Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

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.

Original publication

DOI

10.1074/jbc.M002740200

Type

Journal article

Journal

J Biol Chem

Publication Date

18/08/2000

Volume

275

Pages

25733 - 25741

Keywords

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