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 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.
Cockman
M E
ME
Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom.
Masson
N
N
Mole
D R
DR
Jaakkola
P
P
Chang
G W
GW
Clifford
S C
SC
Maher
E R
ER
Pugh
C W
CW
Ratcliffe
P J
PJ
Maxwell
P H
PH
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D019556:COS Cells; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D015151:Immunoblotting; D008025:Ligases; D009097:Multienzyme Complexes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D015534:Trans-Activators; D014157:Transcription Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D003546:Cysteine Endopeptidases; D004268:DNA-Binding Proteins; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008025:Ligases; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D010100:Oxygen; D046988:Proteasome Endopeptidase Complex; D011506:Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D000099162:Endothelial PAS Domain-Containing Protein 1; C497444:Hif1a protein, rat