Interaction of the PAS B domain with HSP90 accelerates hypoxia-inducible factor-1alpha stabilization.
Hypoxia-inducible factor (HIF) alpha subunits are induced under hypoxic conditions, when limited oxygen supply prevents prolyl hydroxylation-dependent binding of the ubiquitin ligase pVHL and subsequent proteasomal degradation. A short normoxic half-life of HIF-alpha and a very rapid hypoxic protein stabilization are crucial to the cellular adaptation to changing oxygen supply. However, the molecular requirements for the unusually rapid mechanisms of protein synthesis, folding and nuclear translocation are not well understood. We and others previously found that the chaperone heat-shock protein 90 (HSP90) can interact with HIF-1alpha in vitro. Here we show that HSP90 also interacts with HIF-2alpha and HIF-3alpha, suggesting a general involvement of HSP90 in HIF-alpha stabilization. The PAS B domain, common to all three alpha subunits, was required for HSP90 interaction. ARNT competed with HSP90 for binding to the PAS B domain since an excess of either component inhibited the activity of the other. HSP90 as well as the heterocomplex members HSP70 and p23, but not HSP40, were detected in immunoprecipitations of endogenous cellular HIF-1alpha. While HSP90 and HSP70 bound to HIF-1alpha predominantly under normoxic conditions, ARNT bound to HIF-1alpha primarily under hypoxic conditions, suggesting that ARNT displaced HSP90 from HIF-1alpha following nuclear translocation. Hypoxic accumulation of HIF-1alpha was delayed in a novel cell model deficient for HSP90beta as well as after treatment of wild-type cells with the HSP90 inhibitor geldanamycin, suggesting that HSP90 activity is involved in the rapid HIF-1alpha protein induction.
Cell Physiology Group, Medical Faculty, Martin-Luther-University Halle, Germany. doerthe.katschinski@medizin.uni-halle.de
Dörthe M
DM
Katschinski
Lu
L
Le
Susann G
SG
Schindler
Tim
T
Thomas
Anne K
AK
Voss
Roland H
RH
Wenger
D051017:Apoptosis Regulatory Proteins; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D016227:Benzoquinones; D015687:Cell Hypoxia; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047468:Immunoprecipitation; D047029:Lactams, Macrocyclic; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000235:genetics; D011809:Quinones / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D017384:Sequence Deletion; D013234:Stem Cells / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000099162:Endothelial PAS Domain-Containing Protein 1
D051017:Apoptosis Regulatory Proteins; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D016227:Benzoquinones; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047029:Lactams, Macrocyclic; D010100:Oxygen; D011809:Quinones; D018336:Receptors, Aryl Hydrocarbon; D012097:Repressor Proteins; D014157:Transcription Factors; D000099162:Endothelial PAS Domain-Containing Protein 1; C497396:ARNT protein, human; C497442:HIF1A protein, human; C116973:HIF3A protein, human; C001277:geldanamycin