Interaction of the PAS B Domain with HSP90 Accelerates Hypoxia-Inducible Factor-1α Stabilization

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Abstract
Hypoxia-inducible factor (HIF) α 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-α 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-1α in vitro. Here we show that HSP90 also interacts with HIF-2α and HIF-3α, suggesting a general involvement of HSP90 in HIF-α stabilization. The PAS B domain, common to all three α 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-1α. While HSP90 and HSP70 bound to HIF-1α predominantly under normoxic conditions, ARNT bound to HIF-1α primarily under hypoxic conditions, suggesting that ARNT displaced HSP90 from HIF-1α following nuclear translocation. Hypoxic accumulation of HIF-1α was delayed in a novel cell model deficient for HSP90β as well as after treatment of wild-type cells with the HSP90 inhibitor geldanamycin, suggesting that HSP90 activity is involved in the rapid HIF-1α protein induction.

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