SMART MODE:

NORMAL GENOMIC

• Kamura T et al.
• Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase.
• J Biol Chem. 2001; 276: 29748-53
• Display abstract

• The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.

• Ivan M et al.
• HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.
• Science. 2001; 292: 464-8
• Display abstract

• HIF (hypoxia-inducible factor) is a transcription factor that plays a pivotal role in cellular adaptation to changes in oxygen availability. In the presence of oxygen, HIF is targeted for destruction by an E3 ubiquitin ligase containing the von Hippel-Lindau tumor suppressor protein (pVHL). We found that human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. Because proline hydroxylation requires molecular oxygen and Fe(2+), this protein modification may play a key role in mammalian oxygen sensing.

• Bonicalzi ME, Groulx I, de Paulsen N, Lee S
• Role of exon 2-encoded beta -domain of the von Hippel-Lindau tumor suppressor protein.
• J Biol Chem. 2001; 276: 1407-16
• Display abstract

• Sporadic clear cell renal carcinomas frequently harbor inactivating mutations in exon 2 of the von Hippel-Lindau (VHL) tumor suppressor gene. Here, we examine the effect of the loss of exon 2-encoded beta-domain function on VHL biochemical properties. Exon 2-encoded residues are required for VHL-mediated NEDD8 conjugation on cullin-2 and assembly with hypoxia-inducible factor alpha (HIFalpha) and fibronectin. These residues are not essential for VHL ability to assemble with elongin BC/cullin-2, to display E3 ubiquitin ligase activity in vitro and to confer energy-dependent nuclear import properties to a reporter protein. Localization studies in HIF-1alpha-null embryonic cells suggest that exon 2-encoded beta-domain mediates transcription-dependent nuclear/cytoplasmic shuttling of VHL independently of assembly with HIF-1alpha and oxygen concentration. Exon 3-encoded alpha-helical domain is required for VHL complex formation with BC/cullin-2 and E3 ubiquitin ligase activity, for binding to HIFalpha/fibronectin, but this domain is not essential for transcription-dependent nuclear/cytoplasmic trafficking. VHL(-/-) renal carcinoma cells expressing beta-domain mutants failed to produce an extracellular fibronectin matrix and to degrade HIFalpha, which accumulated exclusively in the nucleus of normoxic cells. These results demonstrate that exon 2-encoded residues are involved in two independent functions: substrate protein recognition and transcription-dependent nuclear/cytoplasmic trafficking. They also suggest that beta-domain mutations inactivate VHL function differently than alpha-domain mutations, potentially providing an explanation for the relationship between different mutations of the VHL gene and clinical outcome.

• Yu F, White SB, Zhao Q, Lee FS
• Dynamic, site-specific interaction of hypoxia-inducible factor-1alpha with the von Hippel-Lindau tumor suppressor protein.
• Cancer Res. 2001; 61: 4136-42
• Display abstract

• Hypoxia-inducible factor (HIF)-1alpha is a transcription factor that plays a critical role in regulating genes involved in erythropoiesis and angiogenesis. Recent evidence indicates that the von Hippel-Lindau tumor suppressor protein (VHL) is part of a ubiquitin ligase complex that promotes the degradation of HIF-1alpha under normoxic conditions. Under hypoxic conditions, HIF-1alpha is markedly stabilized. A critical issue in understanding the hypoxic response is the identification of hypoxia-regulated steps. We show here that hypoxia and cobalt treatment modulate the capacity of a HIF-1alpha fragment comprising residues 531-652 to coimmunoprecipitate with VHL. Hypoxia and cobalt both significantly diminish the interaction, and furthermore, normoxia treatment after hypoxia rapidly normalizes it. This HIF-1alpha fragment confers hypoxia and cobalt inducibility on a heterologous protein. Significantly, contained within this fragment is a short 27-residue sequence that behaves identically in all respects noted above. Finally, evidence is provided to show that cobalt and hypoxia both induce a posttranslational modification (or loss of one) in HIF-1alpha that affects its binding to VHL. We propose that dynamic, site-specific interaction of HIF-1alpha with VHL provides one mechanism by which HIF-1alpha can be regulated.

• Ivan M, Kaelin WG Jr
• The von Hippel-Lindau tumor suppressor protein.
• Curr Opin Genet Dev. 2001; 11: 27-34
• Display abstract

• The von Hippel-Lindau tumor suppressor protein (pVHL) has been shown to bind directly to the alpha subunits of the heterodimeric transcription factor HIF (hypoxia inducible factor). pVHL directs the polyubiquitination and, hence, destruction of HIF in the presence of oxygen. Loss of pVHL function leads to deregulation of HIF target genes, which play critical roles in angiogenesis.

• Tanimoto K, Makino Y, Pereira T, Poellinger L
• Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein.
• EMBO J. 2000; 19: 4298-309
• Display abstract

• In normoxic cells the hypoxia-inducible factor-1 alpha (HIF-1 alpha) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1 alpha to a functional form requires protein stabilization. Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1 alpha under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1 alpha. The region of VHL mediating interaction with HIF-1 alpha overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1 alpha and subsequent degradation. Interestingly, the VHL binding site within HIF-1 alpha overlapped with one of the minimal transactivation domains. Protection of HIF-1 alpha against degradation by VHL was a multistep mechanism, including hypoxia-induced nuclear translocation of HIF-1 alpha and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1 alpha during this process. Finally, stabilization of HIF-1 alpha protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.

• Adryan B, Decker HJ, Papas TS, Hsu T
• Tracheal development and the von Hippel-Lindau tumor suppressor homolog in Drosophila.
• Oncogene. 2000; 19: 2803-11
• Display abstract

• von Hippel-Lindau disease is a hereditary cancer syndrome. Mutations in the VHL tumor suppressor gene predispose individuals to highly vascularized tumors. However, VHL-deficient mice die in utero due to a lack of vascularization in the placenta. To resolve the contradiction, we cloned the Drosophila VHL homologue (d-VHL) and studied its function. It showed an overall 50% similarity to the human counterpart and 76% similarity in the crucial functional domain: the elongin C binding site. The putative d-VHL protein can bind Drosophila elongin C in vitro. During embryogenesis, d-VHL is expressed in the developing tracheal regions where tube outgrowth no longer occurs. Reduced d-VHL activity (using RNA interference methodology) caused breakage of the main vasculature accompanied by excessive looping of smaller branches, whereas over-expression caused a general lack of vasculature. Importantly, human VHL can induce the same gain-of-function phenotypes. VHL is likely involved in halting cell migration at the end of vascular tube outgrowth. Loss of VHL activity can therefore lead to disruption of major vasculature (as in the mouse embryo), which requires precise cell movement and tube fusion, or ectopic outgrowth from existing secondary vascular branches (as in the adult tumors). Oncogene (2000) 19, 2803 - 2811

• Ohh M et al.
• Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein.
• Nat Cell Biol. 2000; 2: 423-7
• Display abstract

• von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is characterized by the development of multiple vascular tumors and is caused by inactivation of the von Hippel-Lindau protein (pVHL). Here we show that pVHL, through its beta-domain, binds directly to hypoxia-inducible factor (HIF), thereby targeting HIF for ubiquitination in an alpha-domain-dependent manner. This is the first function to be ascribed to the pVHL beta-domain. Furthermore, we provide the first direct evidence that pVHL has a function analogous to that of an F-box protein, namely, to recruit substrates to a ubiquitination machine. These results strengthen the link between overaccumulation of HIF and development of VHL disease.

• Miyakis S, Sourvinos G, Liloglou TL, Stathopoulos GP, Field JK, Spandidos DA
• The Von Hippel-Lindau (VHL) tumor-suppressor gene is not mutated in sporadic human colon adenocarcinomas.
• Int J Cancer. 2000; 88: 503-5

• Aso T, Yamazaki K, Aigaki T, Kitajima S
• Drosophila von Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin ligase activity.
• Biochem Biophys Res Commun. 2000; 276: 355-61
• Display abstract

• Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene predispose individuals to a variety of human tumors, including renal cell carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. Here we report on the identification and characterization of the Drosophila homolog of VHL. The predicted amino acid sequence of Drosophila VHL protein shows 29% identity and 44% similarity to that of human VHL protein. Biochemical studies have shown that Drosophila VHL protein binds to Elongins B and C directly, and via this Elongin BC complex, associates with Cul-2 and Rbx1. Like human VHL, Drosophila VHL complex containing Cul-2, Rbx1, Elongins B and C, exhibits E3 ubiquitin ligase activity. In addition, we provide evidence that hypoxia-inducible factor (HIF)-1alpha is the ubiquitination target of both human and Drosophila VHL complexes.

• Woodward ER, Buchberger A, Clifford SC, Hurst LD, Affara NA, Maher ER
• Comparative sequence analysis of the VHL tumor suppressor gene.
• Genomics. 2000; 65: 253-65
• Display abstract

• Comparative genome analysis may provide novel insights into gene evolution and function. To investigate the von Hippel-Lindau (VHL) disease tumor suppressor gene, we sequenced the VHL gene in seven primate species. Comparative analysis was performed for human, primate, and rodent VHL genes and for a putative Caenorhabditis elegans VHL homologue identified by database analysis. The VHL gene has two translation initiation sites (at codons 1 and 54); however, the relative importance of the full-length translation product (pVHL30) and that translated from the second internal translation initiation site (pVHL19) is unclear. The N-terminal sequence of pVHL30 contains eight copies of a GXEEX acidic repeat motif in human and higher primates, but only three copies were present in the marmoset, and only one copy was present in rodent VHL genes. Evolutionary analysis suggested that the N-terminal repetitive sequence in pVHL30 was of less functional importance than those regions present in both pVHL30 and pVHL19. The VHL gene product is reported to form complexes with various proteins including elongin B, elongin C, VBP-1, fibronectin, Spl, CUL2, and HIF-1. Although most of the regions in pVHL that had been implicated in binding specific proteins demonstrated evolutionary conservation, the carboxy-terminal putative VBP-1 binding site was less well conserved, suggesting that VBP-1 binding may have less functional significance. Although an amino acid substitution (K171T) close to the pVHL elongin binding region was found in baboon, analysis of the structure of human pVHL suggested that this substitution would not interfere with pVHL/elongin C interaction. In general, there was a good correlation between the pVHL domains that demonstrated most evolutionary conservation and those that were most frequently mutated in tumors. Analysis of human/C. elegans conservation and human germline and somatic mutation patterns identified a highly conserved mutation cluster region between codons 74 and 90. However, this region is likely to be important for the structural integrity of pVHL rather than representing an additional protein binding domain.

• Shuin T, Ashida S, Yao M, Kanno H
• [Von Hippel-Lindau disease]
• Nippon Rinsho. 2000; 58: 1448-54
• Display abstract

• The von Hippel-Lindau (VHL) disease is a multitumor syndrome characterized by CNS hemangioblastoma, pheochromocytoma and renal cell carcinoma. The VHL disease gene is a putative tumor suppressor gene responsible for VHL disease. VHL disease without pheochromocytoma (pheo) and those with pheo are classified as type 1 or type 2, respectively. Germline missense mutations are clustered in 4 regions in the VHL gene. The VHL protein is now regarded as a one of the key protein for ubiquitination and further degradation of certain transcription factors such as hypoxia inducible factor (HIF). Since almost of germline mutations are detectable, presymptomatic diagnosis of the VHL disease with DNA analyses is important to improve the prognosis of VHL disease.

• Soussi T
• [File no. 41. VHL (von Hippel-Lindau). Tumor suppressor gene]
• Bull Cancer. 2000; 87: 771-2

• Schoenfeld AR, Davidowitz EJ, Burk RD
• Elongin BC complex prevents degradation of von Hippel-Lindau tumor suppressor gene products.
• Proc Natl Acad Sci U S A. 2000; 97: 8507-12
• Display abstract

• Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene causes the familial cancer syndrome, VHL disease, characterized by a predisposition to renal cell carcinoma and other tumor types. Loss of VHL gene function also is found in a majority of sporadic renal carcinomas. A preponderance of the tumor-disposing inherited missense mutations detected in VHL disease are within the elongin-binding domain of VHL. This region mediates the formation of a multiprotein VHL complex containing elongin B, elongin C, cul-2, and Rbx1. This VHL complex is thought to function as an E3 ubiquitin ligase. Here, we report that VHL proteins harboring mutations which disrupt elongin binding are unstable and rapidly degraded by the proteasome. In contrast, wild-type VHL proteins are directly stabilized by associating with both elongins B and C. In addition, elongins B and C are stabilized through their interactions with each other and VHL. Thus, the entire VHL/elongin complex is resistant to proteasomal degradation. Because the elongin-binding domain of VHL is frequently mutated in cancers, these results suggest that loss of elongin binding causes tumorigenesis by compromising VHL protein stability and/or potential VHL ubiquitination functions.

• Buchberger A et al.
• Biophysical characterization of elongin C from Saccharomyces cerevisiae.
• Biochemistry. 2000; 39: 11137-46
• Display abstract

• Elongin C (ELC) is an essential component of the mammalian CBC(VHL) E3 ubiquitin ligase complex. As a step toward understanding the role of ELC in assembly and function of CBC-type ubiquitin ligases, we analyzed the quaternary structure and backbone dynamics of the highly homologous Elc1 protein from Saccharomyces cerevisiae. Analytical ultracentrifugation experiments in conjunction with size exclusion chromatography showed that Elc1 is a nonglobular monomer over a wide range of concentrations. Pronounced line broadening in (1)H,(15)N-HSQC NMR spectra and failure to assign peaks corresponding to the carboxy-terminal helix 4 of Elc1 indicated that helix 4 is conformationally labile. Measurement of (15)N NMR relaxation parameters including T(1), T(2), and the (1)H-(15)N nuclear Overhauser effect revealed (i) surprisingly high flexibility of residues 69-77 in loop 5, and (ii) chemical exchange contributions for a large number of residues throughout the protein. Addition of 2,2,2-trifluoroethanol (TFE) stabilized helix 4 and reduced chemical exchange contributions, suggesting that stabilization of helix 4 suppresses the tendency of Elc1 to undergo conformational exchange on a micro- to millisecond time scale. Binding of a peptide representing the major ELC binding site of the von Hippel-Lindau (VHL) tumor suppressor protein almost completely eliminated chemical exchange processes, but induced substantial conformational changes in Elc1 leading to pronounced rotational anisotropy. These results suggest that elongin C interacts with various target proteins including the VHL protein by an induced fit mechanism involving the conformationally flexible carboxy-terminal helix 4.

• Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, Conaway JW
• Activation of HIF1alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex.
• Proc Natl Acad Sci U S A. 2000; 97: 10430-5
• Display abstract

• Mutations in the VHL tumor suppressor gene result in constitutive expression of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1alpha, which in normal cells is rapidly ubiquitinated and degraded by the proteasome under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1/Cdc53-F-box (SCF)-like E3 ubiquitin ligase raised the possibility that VHL may be directly responsible for regulating cellular levels of HIF1alpha by targeting it for ubiquitination and proteolysis. In this report, we test this hypothesis directly. We report development of methods for production of the purified recombinant VHL complex and present direct biochemical evidence that it can function with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1alpha ubiquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for future investigations of the mechanisms underlying VHL regulation of oxygen-dependent gene expression.

• Kamura T et al.
• Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase.
• Science. 1999; 284: 657-61
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• The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in most human kidney cancers. The VHL protein is part of a complex that includes Elongin B, Elongin C, and Cullin-2, proteins associated with transcriptional elongation and ubiquitination. Here it is shown that the endogenous VHL complex in rat liver also includes Rbx1, an evolutionarily conserved protein that contains a RING-H2 fingerlike motif and that interacts with Cullins. The yeast homolog of Rbx1 is a subunit and potent activator of the Cdc53-containing SCFCdc4 ubiquitin ligase required for ubiquitination of the cyclin-dependent kinase inhibitor Sic1 and for the G1 to S cell cycle transition. These findings provide a further link between VHL and the cellular ubiquitination machinery.

• Iwai K et al.
• Identification of the von Hippel-lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex.
• Proc Natl Acad Sci U S A. 1999; 96: 12436-41
• Display abstract

• Mutations of von Hippel-Lindau disease (VHL) tumor-suppressor gene product (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadic clear cell renal carcinomas. The function of the pVHL protein has not been clarified. pVHL has been shown to form a complex with elongin B and elongin C (VBC) and with cullin (CUL)-2. In light of the structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified UbcH5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhances ligase activity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We also found that pVHL associates with two proteins, p100 and p220, which migrate at a similar molecular weight as two major bands in the ubiquitination assay. Furthermore, naturally occurring pVHL missense mutations, including mutants capable of forming a complex with elongin B-elongin C-CUL-2, fail to associate with p100 and p220 and cannot exhibit the E3 ligase activity. These results suggest that pVHL might be the substrate recognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being directly involved in the ubiquitin conjugation system which leads to the targeted degradation of substrate proteins.

• Cohen HT et al.
• An important von Hippel-Lindau tumor suppressor domain mediates Sp1-binding and self-association.
• Biochem Biophys Res Commun. 1999; 266: 43-50
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• VHL is the causative gene for both von Hippel-Lindau (VHL) disease and sporadic clear-cell renal cancer. We showed earlier that VHL downregulates vascular endothelial growth factor transcription by directly binding and inhibiting the transcriptional activator Sp1. We have now mapped the VHL Sp1-binding domain to amino acids 96-122. The 96-122 domain is disproportionately affected by substitution mutations, which interfere with the VHL-Sp1 interaction. Deletion of the 96-122 domain prevents VHL effects on Sp1 DNA binding and on VHL target gene expression, indicating the domain contributes importantly to VHL tumor suppressor activity. Nevertheless, prevention of the VHL-Sp1 interaction only partially abrogates VHL's transcriptional repressor activity, supporting the existence of VHL transcriptional effectors in addition to Sp1. VHL also directly interacts with the Sp1 zinc fingers and self-associates via the 96-122 domain, which furthermore suggest the domain may bind other metalloproteins and contribute to VHL dominant-negative effects.

• Ohh M, Kaelin WG Jr
• The von Hippel-Lindau tumour suppressor protein: new perspectives.
• Mol Med Today. 1999; 5: 257-63
• Display abstract

• von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations of the VHL tumour suppressor gene. The VHL gene product, pVHL, forms multiprotein complexes that contain elongin B, elongin C and Cul-2, and negatively regulates hypoxia-inducible mRNAs. pVHL is suspected to play a role in ubiquitination given the similarity of elongin C and Cul-2 with Skp1 and Cdc53, respectively. pVHL can also interact with fibronectin and is required for the assembly of a fibronectin matrix. Finally, pVHL, at least indirectly, plays a role in the ability of cells to exit the cell cycle. Thus, pVHL is a tumour suppressor protein that regulates angiogenesis, extracellular matrix formation and the cell cycle.

• Ohh M et al.
• Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein.
• J Clin Invest. 1999; 104: 1583-91
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• The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17-50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157-171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.

• Botuyan MV et al.
• Binding of elongin A or a von Hippel-Lindau peptide stabilizes the structure of yeast elongin C.
• Proc Natl Acad Sci U S A. 1999; 96: 9033-8
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• Elongin is a heterotrimeric transcription elongation factor composed of subunits A, B, and C in mammals. Elongin A and C are F-box-containing and SKP1 homologue proteins, respectively, and are therefore of interest for their potential roles in cell cycle-dependent proteolysis. Mammalian elongin C interacts with both elongin A and elongin B, as well as with the von Hippel-Lindau tumor suppressor protein VHL. To investigate the corresponding interactions in yeast, we have utilized NMR spectroscopy combined with ultracentrifugal sedimentation experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A (Ela1) and two peptides from homologous regions of Ela1 and human VHL. Elc1 alone is a homotetramer composed of subunits with a structured N-terminal region and a dynamically unstable C-terminal region. Binding of a peptide fragment of the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL promotes folding of the C-terminal region of Elc1 into two regular helical structures and dissociates Elc1 into homodimers. Moreover, analysis of the complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer. Thus, elongin C is found to oligomerize in solution and to undergo significant structural rearrangements upon binding of two different partner proteins. These results suggest a structural basis for the interaction of an F-box-containing protein with a SKP1 homologue and the modulation of this interaction by the tumor suppressor VHL.

• Wada H, Yeh ET, Kamitani T
• The von Hippel-Lindau tumor suppressor gene product promotes, but is not essential for, NEDD8 conjugation to cullin-2.
• J Biol Chem. 1999; 274: 36025-9
• Display abstract

• We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100-105). Cul-2 has also been reported to form a multiprotein complex, Cul-2.VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elongins B and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2.VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2.

• Feldman DE, Thulasiraman V, Ferreyra RG, Frydman J
• Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC.
• Mol Cell. 1999; 4: 1051-61
• Display abstract

• von Hippel-Lindau (VHL) disease is caused by loss of function of the VHL tumor suppressor protein. Here, we demonstrate that the folding and assembly of VHL into a complex with its partner proteins, elongin B and elongin C (herein, elongin BC), is directly mediated by the chaperonin TRiC/CCT. Association of VHL with TRiC is required for formation of the VHL-elongin BC complex. A 55-amino acid domain of VHL is both necessary and sufficient for binding to TRiC. Importantly, mutation or deletion of this domain is associated with VHL disease. We identified two mutations that disrupt the normal interaction with TRiC and impair VHL folding. Our results define a novel role for TRiC in mediating oligomerization and suggest that inactivating mutations can impair polypeptide function by interfering with chaperone-mediated folding.

• Kaelin WG, Iliopoulos O, Lonergan KM, Ohh M
• Functions of the von Hippel-Lindau tumour suppressor protein.
• J Intern Med. 1998; 243: 535-9
• Display abstract

• Von Hippel-Lindau disease (VHL) is caused by germline mutations in the VHL tumour suppressor gene. Tumour development in this setting is due to loss or inactivation of the remaining wild-type VHL allele. The VHL gene product (pVHL) resides primarily in the cytoplasm. A frequently mutated region of pVHL can bind to complexes containing elongin B, elongin C and Cul2. Loss of pVHL leads to an inappropriate accumulation of hypoxia-inducible mRNAs, such as the mRNA encoding vascular endothelial growth factor (VEGF), under normoxic conditions. This finding is most likely to account for the hypervascular nature of VHL-associated neoplasms. Current studies are focussed on understanding if and how binding to elongins and Cul2 is linked to the ability of pVHL to regulate hypoxia-inducible mRNAs. In this regard, it is perhaps noteworthy that elongin C and Cul2 are homologous to yeast proteins Skp1 and Cdc53. These latter proteins participate in the formation of complexes that target certain proteins for ubiquitination.

• Ohh M et al.
• The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix.
• Mol Cell. 1998; 1: 959-68
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• Fibronectin coimmunoprecipitated with wild-type von Hippel-Lindau protein (pVHL) but not tumor-derived pVHL mutants. Immunofluorescence and biochemical fractionation experiments showed that fibronectin colocalized with a fraction of pVHL associated with the endoplasmic reticulum, and cold competition experiments suggested that complexes between fibronectin and pVHL exist in intact cells. Assembly of an extracellular fibronectin matrix by VHL-/- renal carcinoma cells, as determined by immunofluorescence and ELISA assays, was grossly defective compared with VHL+/+ renal carcinoma cells. Reintroduction of wildtype, but not mutant, pVHL into VHL-/- renal carcinoma cells partially corrected this defect. Finally, extracellular fibronectin matrix assembly by VHL-/- mouse embryos and mouse embryo fibroblasts (MEFs), unlike their VHL+/+ counterparts, was grossly impaired. These data support a direct role of pVHL in fibronectin matrix assembly.

• Iliopoulos O, Ohh M, Kaelin WG Jr
• pVHL19 is a biologically active product of the von Hippel-Lindau gene arising from internal translation initiation.
• Proc Natl Acad Sci U S A. 1998; 95: 11661-6
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• The von Hippel-Lindau (VHL) gene encodes a protein consisting of 213 amino acid residues with an apparent molecular mass of 30 kDa (pVHL30). Here we show that cells also produce a VHL protein (pVHL19) that appears to arise as a result of internal translation from the second methionine within the VHL ORF. pVHL30 resides primarily in the cytosol, with less amounts found in the nucleus or associated with cell membranes. In contrast pVHL19, in biochemical fractionation experiments, is equally distributed between the nucleus and cytosol and is not found in association with membranes. pVHL19, like pVHL30, can bind to elongin B, elongin C, and Hs-Cul2 in coimmunoprecipitation assays and can inhibit the production of hypoxia-inducible proteins such as vascular endothelial growth factor (VEGF) and GLUT1 when reintroduced into renal carcinoma cells that lack a wild-type VHL allele. Thus, cells contain two biologically active VHL gene products.

• Iliopoulos O, Kaelin WG Jr
• The molecular basis of von Hippel-Lindau disease.
• Mol Med. 1997; 3: 289-93

• Levy AP, Levy NS, Iliopoulos O, Jiang C, Kaplin WG Jr, Goldberg MA
• Regulation of vascular endothelial growth factor by hypoxia and its modulation by the von Hippel-Lindau tumor suppressor gene.
• Kidney Int. 1997; 51: 575-8
• Display abstract

• The regulation of VEGF production is mediated by both transcriptional and post-transcriptional mechanisms. A schematic model of elements involved in hypoxic regulation of VEGF is shown in Figure 1.

• Chen F, Wu M
• [Progress in the study of von Hippel-Lindau syndrome]
• Zhonghua Yi Xue Za Zhi. 1997; 77: 236-8

• Takagi Y, Pause A, Conaway RC, Conaway JW
• Identification of elongin C sequences required for interaction with the von Hippel-Lindau tumor suppressor protein.
• J Biol Chem. 1997; 272: 27444-9
• Display abstract

• Elongin C is a 112-amino acid protein that is found in mammalian cells as a positive regulatory subunit of heterotrimeric RNA polymerase II elongation factor Elongin (SIII) and as a component of a multiprotein complex containing the von Hippel-Lindau (VHL) tumor suppressor protein. As a subunit of the Elongin complex, Elongin C interacts directly with the transcriptionally active Elongin A subunit and potently induces its elongation activity; in addition, Elongin C interacts with the ubiquitin-like Elongin B subunit, which regulates the interaction of Elongin C with Elongin A. As a component of the VHL complex, Elongin C interacts directly with both Elongin B and the VHL protein. Binding of the VHL protein to Elongin C was found to prevent Elongin C from interacting with and activating Elongin A in vitro, leading to the proposal that one function of the VHL protein may be to regulate RNA polymerase II elongation by negatively regulating the Elongin complex. In this report, we identify Elongin C sequences required for its interaction with the VHL protein. We previously demonstrated that the ability of Elongin C to bind and activate Elongin A is sensitive to mutations in the C-terminal half of Elongin C, as well as to mutations in an N-terminal Elongin C region needed for formation of the Elongin BC complex. Here we show that interaction of Elongin C with the VHL tumor suppressor protein depends strongly on sequences in the C terminus of Elongin C but is independent of the N-terminal Elongin C region required for binding to Elongin B and for binding and activation of Elongin A. Taken together, our results are consistent with the proposal that the VHL protein negatively regulates Elongin C activation of the Elongin complex by sterically blocking the interaction of C-terminal Elongin C sequences with Elongin A. In addition, our finding that only a subset of Elongin C sequences required for its interaction with Elongin A are critical for binding to VHL may offer the opportunity to develop reagents that selectively interfere with Elongin and VHL function.

• Pause A et al.
• The von Hippel-Lindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins.
• Proc Natl Acad Sci U S A. 1997; 94: 2156-61
• Display abstract

• The inactivation of the von Hippel-Lindau (VHL) gene predisposes affected individuals to VHL syndrome and is an early genetic event associated with sporadic renal cell carcinoma and CNS hemangioblastomas. The VHL protein (pVHL) has been shown to form a stable complex with elongin B and elongin C, two factors that stabilize and activate the transcription elongation factor elongin A. Here, Hs-CUL-2, a member of the recently identified multigene family, the cullins, is shown to specifically associate with the trimeric pVHL-elongin B-C (VBC) complex in vitro and in vivo. Nearly 70% of naturally occurring cancer-predisposing mutations of VHL disrupt this interaction. The pVHL-Hs-CUL-2 association is strictly dependent on the integrity of the trimeric VBC complex. Immunofluorescence studies show Hs-CUL-2 to be a cytosolic protein that can be translocated to the nucleus by pVHL. Recently it has been shown that a yeast Hs-CUL-2 homolog, Cdc53, is part of a ubiquitin protein ligase complex that targets cell cycle proteins for degradation by the ubiquitin proteolytic pathway. In Caenorhabditis elegans, a null mutation of another Hs-cul-2 homolog, Ce-cul-1, results in hyperplasia in all tissues and is required for cell cycle exit. Hence, Hs-cul-2 may be required for VHL function and, therefore, may be a candidate human tumor-suppressor gene.

• Aso T, Haque D, Barstead RJ, Conaway RC, Conaway JW
• The inducible elongin A elongation activation domain: structure, function and interaction with the elongin BC complex.
• EMBO J. 1996; 15: 5557-66
• Display abstract

• The elongin (SIII) complex strongly stimulates the rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along the DNA. Elongin (SIII) is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which bind stably to each other to form a binary complex that interacts with elongin A and strongly induces its transcriptional activity. The elongin (SIII) complex is a potential target for negative regulation by the von Hippel-Lindau (VHL) tumor suppressor protein, which is capable of binding stably to the elongin BC complex and preventing it from activating elongin A. Here, we identify an elongin A domain sufficient for activation of elongation and demonstrate that it is a novel type of inducible activator that targets the RNA polymerase II elongation complex and is evolutionarily conserved in species as distantly related as Caenorhabditis elegans and man. In addition, we demonstrate that both the elongin A elongation activation domain and the VHL tumor suppressor protein interact with the elongin BC complex through a conserved elongin BC binding site motif that is essential for induction of elongin A activity by elongin BC and for tumor suppression by the VHL protein.

• Martin RL, Walpole I, Goldblatt J
• Identification of two sporadically derived mutations in the Von Hippel-Lindau gene.
• Hum Mutat. 1996; 7: 185-185

• Shilatifard A, Lane WS, Jackson KW, Conaway RC, Conaway JW
• An RNA polymerase II elongation factor encoded by the human ELL gene.
• Science. 1996; 271: 1873-6
• Display abstract

• The human ELL gene on chromosome 19 undergoes frequent translocations with the trithorax-like MLL gene on chromosome 11 in acute myeloid leukemias. Here, ELL was shown to encode a previously uncharacterized elongation factor that can increase the catalytic rate of RNA polymerase II transcription by suppressing transient pausing by polymerase at multiple sites along the DNA. Functionally, ELL resembles Elongin (SIII), a transcription elongation factor regulated by the product of the von Hippel-Lindau (VHL) tumor suppressor gene. The discovery of a second elongation factor implicated in oncogenesis provides further support for a close connection between the regulation of transcription elongation and cell growth.

• Lee S, Chen DY, Humphrey JS, Gnarra JR, Linehan WM, Klausner RD
• Nuclear/cytoplasmic localization of the von Hippel-Lindau tumor suppressor gene product is determined by cell density.
• Proc Natl Acad Sci U S A. 1996; 93: 1770-5
• Display abstract

• The product of the von Hippel-Lindau (VHL) tumor suppressor gene, the gene inactivated in VHL disease and in sporadic clear-cell renal carcinomas, has recently been shown to have as a functional target the transcription elongation complex, elongin (also called SIII). Here it is shown that there is a tightly regulated, cell-density-dependent transport of VHL into and/or out of the nucleus. In densely grown cells, the VHL protein is predominantly in the cytoplasm, whereas in sparse cultures, most of the protein can be detected in the nucleus. We have identified a putative nuclear localization signal in the first 60 and first 28 amino acids of the human and rat VHL protein, respectively. Sequences in the C-terminal region of the VHL protein may also be required for localization to the cytosol. These findings provide the initial indication of a novel cell density-dependent pathway that is responsible for the regulation of VHL cellular localization.

• Gnarra JR et al.
• Molecular cloning of the von Hippel-Lindau tumor suppressor gene and its role in renal carcinoma.
• Biochim Biophys Acta. 1996; 1242: 201-10

• Humphrey JS, Klausner RD, Linehan WM
• Von Hippel-Lindau syndrome: hereditary cancer arising from inherited mutations of the VHL tumor suppressor gene.
• Cancer Treat Res. 1996; 88: 13-39

• Takagi Y, Conaway RC, Conaway JW
• Characterization of elongin C functional domains required for interaction with elongin B and activation of elongin A.
• J Biol Chem. 1996; 271: 25562-8
• Display abstract

• The Elongin (SIII) complex stimulates the rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along DNA templates. The Elongin (SIII) complex is composed of a transcriptionally active A subunit, a chaperone-like B subunit, which promotes assembly and enhances stability of the Elongin (SIII) complex, and a regulatory C subunit, which (i) functions as a potent activator of Elongin A transcriptional activity, (ii) interacts specifically with Elongin B to form an isolable Elongin BC complex, and (iii) is bound and negatively regulated in vitro by the product of the von Hippel-Lindau tumor suppressor gene. As part of our effort to understand how Elongin C regulates the activity of the Elongin (SIII) complex, we are characterizing Elongin C functional domains. In this report, we identify Elongin C mutants that fall into multiple functional classes based on their abilities to bind Elongin B and to bind and activate Elongin A under our assay conditions. Characterization of these mutants suggests that Elongin C is composed of multiple overlapping regions that mediate functional interactions with Elongin A and B.

• Bertherat J
• Von Hippel-Lindau tumor suppressor protein and transcription elongation: new insights into regulation of gene expression.
• Eur J Endocrinol. 1996; 134: 157159-157159

• Kibel A, Iliopoulos O, DeCaprio JA, Kaelin WG Jr
• Binding of the von Hippel-Lindau tumor suppressor protein to Elongin B and C.
• Science. 1995; 269: 1444-6
• Display abstract

• Germ-line mutations of the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of human tumors, and somatic mutations of this gene have been identified in sporadic renal cell carcinomas and cerebellar hemangioblastomas. Two transcriptional elongation factors, Elongin B and C, were shown to bind in vitro and in vivo to a short, colinear region of the VHL protein (pVHL) that is frequently mutated in human tumors. A peptide replica of this region inhibited binding of pVHL to Elongin B and C whereas a point-mutant derivative, corresponding to a naturally occurring VHL missense mutation, had no effect. These results suggest that the tumor suppression function of pVHL may be linked to its ability to bind to Elongin B and C.

• Aso T, Lane WS, Conaway JW, Conaway RC
• Elongin (SIII): a multisubunit regulator of elongation by RNA polymerase II.
• Science. 1995; 269: 1439-43
• Display abstract

• The Elongin (SIII) complex activates elongation by mammalian RNA polymerase II by suppressing transient pausing of the polymerase at many sites within transcription units. Elongin is a heterotrimer composed of A, B, and C subunits of 110, 18, and 15 kilodaltons, respectively. Here, the mammalian Elongin A gene was isolated and expressed, and the Elongin (SIII) complex reconstituted with recombinant subunits. Elongin A is shown to function as the transcriptionally active component of Elongin (SIII) and Elongin B and C as regulatory subunits. Whereas Elongin C assembles with Elongin A to form an AC complex with increased specific activity, Elongin B, a member of the ubiquitin-homology gene family, appears to serve a chaperone-like function, facilitating assembly and enhancing stability of the Elongin (SIII) complex.

• Iliopoulos O, Kibel A, Gray S, Kaelin WG Jr
• Tumour suppression by the human von Hippel-Lindau gene product.
• Nat Med. 1995; 1: 822-6
• Display abstract

• A partial cDNA sequence for the gene linked to the von Hippel-Lindau (VHL) syndrome was reported in 1993. Mutation or loss of both VHL alleles has been documented in sporadic renal cell carcinomas and in the neoplasms that arise in von Hippel-Lindau kindreds. We have determined that the protein product of the VHL gene is an approximately 30 kilodalton cytoplasmic protein. The renal carcinoma cell line 786-O is known to harbour a VHL mutation and, as shown here, fails to produce a wild-type VHL protein. Reintroduction of wild-type, but not mutant, VHL into these cells had no demonstrable effect on their growth in vitro but inhibited their ability to form tumours in nude mice.

• Bailly M, Bain C, Favrot MC, Ozturk M
• Somatic mutations of von Hippel-Lindau (VHL) tumor-suppressor gene in European kidney cancers.
• Int J Cancer. 1995; 63: 660-4
• Display abstract

• Somatic mutations of von Hippel Lindau (VHL) tumor-suppressor gene have been identified in kidney cancers from North America and Japan. We studied VHL gene mutation in 31 kidney tumors from France. Of these tumors, 45% (14/31) displayed mutations, 60% of which occurred at A:T base pairs. The frequency and the spectrum of mutations identified in kidney tumors from Europe were similar to those reported for tumors from other geographical locations.

• Gao J, Naglich JG, Laidlaw J, Whaley JM, Seizinger BR, Kley N
• Cloning and characterization of a mouse gene with homology to the human von Hippel-Lindau disease tumor suppressor gene: implications for the potential organization of the human von Hippel-Lindau disease gene.
• Cancer Res. 1995; 55: 743-7
• Display abstract

• The human von Hippel-Lindau disease (VHL) gene has recently been identified and, based on the nucleotide sequence of a partial cDNA clone, has been predicted to encode a novel protein with as yet unknown functions [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. The length of the encoded protein and the characteristics of the cellular expressed protein are as yet unclear. Here we report the cloning and characterization of a mouse gene (mVHLh1) that is widely expressed in different mouse tissues and shares high homology with the human VHL gene. It predicts a protein 181 residues long (and/or 162 amino acids, considering a potential alternative start codon), which across a core region of approximately 140 residues displays a high degree of sequence identity (98%) to the predicted human VHL protein. High stringency DNA and RNA hybridization experiments and protein expression analyses indicate that this gene is the most highly VHL-related mouse gene, suggesting that it represents the mouse VHL gene homologue rather than a related gene sharing a conserved functional domain. These findings provide new insights into the potential organization of the VHL gene and nature of its encoded protein.

• Nelson NJ
• Old protein, new function: VHL tumor suppressor protein inhibits transcription.
• J Natl Cancer Inst. 1995; 87: 1432-3

• Duan DR et al.
• Inhibition of transcription elongation by the VHL tumor suppressor protein.
• Science. 1995; 269: 1402-6
• Display abstract

• Germline mutations in the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of tumors, including renal carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. Here, a cellular transcription factor, Elongin (SIII), is identified as a functional target of the VHL protein. Elongin (SIII) is a heterotrimer consisting of a transcriptionally active subunit (A) and two regulatory subunits (B and C) that activate transcription elongation by RNA polymerase II. The VHL protein was shown to bind tightly and specifically to the Elongin B and C subunits and to inhibit Elongin (SIII) transcriptional activity in vitro. These findings reveal a potentially important transcriptional regulatory network in which the VHL protein may play a key role.

• Seizinger BR
• von Hippel-Lindau disease: a model system for the isolation of "tumor suppressor" genes associated with the primary genetic mechanisms of cancer.
• Adv Nephrol Necker Hosp. 1994; 23: 29-42

• Latif F et al.
• Identification of the von Hippel-Lindau disease tumor suppressor gene.
• Science. 1993; 260: 1317-20
• Display abstract

• A gene discovered by positional cloning has been identified as the von Hippel-Lindau (VHL) disease tumor suppressor gene. A restriction fragment encompassing the gene showed rearrangements in 28 of 221 VHL kindreds. Eighteen of these rearrangements were due to deletions in the candidate gene, including three large nonoverlapping deletions. Intragenic mutations were detected in cell lines derived from VHL patients and from sporadic renal cell carcinomas. The VHL gene is evolutionarily conserved and encodes two widely expressed transcripts of approximately 6 and 6.5 kilobases. The partial sequence of the inferred gene product shows no homology to other proteins, except for an acidic repeat domain found in the procyclic surface membrane glycoprotein of Trypanosoma brucei.

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