HIF-1α binding to VHL is regulated by stimulus-sensitive proline hydroxylation

Hypoxia-inducible factor-1 alpha (HIF-1 alpha)(1) is a global transcriptional regulator of the hypoxic response. Under normoxic conditions, HIF-1 alpha is recognized by the von Hippel-Lindau tumor-suppressor protein (VHL), a component of an E3 ubiquitin ligase complex. This interaction thereby promotes the rapid degradation of HIF-1 alpha. Under hypoxic conditions, HIF-1 alpha is stabilized. We have previously shown that VHL binds in a hypoxia-sensitive manner to a 27-aa segment of HIF-1 alpha, and that this regulation depends on a post-translational modification of HIF-1 alpha. Through a combination of in vivo coimmunoprecipitation assays using VHL and a panel of point mutants of HIF-1 alpha a in this region, as well as MS and in vitro binding assays, we now provide evidence that this modification, which occurs under normoxic conditions, is hydroxylation of Pro-564 of HIF-1 alpha. The data furthermore show that this proline hydroxylation is the primary regulator of VHL binding.