Hypoxia Inducible Factor-α Binding and Ubiquitylation by the von Hippel-Lindau Tumor Suppressor Protein

Abstract

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 ␣ subunits of hypoxia inducible factor-1 (HIF). Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes. Here we investigate the mechanisms underlying these processes and show that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-␣ ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-␣ among a range of substrates tested. Furthermore, HIF-␣ subunits were the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-␣ interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-␣ molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-␣, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the ␤-domain of pVHL and loss of interaction was associated with defective HIF-␣ ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-␣ proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its ␤ domain interacts with short recognition sequences in HIF-␣ subunits.

Evaluation of hypoxia in an experimental rat tumour model by [18F]Fluoromisonidazole PET and immunohistochemistry

“…A difference in location and area of positive staining (Wykoff et al, 2000;Olive et al, 2001 and also the present study) supports this explanation. von Hippel-Lindau mutations, which result in an upregulation of HIF-1a (Cockman et al, 2000) and downstream components like CA IX (Ashida et al, 2002), and the relationship between CA IX expression and acidic pH found in tumours (Stubbs et al, 2000) could potentially contribute to the published differences.…”

Target gene selectivity of hypoxia-inducible factor-α in renal cancer cells is conveyed by post-DNA-binding mechanisms

“…Hs.HIF-2a (Cockman et al, 2000) to introduce novel restriction sites without changing the encoded protein using primers listed in Supplementary Table 1. After confirmation by restriction digestion and DNA sequencing, the sites introduced were used to allow domain exchange by standard recombinant techniques.…”

Gene array of VHL mutation and hypoxia shows novel hypoxia-induced genes and that cyclin D1 is a VHL target gene

“…The VHL gene product pVHL is a critical component of a multiprotein ubiquitin ligase complex that targets the regulatory HIF-a subunits of hypoxia-inducible factor 1 (HIF-1) for oxygen-dependent proteolysis (Iwai et al, 1999;Maxwell et al, 1999;Cockman et al, 2000;Ivan et al, 2001). HIF-1 is expressed in response to hypoxia in most cell types and activates the transcription of genes involved in a variety of physiological and cellular processes including vascular endothelial growth factor (VEGF), glucose transport (glucose transporters), glycolysis (glycolytic enzymes), and cell survival (insulin-like growth factor 2) (Semenza, 1999).…”