Hypoxia-inducible factor-1α (HIF-1α) ¹ is a global transcriptional regulator of the hypoxic response. Under normoxic conditions, HIF-1α 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-1α. Under hypoxic conditions, HIF-1α is stabilized. We have previously shown that VHL binds in a hypoxia-sensitive manner to a 27-aa segment of HIF-1α, and that this regulation depends on a posttranslational modification of HIF-1α. Through a combination of in vivo coimmunoprecipitation assays using VHL and a panel of point mutants of HIF-1α 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-1α. The data furthermore show that this proline hydroxylation is the primary regulator of VHL binding.

Humans have adapted to the chronic hypoxia of high altitude in several locations, and recent genome-wide studies have indicated a genetic basis. In some populations, genetic signatures have been identified in the hypoxia-inducible factor (HIF) pathway, which orchestrates the transcriptional response to hypoxia. In Tibetans, they have been found in the HIF2A (EPAS1) gene, which encodes for HIF-2α, and the prolyl hydroxylase domain protein 2 (PHD2, also known as EGLN1) gene, which encodes for one of its key regulators, PHD2. High-altitude adaptation may be due to multiple genes that act in concert with one another. Unraveling their mechanism of action can offer new therapeutic approaches toward treating common human diseases characterized by chronic hypoxia.

A critical step in the signal-induced activation of the transcription factor NF-κB is the site-specific phosphorylation of its inhibitor, IκB, that targets the latter for degradation by the ubiquitin–proteasome pathway. We have previously shown that mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) can induce both this site-specific phosphorylation of IκBα at Ser-32 and Ser-36 in vivo and the activity of a high molecular weight IκB kinase complex in vitro . Subsequently, others have identified two proteins, IκB kinase α (IKK-α) and IκB kinase β (IKK-β), that are present in a tumor necrosis factor α-inducible, high molecular weight IκB kinase complex. These kinases are believed to directly phosphorylate IκB based on the examination of the kinase activities of IKK immunoprecipitates, but more rigorous proof of this has yet to be demonstrated. We show herein that recombinant IKK-α and IKK-β can, in fact, directly phosphorylate IκBα at Ser-32 and Ser-36, as well as homologous residues in IκBβ in vitro , and thus are bona fide IκB kinases. We also show that MEKK1 can induce the activation of both IKK-α and IKK-β in vivo . Finally, we show that IKK-α is present in the MEKK1-inducible, high molecular weight IκB kinase complex and treatment of this complex with MEKK1 induces phosphorylation of IKK-α in vitro . We conclude that IKK-α and IKK-β can mediate the NF-κB-inducing activity of MEKK1.

Hypoxia-inducible factor (HIF) α, which has three isoforms, is central to the continuous balancing of the supply and demand of oxygen throughout the body. HIF-α is a transcription factor that modulates a wide range of processes, including erythropoiesis, angiogenesis, and cellular metabolism. We describe a family with erythrocytosis and a mutation in the HIF2A gene, which encodes the HIF-2α protein. Our functional studies indicate that this mutation leads to stabilization of the HIF-2α protein and suggest that wild-type HIF-2α regulates erythropoietin production in adults.