Hypoxia inducible factor-α binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein (2000)

Author(s): Cockman ME, Masson N, Mole DR, Jaakkola P, Chang GW, Clifford SC, Maher ER, Pugh CW, Ratcliffe PJ, Maxwell PH

  • : Hypoxia inducible factor-alpha 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 alpha 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-alpha ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-alpha among a range of substrates tested. Furthermore, HIF-alpha 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-alpha interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-alpha, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the beta-domain of pVHL and loss of interaction was associated with defective HIF-alpha 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-alpha proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its beta domain interacts with short recognition sequences in HIF-alpha subunits.

Notes: Cockman, M E Masson, N Mole, D R Jaakkola, P Chang, G W Clifford, S C Maher, E R Pugh, C W Ratcliffe, P J Maxwell, P H Research Support, Non-U.S. Gov't United states The Journal of biological chemistry J Biol Chem. 2000 Aug 18;275(33):25733-41.

  • Short Title: Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein
  • Date: 22-05-2000
  • Journal: Journal of Biological Chemistry
  • Volume: 275
  • Issue: 33
  • Pages: 25733-25741
  • Publisher: American Society for Biochemistry and Molecular Biology, Inc.
  • Publication type: Article
  • Bibliographic status: Published

Keywords: Animals Basic Helix-Loop-Helix Transcription Factors COS Cells Cysteine Endopeptidases/metabolism DNA-Binding Proteins/chemistry/ metabolism Hypoxia-Inducible Factor 1 Hypoxia-Inducible Factor 1, alpha Subunit Immunoblotting Ligases Multienzyme Complexes/metabolism Mutagenesis, Site-Directed Mutation, Missense Nuclear Proteins/chemistry/ metabolism Oxygen/metabolism Plasmids/metabolism Precipitin Tests Proteasome Endopeptidase Complex Protein Binding Protein Biosynthesis Protein Structure, Tertiary Proteins/chemistry/genetics/ metabolism/physiology Rats Reticulocytes/metabolism Substrate Specificity Time Factors Trans-Activators Transcription Factors Transfection Tumor Suppressor Proteins Ubiquitin-Protein Ligases Ubiquitins/ metabolism Von Hippel-Lindau Tumor Suppressor Protein


Professor Steven Clifford
Prof of Molecular Paediatric Oncology