Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein

The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of alpha subunits of hypoxia inducible factor -1 (HIF), Mutations in VHL cause a hereditary cancer syndrome associated wi th dysregulated angiogenesis, and up-regulation of hypoxia inducible genes, Here we investigate the mechanisms underlying these processes and show tha t extracts from VHL-deficient renal carcinoma cells have a defect in HIF-al pha ubiquitylation activity which is complemented by exogenous pVHL, This d efect was specific for HIF-alpha among a range of substrates tested. Furthe rmore, HIF-alpha subunits were the only pVHL-associated proteasomal substra tes identified by comparison of metabolically labeled anti-pVHL immunopreci pitates from proteosomally inhibited cells and normal cells. Analysis of pV HL/HIF-alpha interactions defined short sequences of conserved residues wit hin the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL, In contrast, while full-length pVHL and the p19 va riant interact with HIF-alpha, the association was abrogated by further N-t erminal and C-terminal truncations. The interaction was also disrupted by t umor-associated mutations in the beta-domain of pVHL and loss of interactio n was associated with defective HIF-alpha ubiquitylation and regulation, de fining a mechanism by which these mutations generate a constitutively hypox ic pattern of gene expression promoting angiogenesis, The findings indicate that pVHL regulates HIF-alpha proteolysis by acting as the recognition com ponent of a ubiquitin ligase complex, and support a model in which its beta domain interacts with short recognition sequences in HIF-alpha subunits.