-27512. 50. Pal S, Claffey K, Cohen H, Mukhopadhyay D: Activation of Sp1-mediated vascular permeability factor/vascular endothelial growth factor transcription requires speci®c interaction with protein kinase C f. J Biol Chem 1998, 273:26277-26280. 51. de Paulsen N, Brychzy A, Fournier M-C, Klausner RD, Gnarra JR, Pause A, Lee S: Role of transforming growth factor-alpha in VHL / clear cell renal carcinoma cell proliferation: a possible mechanism coupling von Hippel±Lindau tumor suppressor inactivation and tumorigenesis. Proc Natl Acad Sci USA 2001, 13:1387-1392.
Current Opinion in Genetics & Development 2003, 13:55±60
54. Aiello L, George D, Cahill M, Wong J, Cavallerano J, Hannah A, Kaelin WG: Rapid and durable recovery of visual function in a patient with von Hippel±Lindau syndrome and optic nerve head hemangioma following systemic treatment with the VEGF receptor inhibitor SU5416. Ophthalmology 2002, 109:1745-1751. 55. Richard S, Croisille L, Yvart J, Casadeval N, Eschwege P, Aghakhani N, David P, Gaudric A, Scigalla P, Hermine O: Paradoxical secondary polycythemia in von Hippel±Lindau patients treated with anti-vascular endothelial growth factor receptor therapy. Blood 2002, 99:3851-3853. 56. Yang J, Haworth S, Steinberg S, Rosenberg S, Novotny W: A randomized double-blind placebo-controlled trial of bevacizumab (anti-VEGF antibody) demonstrating a prolongation in time to progression in patients with metastatic renal cancer. Proc Am Soc Clin Oncol 2002, 21:15. 57. Fabbro D, Parkinson D, Matter A: Protein tyrosine kinase inhibitors: new treatment modalities? Curr Opin Pharmacol 2002, 2:374-381. 58. Vincent K, Shyu K, Luo Y, Magner M, Tio R, Jiang C, Goldberg M, Akita G, Gregory R, Isner J: Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1alpha/VP16 hybrid transcription factor. Circulation 2000, 102:2255-2261. 59. Elson D, Thurston G, Huang L, Ginzinger D, McDonald D, Johnson R, Arbeit J: Induction of hypervascularity without leakage or in¯ammation in transgenic mice overexpressing hypoxia-inducible factor-1a. Genes Dev 2001, 15:2520-2532. 60. Nwogu JI, Geenen D, Bean M, Brenner MB, Huang X, Buttrick PM: Inhibition of collagen synthesis with prolyl 4-hydroxylase inhibitor improves left ventricular function and alters the pattern of left ventricular dilation after myocardial infarction. Circulation 2001, 104:2216-2221.
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The von Hippel±Lindau tumor suppressor protein: new insights into oxygen sensing and cancer William Kim and William G Kaelin Jr The von Hippel±Lindau tumor suppressor protein (pVHL) is the substrate-recognition module of an E3 ubiquitin ligase that targets the alpha subunits of hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. Recognition of HIF by pVHL is linked to enzymatic hydroxylation of conserved prolyl residues in the HIF alpha subunits by members of the EGLN family. Dysregulation of HIF-target genes such as vascular endothelial growth factor and transforming growth factor a has been implicated in the pathogenesis of renal cell carcinomas and of hemangioblastomas, both of which frequently lack pVHL function. Addresses Howard Hughes Medical Institute, Dana-Farber Cancer Institute and Brigham and Women's Hospital, 44 Binney St, Boston, Massachusetts 02115, USA e-mail: [email protected]
Current Opinion in Genetics & Development 2003, 13:55±60 This review comes from a themed issue on Oncogenes and cell proliferation Edited by Frank McCormick and Kevin Shannon 0959-437X/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-437X(02)00010-2 Abbreviations ARNT aryl hydrocarbon receptor nuclear translocator CCT cytosolic chaperonin containing TCP-1 CTAD C-terminal transactivation domain EGFR epidermal growth factor receptor EGLN egg laying nine FIH-1 factor inhibiting HIF-1 HIF hypoxia-inducible factor NTAD N-terminal transactivation domain ODD oxygen-dependent degradation domain PDGF platelet-derived growth factor pVHL von Hippel±Lindau protein TGF transforming growth factor VEGF vascular endothelial growth factor
Introduction
von Hippel±Lindau (VHL) disease is caused by germline mutation of the VHL tumor suppressor gene and is characterized by the development of multiple tumors, including those of the blood vessels (hemangioblastomas) of the retina and central nervous system, clear-cell carcinoma of the kidney, and adrenal-gland tumors (pheochromocytomas) [1]. Tumor development in this setting is linked to somatic inactivation of the remaining wild-type VHL allele. In keeping with the Knudson two-hit model www.current-opinion.com
[1], biallelic VHL inactivation (as a result of mutation or hypermethylation) is also common in sporadic hemangioblastomas and clear-cell carcinomas. Surprisingly, however, somatic VHL mutations are rare in sporadic pheochromocytomas despite the fact that certain germline VHL mutations cause them. Indeed, some germline VHL mutations (e.g. those found in Type 2C VHL disease) cause familial pheochromocytomas without any of the other stigmata that are typical of VHL disease [2,3]. The VHL gene is ubiquitously expressed and VHL orthologs have been identi®ed in mouse, Drosophila and Caenorhabditis elegans. VHL gene encodes two different protein products with apparent molecular weights of 30 kD and 19 kD. The smaller form is biologically active and results from translational initiation from an internal, in-frame methionine. Why cells produce two forms of the VHL protein is still not clear and, for simplicity, `pVHL' will be used below when referring to the two isoforms generically. This review will focus on recent studies that link tumor suppression by pVHL to its ability to downregulate the heterodimeric transcription factor hypoxia-inducible factor (HIF), which is the key regulator of hypoxia-inducible genes, as well as the unanticipated discovery that HIF is enzymatically hydroxylated on speci®c prolyl and asparagine residues in an oxygen-dependent manner. The former regulates pVHL-binding and the latter regulates coactivator recruitment.
pVHL regulates HIF
Earlier studies showed that pVHL-defective tumor cells overproduce hypoxia-inducible mRNAs, such as the vascular endothelial growth factor mRNA, whether or not there are high levels of oxygen present [1]. Many hypoxiainducible genes are under the control of HIF, which consists of an alpha subunit encoded by one of three genes (HIF1a, HIF2a, or HIF3a) and a beta subunit HIF1b (also called aryl hydrocarbon receptor nuclear translocator or ARNT). HIF alpha subunits are normally polyubiquitinated and degraded if oxygen is present. In cells lacking pVHL, this polyubiquitination does not take place, which leads to the inappropriate accumulation of HIF and its downstream targets. Subsequently it was shown that pVHL is a component of a ubiquitin ligase complex (or E3) that polyubiquitinates HIF alpha subunits in the presence of oxygen. The pVHL ubiquitin ligase complex contains additional proteins called elongin B, elongin C, Cullin 2, and Rbx1 (also called ROC1 or Hrt1), and resembles the SCF-like ubiquitin ligase comCurrent Opinion in Genetics & Development 2003, 13:55±60
56 Oncogenes and cell proliferation
plexes (Skp1/Cdc53/F-box protein) that were ®rst described in yeast. The binding of pVHL to elongin B and elongin C is facilitated by CCT (cytosolic chaperonin containing TCP-1), and protects pVHL from autoubiquitination [4±7]. As is true for SCF complexes, pVHL ubiquitin ligase activity requires ongoing neddylation of the cullin component [8±11]. Cullin neddylation is thought to in¯uence the recruitment of an E2 ubiquitin conjugating enzyme [12]. pVHL binds directly to HIF alpha subunits and thus serves as a substrate recognition module in a manner similar to that of an F-box protein in an SCF complex. pVHL contains two subdomains, alpha and beta, that are frequently affected by disease-associated VHL mutations. The beta domain binds to HIF whereas the alpha domain binds to the elongins, which recruit the rest of the complex. Where polyubiquitination of HIF occurs in the cell is still unclear. pVHL shuttles between the nucleus and the cytoplasm in a transcription-dependent manner. One recent report indicated that pVHL polyubiquitinates HIF in the nucleus and transports it to the cytoplasm for proteasomal degradation [13]; however, a HIF variant carrying a heterologous membrane-targeting motif was ef®ciently polyubiquitinated in cells [14].
Hydroxylation of HIFa
Under hypoxic conditions HIF alpha subunits accumulate, bind to ARNT, and activate the transcription of hypoxia-inducible genes bearing canonical HIF DNAbinding sites. This implies that the recognition of HIF by pVHL, and therefore the functional consequences of such recognition, is linked to whether or not oxygen is present. In early 2001, several groups reported that the binding of pVHL to HIFa was dependent upon the hydroxylation of conserved prolyl residues within a region of HIF called the oxygen-dependent degradation domain (ODD) [15±17,18±20] (Figure 1). This modi®cation is inherently linked to oxygen availability as the hydroxyl oxygen atom is derived from molecular oxygen. The crystal structure of pVHL and a hydroxylated HIF peptide when bound together has been elucidated and reveals that the HIF prolyl hydroxy group forms two critical hydrogen bonds with hydrophilic residues within the pVHL beta domain [21,22]. Prolyl hydroxylases belong to a larger family of irondependent, 2-oxoglutarate-dependent dioxygenases [23, 24,25]. The classic prolyl hydroxylases are located in the endoplasmic reticulum and modify collagen. In contrast, HIF is an intracellular protein and the HIF prolyl hydroxylation sites do not resemble the sites that are modi®ed in collagen. It is now clear that the HIF prolyl hydroxylases belong to the EGLN family, which in human cells comprise EGLN1, EGLN2, and EGLN3 [25,26,27±29]. Disruption of the single EGLN family member (EGL9) in C. elegans or Drosophila leads to the Current Opinion in Genetics & Development 2003, 13:55±60
stabilization of transcriptionally active HIF [27,28]. Inhibition of mammalian EGLN with small molecule antagonists likewise leads to HIF stabilization [28,29]. HIF is regulated at multiple levels in addition to the protein turnover level. In particular, HIF contains two modular transcriptional activation domains: the N-terminal transactivation domain (NTAD), which overlaps with the ODD, and the C-terminal transactivation domain (CTAD). Transcriptional activation by the CTAD as an isolated polypeptide (e.g. when it is fused to a heterologous DNA-binding domain) is induced by hypoxia without a concomitant change in CTAD's abundance [30]. A speci®c asparagine residue within the CTAD is hydroxylated by a protein called FIH-1 (factor inhibiting HIF-1) in the presence of oxygen, which in turn prevents the CTAD from recruiting the coactivator proteins p300 and CBP [31±33,34±36] (Figure 1). FIH-1, like the EGLN family members, is an iron-dependent and 2-oxoglutarate dependent dioxygenase. In summary, hydroxylation affects HIF turnover as well as HIF transcriptional activation capability. The former is linked to the recognition of HIF by the pVHL ubiquitin ligase complex and the latter to the regulated interaction of the CTAD with p300 and CBP coactivator proteins. On the other hand, the inactivation of pVHL, either genetically or with peptidic inhibitors [37], is suf®cient to activate HIF target genes in the presence of oxygen. Thus, the presumed failure of the CTAD to recruit p300 and CBP does not translate into failure to activate transcription under all conditions. It is possible, for example, that activation of HIF target genes following pVHL inactivation is caused by NTAD, which can function in the absence of the CTAD as shown by the analysis of naturally occurring HIF splice variants [38].
Additional functions of pVHL
pVHL is suspected of having other functions in addition to its role in regulating HIF. For example, pVHL has been implicated in the control of extracellular-matrix deposition and turnover, epithelial differentiation, and cell-cycle exit, although some of these functions may, in time, be linked to HIF. Recent studies provided evidence that pVHL can downregulate Cyclin D1, perhaps accounting for the earlier ®nding that pVHL-defective cells fail to exit the cell-cycle normally under certain experimental conditions [39±41]. pVHL also affects ®bronectin matrix assembly and the expression of both metalloproteinases and tissue inhibitors of metalloproteinases. Perhaps the best evidence for HIF-independent pVHL functions has come from the study of VHL genotype± phenotype correlations. VHL mutations linked to Type 2C VHL disease encode proteins that seemingly retain the ability to recognize and polyubiquitinate HIF [2,3]. Although a quantitative defect in HIF regulation has not www.current-opinion.com
The von Hippel±Lindau tumor suppressor protein Kim and Kaelin 57
Figure 1
Nedd8 Elongin C Elongin B
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Ubc12
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Oxygen EGLN FIH-1
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Oxygen-dependent regulation of HIF. CCT facilitates the formation of the pVHL±elongin B±elongin C complex, which then binds to Cul2 and Rbx1 (also called ROC1 or Hrt1). Cul2 is neddylated by the Nedd8 conjugating enzyme Ubc12. HIFa contains two transactivation domains called the NTAD and CTAD. In the presence of oxygen, the NTAD and CTAD are hydroxylated at conserved proly and asparagine residues by an EGLN family member and FIH-1 respectively. For simplicity, only one of two potential prolyl hydroxylation sites in the NTAD is shown. Hydroxylation of the CTAD displaces the p300 and CBP coactivators whereas hydroxylation of the NTAD creates a binding site for pVHL, which then directs the polyubiquitination of HIF.
been excluded, this observation raises the possibility that pVHL interacts biochemically with additional targets. With regard to this, pVHL has been reported to interact with atypical protein kinase C isoforms, VHL-interacting deubiquitinating enzyme 1, and the plant homeodomain protein Jade-1 [42±50].
Therapeutic modulation of the pVHL pathway
The pVHL mutants that are linked to hemangioblastoma and renal cell carcinoma are invariably defective with respect to HIF polyubiquitination. Accordingly, these two tumor types are highly vascular and frequently overproduce growth factors that are either directly or indirectly under the control of HIFs such as VEGF, plateletderived growth factor B (PDGF B), and transforming growth factor a (TGFa). VEGF and PDGF B have been linked to angiogenesis whereas TGFa and its receptor www.current-opinion.com
EGFR have been implicated in the control of renal epithelial proliferation [1,51]. Two groups recently reported that HIFa variants that escaped pVHL control by virtue of mutations within their pVHL-binding motifs could override certain aspects of pVHL tumor suppressor function in vitro and in vivo [52,53]. This establishes that suppression of HIF is necessary for pVHL tumor suppressor activity. For these reasons, agents that are directed against HIFresponsive growth factors such as VEGF, PDGF B, and TGFa, and their cognate receptors, warrant investigation as potential treatments for hemangioblastomas and renal cell carcinomas. There is now anecdotal evidence that the VEGF inhibitor SU5416 can cause symptomatic improvement in VHL patients with hemangioblastomas without inducing frank tumor regression [54,55]. Symptomatic Current Opinion in Genetics & Development 2003, 13:55±60
58 Oncogenes and cell proliferation
improvement in this setting might be at least partly attributable to the decreased peritumoral edema that results from blocking the vascular-permeability effects of VEGF. A recent Phase II trial of a neutralizing VEGF antibody (AvastinTM, as manufactured by Genentech; generic name Bevacizumab) for the treatment of recurrent renal cancer showed a modest, but statistically signi®cant, prolongation in time to progression [56]. Additional VEGF inhibitors, some of which also block PDGF signaling, are being developed [57]. Theoretically, such agents could be combined with agents that block TGFa or epidermal growth factor receptor. In theory, activation of HIF target genes might promote cell survival in the context of acute or chronic hypoxia. In this regard, it is worth noting that there is no evidence that HIF is suf®cient to induce tumorigenesis and some evidence that it is not [58,59]. In any event, acute administration of a HIF agonist to patients with conditions such as myocardial infarction or stroke would not be expected to cause tumors. The HIF prolyl hydroxylase appears susceptible to pharmacological attack and the same would be expected to be true of FIH-1 [28,29]. One compound, FG-0041 (manufactured by Fibrogen), was shown to preserve left ventricular function when administered after acute myocardial infarction in rats [60]. Although this study was motivated by the ability of FG-0041 to inhibit collagen prolyl hydroxylase (and hence ®brosis), it now appears that the bene®cial effects that were observed may have been caused by inhibition of EGLN and the resulting stabilization of transcriptionally active HIF in the perinfarct zone [29]. It will be of interest to characterize more fully the biological effects of small molecules that inhibit the VHL ubiquitin ligase [37] or the HIF hydroxylases.
Conclusions and future prospects
The study of pVHL and its interaction with HIF has led to the discovery of a critical component of the mammalian oxygen-sensing pathway and, more broadly, of a previously unappreciated role for protein hydroxylation in intracellular signaling. It will be of interest to determine whether all three EGLN family members hydroxylate HIF in vivo and whether any of these proteins have substrates in addition to, or perhaps instead of, HIF. A related issue is whether the interaction of pVHL with any other cellular protein is likewise governed by prolyl hydroxylation. The available evidence suggests that downregulation of HIF target genes is necessary for suppression of renal cell carcinoma and hemangioblastoma by pVHL. From a therapeutic viewpoint, it will be more important to establish whether suppression of HIF is also suf®cient for tumor suppression in these settings and, if so, to identify which HIF target genes are required for the development of these tumors in vivo. Similarly, it will be important to Current Opinion in Genetics & Development 2003, 13:55±60
determine whether HIF dysregulation is suf®cient to cause tumor growth or whether the oncogenic effects of HIF are dependent upon the loss of other pVHL functions, perhaps in conjunction with mutations in genes other than VHL. Finally, preliminary data suggest that drugs that activate, rather than inhibit, HIF might one day play a role in the management of diseases characterized by tissue ischemia such as myocardial infarction and stroke.
Acknowledgements
We thank the members of the Kaelin Laboratory for many helpful discussions. We apologise to colleagues whose work was not cited because of space limitations or our oversight. Please bring signi®cant errors or omissions to our attention.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Wada H, Yeh E, Kwamitani T: Identi®cation of NEDD8-conjugation site in human cullin-2. Biochem Biophys Res Commun 1999, 257:100-105.
10. Liakopoulos D, Busgen T, Brychzy A, Jentsch S, Pause A: Conjugation of the ubiquitin-like protein NEDD8 to cullin-2 is linked to von Hippel±Lindau tumor suppressor function. Proc Natl Acad Sci USA 1999, 96:5510-5515. 11. Hori T, Osaka F, Chiba T, Miyamoto C, Okabayashi K, Shimbara N, Kato S, Tanaka K: Covalent modi®cation of all members of human cullin family proteins by NEDD8. Oncogene 1999, 18:6829-6834. 12. Kawakami T, Chiba T, Suzuki T, Iwai K, Yamanaka K, Minato N, Suzuki H, Shimbara N, Hidaka Y, Osaka F et al.: NEDD8 recruits E2-ubiquitin to SCF E3 ligase. EMBO J 2001, 20:4003-4012. www.current-opinion.com
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13. Groulx I, Lee S: Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic traf®cking of the von Hippel±Lindau tumor suppressor protein. Mol Cell Biol 2002, 22:5319-5336. 14. Berra E, Roux D, Richard D, Pouyssegur J: Hypoxia-inducible factor-1a (HIF-1a) escapes O2-driven proteasomal degradation irrespective of its subcellular localization: nucleus or cytoplasm. EMBO Rep 2001, 2:615-620. 15. Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara J, Lane W, Kaelin WG: HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 2001, 292:464-468. See annotation [17]. 16. Jaakkola P, Mole D, Tian Y, Wilson M, Gielbert J, Gaskell S, Kriegsheim A, Hebestreit H, Mukherji M, Scho®eld C et al.: Targeting of HIF-alpha to the von Hippel±Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001, 292:468-472. See annotation [17]. 17. Yu F, White S, Zhao Q, Lee F: HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc Natl Acad Sci USA 2001, 98:9630-9635. This paper, and [15,16], show that binding of pVHL to HIF is regulated by enzymatic hydroxylation of a conserved prolyl residue within the HIF oxygen-dependent degradation domain. As oxygen is required for hydroxylation, these papers provide a satisfying explanation for why HIF turnover is oxygen-dependent. Moreover, these studies provided the ®rst evidence for a role of protein hydroxylation in intracellular signaling. 18. Yu F, White S, Zhao Q, Lee F: Dynamic, site-speci®c interaction of hypoxia-inducible factor-1alpha with the von Hippel±Lindau tumor suppressor protein. Cancer Res 2001, 61:4136-4142. 19. Masson N, Willam C, Maxwell P, Pugh C, Ratcliffe P: Independent function of two destruction domains in hypoxia-inducible factor-a chains activated by prolyl hydroylation. EMBO 2001, 20:5197-5206. 20. Kaelin WG Jr: How oxygen makes its presence felt. Genes Dev 2002, 16:1441-1445. 21. Min JH, Yang H, Ivan M, Gertler F, Kaelin WG Jr, Pavletich NP: Structure of an HIF-1alpha-pVHL complex: hydroxyproline recognition in signaling. Science 2002, 296:1886-1889. See annotation [22]. 22. Hon WC, Wilson MI, Harlos K, Claridge TD, Scho®eld CJ, Pugh CW, Maxwell PH, Ratcliffe PJ, Stuart DI, Jones EY: Structural basis for the recognition of hydroxyproline in HIF-1alpha by pVHL. Nature 2002, 417:975-978. This paper, and [21], reveal why binding of pVHL to HIF depends upon prolyl hydroxylation. The prolyl hydroxy group forms critical hydrogen bonds with two hydrophilic pVHL side-chains located in the otherwise hydrophobic pVHL b domain. In the unbound state these two side chains are partially solvent-exposed. Binding to HIF leads to the removal of the solvent groups, which would destabilize the pVHL structure were it not for these two hydrogen bonds, which cannot form with unmodi®ed proline. 23. Scho®eld CJ, Zhang Z: Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes. Curr Opin Struct Biol 1999, 9:722-731. 24. Kivirikko KI, Myllyharju J: Prolyl 4-Hydroxylases and their protein disul®de isomerase subunit. Matrix Biol 1998, 16:357-368. 25. Aravind L, Koonin EV: The DNA-repair protein AlkB, EGL-9, and leprecan de®ne new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome Biol 2001, 2:reasearch0007.0001-0007.0008. This paper was the ®rst to predict that EGL-9 might encode a novel prolyl hydroxylase. Moreover, this paper identi®ed a number of other potential protein hydroxylases, which suggests that protein hydroxylation may be more common than is appreciated. 26. Taylor MS: Characterization and comparative analysis of the EGLN gene family. Gene 2001, 275:125-132. 27. Bruick R, McKnight S: A conserved family of prolyl-4 hydroxylases that modify HIF. Science 2001, 294:1337-1340. See annotation [28]. www.current-opinion.com
28. Epstein A, Gleadle J, McNeill L, Hewitson K, O'Rourke J, Mole D, Mukherji M, Metzen E, Wilson M, Dhanda A et al.: C. elegans EGL-9 and mammalian homologs de®ne a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001, 107:43-54. This paper and [27] showed that Egl9 is the HIF prolyl hydroxylase using bioinformatic and genetic approaches, followed by biochemical validation experiments. 29. Ivan M, Haberberger T, Gervasi D, Michelson K, Gunzler V, Kondo K, Yang H, Sorokina I, Conaway R, Conaway J et al.: Biochemical puri®cation and pharmacological inhibition of a mammalian HIF prolyl hydroxylase. Proc Natl Acad Sci USA 2002, 99:13459-13464. The authors of this paper identify a series of structurally diverse smallmolecule EGLN antagonists. One such compound, FG-0041, activated HIF and HIF target genes in cell-based assays and was shown earlier [60] to be protective in a rat model of myocardial ischemia. 30. Sang N, Fang J, Srinivas V, Leshchinsky I, Caro J: Carboxyl-terminal transactivation activity of hypoxia-inducible factor 1alpha is governed by a von Hippel±Lindau protein-independent, hydroxylation-regulated association with p300/CBP. Mol Cell Biol 2002, 22:2984-2992. 31. Lando D, Peet D, Whelan D, Gorman J, Whitelaw M: Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science 2002, 295:858-861. This paper is the ®rst to demonstrate that the transcriptional-activation function of the HIF CTAD is regulated by oxygen-dependent hydroxylation of a conserved asparagine residue. 32. Lando D, Peet D, Gorman J, Whelan D, Whitelaw M, Bruick R: FIH-1 is a an asparaginyl hydroxylase that regulates the transcriptional activity of hypoxia inducible factor. Genes Dev 2002, 16:1466-1471. See annotation [33]. 33. Hewitson KS, McNeill LA, Riordan MV, Tian YM, Bullock AN, Welford RW, Elkins JM, Oldham NJ, Bhattacharya S, Gleadle JM et al.: Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family. J Biol Chem 2002, 277:26351-26355. This paper and [32] show that FIH-1, the HIF-interacting protein identi®ed earlier by Mahon et al. [36], is the enzyme responsible for the asparaginyl hydroxylation of HIF. 34. Freedman S, Sun Z, Poy F, Kung A, Livingston D, Wagner G, Eck M: Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1alpha. Proc Natl Acad Sci USA 2002, 99:5367-5372. 35. Dames S, Martinez-Yamout M, De Guzman R, Dyson H, Wright P: From the cover: structural basis for Hif-1alpha /CBP recognition in the cellular hypoxic response. Proc Natl Acad Sci USA 2002, 99:5271-5276. 36. Mahon P, Hirota K, Semenza G: FIH-1: a novel protein that interacts with HIF-1a and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev 2001, 15:2675-2686. 37. Willam C, Masson N, Tian YM, Mahmood SA, Wilson MI, Bicknell R, Eckardt KU, Maxwell PH, Ratcliffe PJ, Pugh CW: Peptide blockade of HIFalpha degradation modulates cellular metabolism and angiogenesis. Proc Natl Acad Sci USA 2002, 99:10423-10428. 38. Gothie E, Richard D, Berra E, Pages G, Pouyssegur J: Identi®cation of alternative spliced variants of human hypoxia-inducible factor-1alpha. J Biol Chem 2000, 275:6922-6927. 39. Bindra RS, Vasselli JR, Stearman R, Linehan WM, Klausner RD: VHL-mediated hypoxia regulation of cyclin D1 in renal carcinoma cells. Cancer Res 2002, 62:3014-3019. 40. Zatyka M, da Silva NF, Clifford SC, Morris MR, Wiesener MS, Eckardt KU, Houlston RS, Richards FM, Latif F, Maher ER: Identi®cation of cyclin D1 and other novel targets for the von Hippel±Lindau tumor suppressor gene by expression array analysis and investigation of cyclin D1 genotype as a modi®er in von Hippel±Lindau disease. Cancer Res 2002, 62:3803-3811. 41. Davidowitz E, Schoenfeld A, Burk R: VHL induces renal cell differentiation and growth arrest through integration of cell-cell and cell-extracellular matrix signaling. Mol Cell Biol 2001, 21:865-874. Current Opinion in Genetics & Development 2003, 13:55±60
60 Oncogenes and cell proliferation
42. Okuda H, Saitoh K, Hirai S, Iwai K, Takaki Y, Baba M, Minato N, Ohno S, Shuin T: The von Hippel±Lindau tumor suppressor protein mediates ubiquitination of activated atypical protein kinase C. J Biol Chem 2001, 276:43611-43617.
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