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The Elongin BC complex and the von Hippel–Lindau tumor suppressor protein
Biochimica et Biophysica Acta 1377 Ž1998. M49–M54
Mini review
The Elongin BC complex and the von Hippel–Lindau tumor suppressor protein Joan Weliky Conaway
a,b,c
, Takumi Kamura b, Ronald C. Conaway
b,)
a Howard Hughes Medical Institute, Oklahoma City, OK 73104, USA Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA Department of Biochemistry and Molecular Biology, UniÕersity of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA b
c
Received 1 October 1997; accepted 1 October 1997
Keywords: Elongins; RNA polymerase II; von Hippel–Lindau tumor suppressor; Vascular endothelial growth factor
1. The Elongins The Elongins were initially identified as components of the 3-subunit Elongin complex, 1 which is one of several transcription factors that are capable of activating the overall rate of elongation by RNA polymerase II in vitro by suppressing transient pausing by polymerase at many sites along the DNA template w1,2x. The Elongin complex is composed of a transcriptionally active A subunit of ; 770 amino acids w3x and two small ; 110 amino acid B and C regulatory subunits w4,5x. Elongin B and C form an isolable Elongin BC complex that functions as a potent inducer of Elongin A transcriptional activity. Elongin B and C perform different functions in regulation of Elongin A activity w3x: Elongin C functions as the inducing ligand and is capable of binding directly to a binding site in the Elongin A elongation activation domain to form a relatively unstable, but
)
Corresponding author. Tel.: q1-405-271-1950; fax: q1-405271-1580. 1 Originally referred to as SII.
highly active Elongin AC complex; Elongin B, via its N-terminal ubiquitin homology Ž UbH. domain, binds directly to Elongin C and promotes formation of the highly stable Elongin ABC complex w3,6x.
2. The Elongin BC complex and the von Hippel– Lindau tumor suppressor protein An expanded role for the Elongin BC complex in cell regulation was recently brought to light by the discovery that Elongins B and C are integral components of a multi-protein complex containing the product of the Õon Hippel–Lindau (VHL) tumor suppressor gene w7–9x. The VHL gene on chromosome 3p25.5 is mutated in the majority of sporadic clear cell renal carcinomas and in VHL disease, an autosomal dominant familial cancer syndrome that predisposes affected individuals to a variety of tumors including clear cell renal carcinomas, cerebellar hemangioblastomas and hemangiomas, retinal angiomata, and pheochromocytomas w10–16x. Evidence that Elongin BC plays an important role in tumor suppression by pVHL has come from analy-
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complex is unlikely to account for pVHL tumor suppressor activity.
3. A role for the pVHL-Elongin BC complex in negative regulation of VEGF and other hypoxiainducible genes Fig. 1. The Elongin BC binding site motif in Elongin A and pVHL from different species. Elo A, Elongin A.
sis of naturally-occurring pVHL mutants. Elongin A and pVHL share a conserved Elongin BC binding site m o tif w ith consensus sequence ŽT,S.LxxxŽC,S.xxxŽL,V,I. ŽFig. 1.; this binding site is the only region of significant similarity between the two proteins. More than 70% of VHL mutations found in VHL kindreds and sporadic clear cell renal carcinomas lead to mutation or deletion of the pVHL Elongin BC binding site, and, in all cases tested, these pVHL mutants exhibit substantially reduced binding to Elongin BC w7–9x. The observation that Elongin A and pVHL share a conserved Elongin BC binding site initially led to the suggestion that pVHL may function in cells as a negative regulator of Elongin transcriptional activity by competing with Elongin A for binding to the Elongin BC complex. Two predictions of this simple ‘competition’ model are Ži. that pVHL is capable of inhibiting the activity of the Elongin BC complex and Žii. that the concentration of the Elongin BC complex in cells is limiting relative to those of either Elongin A or pVHL. Biochemical studies have shown that binding of Elongin A and pVHL to the Elongin BC complex is mutually exclusive and, further, that pVHL is capable of inhibiting Elongin transcriptional activity in vitro by binding tightly to the Elongin BC complex and preventing it from interacting with Elongin A w7x. We observe, however, that the Elongin BC complex is 100- to 1000-fold more abundant than Elongin A and pVHL in cell extracts Ž T.K., J.W.C., R.C.C., unpublished results.. Although we do not yet know what fraction of the excess cellular Elongin BC is free and available for binding to Elongin A and pVHL, these results suggest that, in its simplest form, the model that pVHL regulates Elongin transcriptional activity by competing for the Elongin BC
The function of the pVHL–Elongin BC complex in cells has not been established. A potentially important clue has come from recent findings suggesting that the pVHL–Elongin BC complex is involved in negative regulation of expression of Õascular endothelial growth factor (VEGF) and other hypoxiainducible genes. VEGF expression is believed to play a critical role in the neovascularization and resulting malignant growth of many solid tumors. In normal cells and most cell lines, VEGF expression is repressed under normal growth conditions, but is strongly induced in cells deprived of oxygen or serum. In contrast, VEGF is expressed at high levels in many tumors including VHL disease-associated and sporadic hemangioblastomas and clear cell renal carcinomas w17–20x, and VEGF and other hypoxia-inducible genes including platelet-deriÕed growth factor B (PDGF-B) and glucose transporter 1 (GLUT1) are expressed constitutively by clear cell renal carcinoma cell lines lacking a functional VHL gene w21– 23x. In a series of elegant experiments carried out in several laboratories, it was discovered that introduction of the wild type VHL gene into a clear cell renal carcinoma cell line was sufficient to repress VEGF, PDGF-B, and GLUT1 expression under normoxic conditions and to restore their normal regulation by hypoxia w22,23x. In addition, it was found that introduction of the wild type VHL gene into several other clear cell renal carcinoma cell lines was sufficient to restore normal VEGF regulation by serum deprivation w21x. Finally, evidence arguing strongly that the pVHL–Elongin BC interaction is critical for negative regulation of the VEGF, PDGF-B, and GLUT1 genes came from findings indicating that normal regulation of these genes by hypoxia or serum deprivation could not be restored to clear cell renal carcinoma cell lines by introduction of genes encoding mutant pVHLs that fail to interact with the Elongin BC complex w21–24x.
J.W. Conaway et al.r Biochimica et Biophysica Acta 1377 (1998) M49–M54
4. The mechanism of regulation of hypoxia-inducible genes by the pVHL-Elongin BC complex Although the evidence described above suggests that the pVHL–Elongin BC complex may function to maintain hypoxia-inducible genes in a repressed state under normal cell growth conditions, the mechanism of repression is not understood. Expression of hypoxia-inducible genes can be regulated by at least two mechanisms. The DNA binding transcriptional activator hypoxia inducible factor 1 ŽHIF1. activates transcription through interactions with enhancers located 5X or 3X of hypoxia-inducible genes w25–28x. HIF1 functions as a heterodimer composed of HIF1a , a novel member of the bHLH PAS Ž PER-ARNTSIM. family of transcription factors, and HIF1-b , another bHLH PAS family member, which was previously identified as the aryl hydrocarbon receptor nuclear translocator protein Ž ARNT. w29,30x. Hypoxic activation of HIF1 activity results in large part from an increase in cellular concentrations of HIF1-a , which is rapidly degraded under normoxic conditions by the ubiquitin-dependent proteasome system, but is stabilized by hypoxia w31,32x. Expression of hypoxia-inducible genes can also be activated in a poorly understood process that results in increases in the stabilities of their mRNAs. The 3X-UTRs of hypoxia-inducible and other normally unstable mRNAs contain AU-rich elements that appear to target them for rapid degradation w33–36x. Hypoxia-inducible RNA-binding activities that interact with AU-rich elements in the VEGF 3X-UTR in vitro assays have been identified w34x, although it remains to be determined whether these activities play a role in regulating VEGF mRNA stability in vivo. On the basis of studies carried out with cells lacking functional HIF1 w37,38x, it has been proposed that hypoxia-inducible genes fall into two classes: those, such as PDGF-B, which are induced solely at the transcriptional level via the action of HIF1, and those, such as VEGF and GLUT1, which are induced both by HIF1 and by increases in the stabilities of their mRNAs. To date, efforts to determine whether the pVHL– Elongin BC complex represses expression of VEGF and other hypoxia-inducible genes by decreasing their transcription rates, by decreasing the stabilities of their mRNAs, or by both mechanisms have yielded
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conflicting results. In one study, expression of the wild type VHL gene in the 786-0 clear cell renal carcinoma cell line led to a decrease in the half-life of VEGF mRNA under normoxic conditions without affecting expression of a reporter gene driven by the VEGF promoter w22x. In this same study, expression of the wild type VHL gene in the same cell line substantially reduced PDGF-B mRNA levels under normoxic conditions. As described above, PDGF-B mRNA levels are believed to be regulated solely at the transcriptional level via HIF1; it will, therefore, be informative to determine whether expression of VHL affects PDGF-B transcription rates, mRNA stability, or both. In a second study, expression of the wild type VHL gene in several clear cell renal carcinoma cell lines significantly reduced VEGF mRNA levels under normal growth conditions without detectably affecting either VEGF transcription rates measured in nuclear run-on assays or the half-life of VEGF mRNA w21x. In contrast, a third study found that expression of the wild type VHL gene in the 786-0 clear cell renal carcinoma cell line reduced VEGF transcription rates measured in nuclear run-on assays, while over-expression of wild type pVHL in several non-renal carcinoma cell lines strongly reduced VEGF promoter activity in transient transfection assays w24x. Based on the additional findings Ž i. that negative regulation of VEGF promoter activity by pVHL depends on transcription factor Sp1, as well as on the presence of a functional Sp1 binding site in the promoter, and Žii. that pVHL can bind to Sp1 in vitro, the authors of this study proposed that pVHL may repress VEGF expression under normal growth conditions at least in part by negatively regulating VEGF transcription through direct interactions with Sp1. Finally, evidence from a fourth study suggests that pVHL may play a role in activation of VEGF expression during placental development. Contrary to what would be predicted based on the observation that pVHL negatively regulates VEGF expression in clear cell renal carcinoma cell lines, VHLŽyry . mice fail to express sufficient VEGF protein to support development of the placental vasculature and therefore die in utero after 10.5 to 12.5 days of gestation w39x. Considered together, the results of these studies are difficult to reconcile with a single model of
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pVHL function. Although some of the apparent discrepancies among these results could be attributable to differences in the cell lines or specific assay conditions used, it may ultimately turn out that the pVHL–Elongin BC complex is capable of regulating gene expression at several levels, through effects on both transcription and mRNA stability.
5. Future directions It is likely that an understanding of the functionŽ s. of the pVHL–Elongin BC complex and its role in tumor suppression will require a knowledge of the complete repertoire of cellular proteins that interact with the complex and with its individual components, as well as the development of new assays for defining its role in such processes as transcription and mRNA stability. A significant advance in efforts to identify cellular proteins that interact with the pVHL–Elongin BC complex was the recent discovery that the product of the Cul2 gene, a member of a gene family that includes C. elegans cul-1 and S. cereÕisiae CDC53, is a stoichiometric component of the complex w40x. Interaction of the Cul2 protein with the pVHL–Elongin BC complex depends on Elongin BC. Although the function of the Cul2 protein in cells is not known, C. elegans cul-1 and S. cereÕisiae CDC53 mutants exhibit cell cycle phenotypes. The cul-1 protein is required for transition of C. elegans cells from G1 to G0 or from G1 to the apoptotic pathway, and null mutations of cul-1 lead to hyperplasia of all C. elegans tissues w41x. The CDC53 protein, which has been proposed to function as an E3 ubiquitin-protein ligase, is required for ubiquitination of G1 cyclins prior to their degradation by the ubiquitin-dependent proteosome system w42x. Whether the Cul2 protein also functions in the ubiquitin-dependent proteolytic pathway is not known. It is tempting to speculate that the pVHL–Elongin BCCul2 complex could regulate ubiquitin-dependent proteolysis of proteins that control the transcription andror stability of hypoxia-inducible genes. In this regard, it is noteworthy that HIF1 levels are also controlled by the ubiquitin-dependent proteosome system w31,32x, and it will be interesting to test the
possibility that HIF1 activity could, under some conditions, be affected by VHL expression. Understanding what role the pVHL–Elongin BC complex plays in regulating transcription will require further investigations both of the mechanism by which pVHL inhibits Sp1-dependent transcription and of the functional relationship between pVHL and Elongin A. As discussed above, the model that pVHL negatively regulates Elongin transcriptional activity simply by competing with Elongin A for binding to the Elongin BC complex is unlikely to account for pVHL tumor suppressor activity, since we find that the Elongin BC complex is substantially more abundant than either pVHL or Elongin A in cell extracts. It is still possible, however, that pVHL could control the amount of Elongin BC available for binding to Elongin A by other mechanisms. Results of biochemical fractionations indicate that Elongin A is a nuclear protein, whereas the bulk of Elongin BC is present in post-nuclear supernatants Ž T.K., J.W.C., R.C.C., unpublished data.. In light of evidence that the nuclear localization of pVHL, Elongin BC, and the Cul2 protein is regulated by cell density-dependent w43x andror cell cycle-dependent mechanisms Ž B.R.G. Williams, personal communication. , it is conceivable that pVHL could regulate Elongin activity by controlling the amount of Elongin BC that is present in the nucleus and available for interaction with Elongin A. Understanding what role the pVHL–Elongin BC complex plays in regulating mRNA stability will require development of biochemical assays for identifying the enzymes and proteins that control mRNA stability and for elucidating their mechanisms of action. Notably, the hypoxia-induced RNA-binding activity that interacts with AU-rich elements in the VEGF 3X-UTR is elevated in clear cell renal carcinoma cells lacking wild type pVHL w44x. Further characterization of this novel activity should shed light on the cellular mechanisms underlying regulation of VEGF mRNA stability. Finally, by systematically fractionating cell extracts, we have discovered that Elongins B and C are components of multiple, chromatographically distinct species, in addition to the Elongin A and pVHL– Elongin BC complexes ŽT.K., J.W.C., R.C.C., unpublished results. . Because it is likely that an understanding of the complete range of cellular functions of the Elongin BC complex will be essential for an under-
J.W. Conaway et al.r Biochimica et Biophysica Acta 1377 (1998) M49–M54
standing of its role in the pVHL–Elongin BC complex, efforts to identify these additional Elongin BCassociated proteins and to establish their functions are underway.
Acknowledgements Work in the authors’ laboratory is supported by National Institutes of Health Grant GM41628 and by funds provided to the Oklahoma Medical Research Foundation by the H.A. and Mary K. Chapman Charitable Trust. J.W.C. is an Associate Investigator of the Howard Hughes Medical Institute.
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Mini review
The Elongin BC complex and the von Hippel–Lindau tumor suppressor protein Joan Weliky Conaway
a,b,c
, Takumi Kamura b, Ronald C. Conaway
b,)
a Howard Hughes Medical Institute, Oklahoma City, OK 73104, USA Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA Department of Biochemistry and Molecular Biology, UniÕersity of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA b
c
Received 1 October 1997; accepted 1 October 1997
Keywords: Elongins; RNA polymerase II; von Hippel–Lindau tumor suppressor; Vascular endothelial growth factor
1. The Elongins The Elongins were initially identified as components of the 3-subunit Elongin complex, 1 which is one of several transcription factors that are capable of activating the overall rate of elongation by RNA polymerase II in vitro by suppressing transient pausing by polymerase at many sites along the DNA template w1,2x. The Elongin complex is composed of a transcriptionally active A subunit of ; 770 amino acids w3x and two small ; 110 amino acid B and C regulatory subunits w4,5x. Elongin B and C form an isolable Elongin BC complex that functions as a potent inducer of Elongin A transcriptional activity. Elongin B and C perform different functions in regulation of Elongin A activity w3x: Elongin C functions as the inducing ligand and is capable of binding directly to a binding site in the Elongin A elongation activation domain to form a relatively unstable, but
)
Corresponding author. Tel.: q1-405-271-1950; fax: q1-405271-1580. 1 Originally referred to as SII.
highly active Elongin AC complex; Elongin B, via its N-terminal ubiquitin homology Ž UbH. domain, binds directly to Elongin C and promotes formation of the highly stable Elongin ABC complex w3,6x.
2. The Elongin BC complex and the von Hippel– Lindau tumor suppressor protein An expanded role for the Elongin BC complex in cell regulation was recently brought to light by the discovery that Elongins B and C are integral components of a multi-protein complex containing the product of the Õon Hippel–Lindau (VHL) tumor suppressor gene w7–9x. The VHL gene on chromosome 3p25.5 is mutated in the majority of sporadic clear cell renal carcinomas and in VHL disease, an autosomal dominant familial cancer syndrome that predisposes affected individuals to a variety of tumors including clear cell renal carcinomas, cerebellar hemangioblastomas and hemangiomas, retinal angiomata, and pheochromocytomas w10–16x. Evidence that Elongin BC plays an important role in tumor suppression by pVHL has come from analy-
0304-419Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 9 X Ž 9 7 . 0 0 0 3 5 - 8
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J.W. Conaway et al.r Biochimica et Biophysica Acta 1377 (1998) M49–M54
complex is unlikely to account for pVHL tumor suppressor activity.
3. A role for the pVHL-Elongin BC complex in negative regulation of VEGF and other hypoxiainducible genes Fig. 1. The Elongin BC binding site motif in Elongin A and pVHL from different species. Elo A, Elongin A.
sis of naturally-occurring pVHL mutants. Elongin A and pVHL share a conserved Elongin BC binding site m o tif w ith consensus sequence ŽT,S.LxxxŽC,S.xxxŽL,V,I. ŽFig. 1.; this binding site is the only region of significant similarity between the two proteins. More than 70% of VHL mutations found in VHL kindreds and sporadic clear cell renal carcinomas lead to mutation or deletion of the pVHL Elongin BC binding site, and, in all cases tested, these pVHL mutants exhibit substantially reduced binding to Elongin BC w7–9x. The observation that Elongin A and pVHL share a conserved Elongin BC binding site initially led to the suggestion that pVHL may function in cells as a negative regulator of Elongin transcriptional activity by competing with Elongin A for binding to the Elongin BC complex. Two predictions of this simple ‘competition’ model are Ži. that pVHL is capable of inhibiting the activity of the Elongin BC complex and Žii. that the concentration of the Elongin BC complex in cells is limiting relative to those of either Elongin A or pVHL. Biochemical studies have shown that binding of Elongin A and pVHL to the Elongin BC complex is mutually exclusive and, further, that pVHL is capable of inhibiting Elongin transcriptional activity in vitro by binding tightly to the Elongin BC complex and preventing it from interacting with Elongin A w7x. We observe, however, that the Elongin BC complex is 100- to 1000-fold more abundant than Elongin A and pVHL in cell extracts Ž T.K., J.W.C., R.C.C., unpublished results.. Although we do not yet know what fraction of the excess cellular Elongin BC is free and available for binding to Elongin A and pVHL, these results suggest that, in its simplest form, the model that pVHL regulates Elongin transcriptional activity by competing for the Elongin BC
The function of the pVHL–Elongin BC complex in cells has not been established. A potentially important clue has come from recent findings suggesting that the pVHL–Elongin BC complex is involved in negative regulation of expression of Õascular endothelial growth factor (VEGF) and other hypoxiainducible genes. VEGF expression is believed to play a critical role in the neovascularization and resulting malignant growth of many solid tumors. In normal cells and most cell lines, VEGF expression is repressed under normal growth conditions, but is strongly induced in cells deprived of oxygen or serum. In contrast, VEGF is expressed at high levels in many tumors including VHL disease-associated and sporadic hemangioblastomas and clear cell renal carcinomas w17–20x, and VEGF and other hypoxia-inducible genes including platelet-deriÕed growth factor B (PDGF-B) and glucose transporter 1 (GLUT1) are expressed constitutively by clear cell renal carcinoma cell lines lacking a functional VHL gene w21– 23x. In a series of elegant experiments carried out in several laboratories, it was discovered that introduction of the wild type VHL gene into a clear cell renal carcinoma cell line was sufficient to repress VEGF, PDGF-B, and GLUT1 expression under normoxic conditions and to restore their normal regulation by hypoxia w22,23x. In addition, it was found that introduction of the wild type VHL gene into several other clear cell renal carcinoma cell lines was sufficient to restore normal VEGF regulation by serum deprivation w21x. Finally, evidence arguing strongly that the pVHL–Elongin BC interaction is critical for negative regulation of the VEGF, PDGF-B, and GLUT1 genes came from findings indicating that normal regulation of these genes by hypoxia or serum deprivation could not be restored to clear cell renal carcinoma cell lines by introduction of genes encoding mutant pVHLs that fail to interact with the Elongin BC complex w21–24x.
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4. The mechanism of regulation of hypoxia-inducible genes by the pVHL-Elongin BC complex Although the evidence described above suggests that the pVHL–Elongin BC complex may function to maintain hypoxia-inducible genes in a repressed state under normal cell growth conditions, the mechanism of repression is not understood. Expression of hypoxia-inducible genes can be regulated by at least two mechanisms. The DNA binding transcriptional activator hypoxia inducible factor 1 ŽHIF1. activates transcription through interactions with enhancers located 5X or 3X of hypoxia-inducible genes w25–28x. HIF1 functions as a heterodimer composed of HIF1a , a novel member of the bHLH PAS Ž PER-ARNTSIM. family of transcription factors, and HIF1-b , another bHLH PAS family member, which was previously identified as the aryl hydrocarbon receptor nuclear translocator protein Ž ARNT. w29,30x. Hypoxic activation of HIF1 activity results in large part from an increase in cellular concentrations of HIF1-a , which is rapidly degraded under normoxic conditions by the ubiquitin-dependent proteasome system, but is stabilized by hypoxia w31,32x. Expression of hypoxia-inducible genes can also be activated in a poorly understood process that results in increases in the stabilities of their mRNAs. The 3X-UTRs of hypoxia-inducible and other normally unstable mRNAs contain AU-rich elements that appear to target them for rapid degradation w33–36x. Hypoxia-inducible RNA-binding activities that interact with AU-rich elements in the VEGF 3X-UTR in vitro assays have been identified w34x, although it remains to be determined whether these activities play a role in regulating VEGF mRNA stability in vivo. On the basis of studies carried out with cells lacking functional HIF1 w37,38x, it has been proposed that hypoxia-inducible genes fall into two classes: those, such as PDGF-B, which are induced solely at the transcriptional level via the action of HIF1, and those, such as VEGF and GLUT1, which are induced both by HIF1 and by increases in the stabilities of their mRNAs. To date, efforts to determine whether the pVHL– Elongin BC complex represses expression of VEGF and other hypoxia-inducible genes by decreasing their transcription rates, by decreasing the stabilities of their mRNAs, or by both mechanisms have yielded
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conflicting results. In one study, expression of the wild type VHL gene in the 786-0 clear cell renal carcinoma cell line led to a decrease in the half-life of VEGF mRNA under normoxic conditions without affecting expression of a reporter gene driven by the VEGF promoter w22x. In this same study, expression of the wild type VHL gene in the same cell line substantially reduced PDGF-B mRNA levels under normoxic conditions. As described above, PDGF-B mRNA levels are believed to be regulated solely at the transcriptional level via HIF1; it will, therefore, be informative to determine whether expression of VHL affects PDGF-B transcription rates, mRNA stability, or both. In a second study, expression of the wild type VHL gene in several clear cell renal carcinoma cell lines significantly reduced VEGF mRNA levels under normal growth conditions without detectably affecting either VEGF transcription rates measured in nuclear run-on assays or the half-life of VEGF mRNA w21x. In contrast, a third study found that expression of the wild type VHL gene in the 786-0 clear cell renal carcinoma cell line reduced VEGF transcription rates measured in nuclear run-on assays, while over-expression of wild type pVHL in several non-renal carcinoma cell lines strongly reduced VEGF promoter activity in transient transfection assays w24x. Based on the additional findings Ž i. that negative regulation of VEGF promoter activity by pVHL depends on transcription factor Sp1, as well as on the presence of a functional Sp1 binding site in the promoter, and Žii. that pVHL can bind to Sp1 in vitro, the authors of this study proposed that pVHL may repress VEGF expression under normal growth conditions at least in part by negatively regulating VEGF transcription through direct interactions with Sp1. Finally, evidence from a fourth study suggests that pVHL may play a role in activation of VEGF expression during placental development. Contrary to what would be predicted based on the observation that pVHL negatively regulates VEGF expression in clear cell renal carcinoma cell lines, VHLŽyry . mice fail to express sufficient VEGF protein to support development of the placental vasculature and therefore die in utero after 10.5 to 12.5 days of gestation w39x. Considered together, the results of these studies are difficult to reconcile with a single model of
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pVHL function. Although some of the apparent discrepancies among these results could be attributable to differences in the cell lines or specific assay conditions used, it may ultimately turn out that the pVHL–Elongin BC complex is capable of regulating gene expression at several levels, through effects on both transcription and mRNA stability.
5. Future directions It is likely that an understanding of the functionŽ s. of the pVHL–Elongin BC complex and its role in tumor suppression will require a knowledge of the complete repertoire of cellular proteins that interact with the complex and with its individual components, as well as the development of new assays for defining its role in such processes as transcription and mRNA stability. A significant advance in efforts to identify cellular proteins that interact with the pVHL–Elongin BC complex was the recent discovery that the product of the Cul2 gene, a member of a gene family that includes C. elegans cul-1 and S. cereÕisiae CDC53, is a stoichiometric component of the complex w40x. Interaction of the Cul2 protein with the pVHL–Elongin BC complex depends on Elongin BC. Although the function of the Cul2 protein in cells is not known, C. elegans cul-1 and S. cereÕisiae CDC53 mutants exhibit cell cycle phenotypes. The cul-1 protein is required for transition of C. elegans cells from G1 to G0 or from G1 to the apoptotic pathway, and null mutations of cul-1 lead to hyperplasia of all C. elegans tissues w41x. The CDC53 protein, which has been proposed to function as an E3 ubiquitin-protein ligase, is required for ubiquitination of G1 cyclins prior to their degradation by the ubiquitin-dependent proteosome system w42x. Whether the Cul2 protein also functions in the ubiquitin-dependent proteolytic pathway is not known. It is tempting to speculate that the pVHL–Elongin BCCul2 complex could regulate ubiquitin-dependent proteolysis of proteins that control the transcription andror stability of hypoxia-inducible genes. In this regard, it is noteworthy that HIF1 levels are also controlled by the ubiquitin-dependent proteosome system w31,32x, and it will be interesting to test the
possibility that HIF1 activity could, under some conditions, be affected by VHL expression. Understanding what role the pVHL–Elongin BC complex plays in regulating transcription will require further investigations both of the mechanism by which pVHL inhibits Sp1-dependent transcription and of the functional relationship between pVHL and Elongin A. As discussed above, the model that pVHL negatively regulates Elongin transcriptional activity simply by competing with Elongin A for binding to the Elongin BC complex is unlikely to account for pVHL tumor suppressor activity, since we find that the Elongin BC complex is substantially more abundant than either pVHL or Elongin A in cell extracts. It is still possible, however, that pVHL could control the amount of Elongin BC available for binding to Elongin A by other mechanisms. Results of biochemical fractionations indicate that Elongin A is a nuclear protein, whereas the bulk of Elongin BC is present in post-nuclear supernatants Ž T.K., J.W.C., R.C.C., unpublished data.. In light of evidence that the nuclear localization of pVHL, Elongin BC, and the Cul2 protein is regulated by cell density-dependent w43x andror cell cycle-dependent mechanisms Ž B.R.G. Williams, personal communication. , it is conceivable that pVHL could regulate Elongin activity by controlling the amount of Elongin BC that is present in the nucleus and available for interaction with Elongin A. Understanding what role the pVHL–Elongin BC complex plays in regulating mRNA stability will require development of biochemical assays for identifying the enzymes and proteins that control mRNA stability and for elucidating their mechanisms of action. Notably, the hypoxia-induced RNA-binding activity that interacts with AU-rich elements in the VEGF 3X-UTR is elevated in clear cell renal carcinoma cells lacking wild type pVHL w44x. Further characterization of this novel activity should shed light on the cellular mechanisms underlying regulation of VEGF mRNA stability. Finally, by systematically fractionating cell extracts, we have discovered that Elongins B and C are components of multiple, chromatographically distinct species, in addition to the Elongin A and pVHL– Elongin BC complexes ŽT.K., J.W.C., R.C.C., unpublished results. . Because it is likely that an understanding of the complete range of cellular functions of the Elongin BC complex will be essential for an under-
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standing of its role in the pVHL–Elongin BC complex, efforts to identify these additional Elongin BCassociated proteins and to establish their functions are underway.
Acknowledgements Work in the authors’ laboratory is supported by National Institutes of Health Grant GM41628 and by funds provided to the Oklahoma Medical Research Foundation by the H.A. and Mary K. Chapman Charitable Trust. J.W.C. is an Associate Investigator of the Howard Hughes Medical Institute.
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