Abstract
pVHL (von Hippel-Lindau tumor suppressor protein) is the substrate recognition subunit of the CBC(VHL) ubiquitin ligase complex promoting the degradation of hypoxia-inducible factor subunits, HIF-1/2alpha. Mutational inactivation of pVHL causes the hereditary von Hippel-Lindau tumor syndrome, which predisposes affected individuals to hemangioblastomas, renal cell carcinomas, and pheochromocytomas. Whereas the development of hemangioblastomas and renal cell carcinomas has been attributed to impaired HIF-1/2alpha down-regulation by pVHL mutant proteins, the molecular defects underlying the development of pheochromocytomas are still unknown. Here, we present a detailed biochemical analysis of pVHL mutant proteins linked to type 2C (pheochromocytoma only) von Hippel-Lindau disease. Type 2C-associated mutations caused extensive structural perturbations of pVHL, as revealed by the reduced stability, increased proteolytic susceptibility, and dramatically altered NMR spectrum of recombinant, mutant pVHL-ElonginC-ElonginB complexes in vitro. In human cell lines, type 2C-linked mutations destabilized the CBC(VHL) ubiquitin ligase complex and resulted in reduced cellular pVHL levels. Together, our data reveal unexpectedly strong structural defects of type 2C-associated pVHL mutant proteins that are likely to affect both HIF-1/2alpha-related and -unrelated pVHL functions in the pathogenesis of pheochromocytomas.
Highlights
Von Hippel-Lindau disease (OMIM 193300) is an autosomal dominant, hereditary cancer syndrome caused by germ line mutations in the VHL tumor suppressor gene [1, 2]
Type 1-associated pVHL mutant proteins fail to assemble into a functional CBCVHL E3 ubiquitin ligase complex [14], whereas type 2Aand type 2B-associated pVHL mutant proteins were shown to be differentially affected in HIF-1/2␣ binding [15,16,17]
We show that type 2C-associated pVHL mutant proteins are mildly defective in HIF-1␣ regulation, suggesting the possibility that a low level of HIF-1/2␣ dysregulation contributes to the pathogenesis of pheochromocytomas
Summary
Plasmids—pVB and pBB75 [14], pST39-HisTrxN-VHLElonginB-ElonginC [39], and pcDNA3-HA-VHL [23] were gifts of N. RCC4-derived cell pools expressing wild type or mutant VHL were generated by infection with retroviruses obtained by transfecting LinX-A cells with pBabe-Puro-VHL using FuGENE (Roche Applied Science). Stable cell pools were selected and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 g/ml puromycin. Protein Purification—Expression and purification of recombinant VCB complex were performed exactly as described [15]. For isotopically enriched protein preparations, VCB or ElonginBC complexes were co-expressed in minimal medium containing 15NH4Cl and purified as will be published elsewhere.. Protein levels in RCC4-derived cell pools were analyzed by immunoblots using antibodies directed against pVHL Quantitative PCR—To quantify mRNA levels in RCC4-derived cell pools, total RNA from 106 cells was purified using a High Pure RNA isolation kit (Roche Applied Science).
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