The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in both sporadic and inherited clear cell renal carcinoma associated with VHL disease. We have identified two distinct native products of the human VHL gene, with apparent molecular masses of 24 and 18 kDa. The 18-kDa VHL protein was more abundant in nearly all cell lines examined. Reintroduction of the 18-kDa VHL gene product into renal carcinoma cells lacking wild-type VHL protein led to down-regulation of vascular endothelial growth factor (VEGF) mRNA and glucose transporter GLUT1 protein and suppressed tumor formation in nude mice. The 18-kDa VHL protein also demonstrated binding to elongins B and C. In an in vitro assay, the second in-frame AUG codon present in VHL mRNA was shown to be necessary and sufficient for production of the 18-kDa VHL protein, consistent with an internal translation mechanism. These data provide evidence for a second major VHL gene product, which contains the functional domains of the VHL gene. Moreover, these results indicate that internal translation initiation is an important mechanism for production of the major VHL protein.

Individuals carrying a germ line mutation of the breast cancer susceptibility gene BRCA2 are predisposed to breast, ovarian, and other types of cancer. The BRCA2 protein has been proposed to function in the repair of DNA double-strand breaks. Using an immunopurification-mass spectrometry approach to identify novel proteins that associate with the BRCA2 gene product, we found that a deubiquitinating enzyme, USP11, formed specific complexes with BRCA2. Moreover, BRCA2 was constitutively ubiquitinated in vivo in the absence of detectable proteasomal degradation. Mitomycin C (MMC) led to decreased BRCA2 protein levels associated with increased ubiquitination, consistent with proteasome-dependent degradation. While BRCA2 could be deubiquitinated by USP11 in transient overexpression assays, a catalytically inactive USP11 mutant had no effect on BRCA2 ubiquitination or protein levels. Antagonism of USP11 function either through expression of this mutant or through RNA interference increased cellular sensitivity to MMC in a BRCA2-dependent manner. All of these results imply that BRCA2 expression levels are regulated by ubiquitination in the cellular response to MMC-induced DNA damage and that USP11 participates in DNA damage repair functions within the BRCA2 pathway independently of BRCA2 deubiquitination.Individuals who carry a germ line mutation in the BRCA2 gene are predisposed to breast, ovarian, and certain other cancers (42,49). Loss of function of the wild-type BRCA2 allele is observed in breast tumors that arise in these individuals (10), defining BRCA2 as a tumor suppressor gene. Additionally, certain biallelic BRCA2 mutations have been linked to the D1 and B subtypes of the cancer susceptibility disorder Fanconi anemia (19). The BRCA2 gene encodes a 3,418-amino-acid nuclear protein (42) with an approximate molecular mass of 460 kDa (24). The BRCA2 protein has been shown to bind to the mammalian homolog of the RecA recombinase, Rad51 (9,26,37,48). Hence, a BRCA2 function in the repair of DNA double-strand breaks through homologous recombination has been proposed (37). In support of this notion, mammalian cells lacking functional BRCA2 are sensitive to DNAdamaging agents (9, 32, 37, 52), show genomic instability (32,45,52), and are deficient in homology-directed DNA repair (28,44). Furthermore, BRCA2 has been shown to possess single-stranded DNA binding ability (51) and can influence Rad51 DNA binding and recombination activities (11). However, the precise role of BRCA2 in homologous recombination and/or DNA repair has not been elucidated.The study of interacting proteins is an important approach toward understanding the biological functions of proteins. Toward this end, we immunopurified a C-terminal region of BRCA2 and subjected copurifying proteins to mass spectrometry analysis. Here we report the identification of a novel BRCA2-interacting protein, USP11, and characterize its role in BRCA2-mediated DNA damage repair.

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Mutations in the vonMutations of the von Hippel-Lindau (VHL) gene are involved in the family cancer syndrome for which it is named and the development of sporadic renal cancer and renal cystic disease (for review, see reference 15). VHL has no significant homology to previously identified proteins (21). Insights into the biochemistry of VHL have come predominantly from the identification of proteins that associate with VHL products (9,17,19,26,29,34). These include elongins B and C (9, 12, 19, 37), cul-2 (26, 34), and Rbx1 (17), which are similar to components of a yeast E3 ubiquitin ligase complex (2,16,26,34). A current hypothesis for VHL activity is that it functions as an F-box protein, directing specific substrates for ubiquitination. Indeed, VHL has been shown to have in vitro ubiquitin ligase activity (13,25) and to target HIF-1␣, a hypoxia inducible transcription factor, for proteasomal degradation (7,18,28,38). However, the exact biochemical function(s) of VHL that is disrupted in VHL disease and which results in susceptibility to clear renal cell cancer (RCC) remains elusive.Alterations of cell-extracellular matrix (ECM) interactions are associated with renal cystic disease (for review, see references 4, 10, and 40). A role for VHL in the synthesis and degradation of ECM has begun to emerge. VHL was found to associate with intracellular fibronectin and was required for assembly of extracellular fibronectin (29). VHL also controls matrix degradation by regulating both matrix metalloproteinases 2 and 9 and their inhibitors (20), as well as the urokinasetype plasminogen activator system (27). (11,33,37). In addition, VHLdeficient RCC cells ectopically expressing VHL demonstrated morphological differentiation and growth arrest when grown as multicellular tumor spheroids, but not under standard culture conditions (23). These studies suggest the importance of the extracellular milieu to elicit biological functions of VHL. Previous studies indicated that reintroduction of VHL into carcinoma cells lacking functional VHL [VHL(Ϫ)] leads to growth suppression in nude mice but not in cells grown under standard culture conditionsIn this report, we describe VHL function in the context of cell-cell and cell-ECM interactions. These studies demonstrate that VHL-dependent morphological and biochemical differentiation requires the establishment of high-density cell-cell contact and, in combination with cell-ECM interactions, results in VHL-dependent growth arrest. MATERIALS AND METHODSCell lines and culture. The VHL(Ϫ) cell lines consist of the parental renal carcinoma cell line, 786-O, and its derivative lines containing either the empty expression vector pCR3 (Invitrogen, Carlsbad, Calif.) or a nonfunctional VHL deletion construct, VHL(MPR)del(114-178) (37). The VHL(ϩ) cell lines consist of 786-O derivative lines stably expressing the VHLp24(MPR) or the VHLp18 (MEA) constructs as previously described (37). At least two independent clones were analyzed for each construct. Cells were grown in 10-cm-diameter dishes in a h...

BackgroundIn von Hippel-Lindau (VHL) disease, germline mutations in the VHL tumor suppressor gene cause clear cell renal carcinomas, hemangioblastomas, and pheochromocytomas. The VHL gene product is part of an ubiquitin E3 ligase complex and hypoxia-inducible factor alpha (HIF-α) is a key substrate, although additional VHL functions have been described. A genotype-phenotype relationship exists in VHL disease such that specific VHL mutations elicit certain subsets of these tumors. Here, we examine VHL genotype-phenotype correlations at the cellular level, focusing on the regulation of tight junctions and cell morphology.MethodsWild-type and various mutant VHL proteins representing VHL disease subtypes were stably expressed in 3 VHL-negative renal carcinoma cell lines. Using these cell lines, the roles of various VHL-associated cellular functions in regulation of cell morphology were investigated.ResultsAs a whole, type 1 mutants varied greatly from type 2 mutants, demonstrating high levels of HIF-2α, cyclin D1 and α5 integrin, lower p27 levels, and a spindly, fibroblastic cellular appearance. Type 2 mutations demonstrated an epithelial morphology similar to wild-type VHL in the majority of the renal cell lines used. Knockdown of p27 in cells with wild-type VHL led to perturbations of both epithelial morphology and ZO-1 localization to tight junctions. ZO-1 localization correlated well with VHL disease subtypes, with greater mislocalization observed for genotypes associated with a higher risk of renal carcinoma. HIF-2α knockdown in 786-O partially restored ZO-1 localization, but did not restore an epithelial morphology.ConclusionVHL has both HIF-α dependent and HIF-α independent functions in regulating tight junctions and cell morphology that likely impact the clinical phenotypes seen in VHL disease.