Oxygen-Dependent Changes In Binding Partners And Post-Translational Modifications Regulate The Abundance And Activity Of Hif-1 Alpha/2 Alpha

Cellular adaptation to low-oxygen environments is mediated in part by the hypoxia-inducible factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs-and consequently, the hypoxic response-are regulated by post-translational modifications (PTMs) and changes in protein-protein interactions. Our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment-based studies typically validated in fragment-expressing cells treated with hypoxia-mimicking compounds. Here, we used immunoprecipitation-based mass spectrometry to characterize the PTMs and binding partners for full-length HIF-1 alpha and HIF-2 alpha under normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. Hypoxia substantially altered the complexity and composition of the HIF alpha protein interaction networks, particularly for HIF-2 alpha, with the hypoxic networks of both isoforms being enriched for mitochondrial proteins. Moreover, both HIF alpha isoforms were heavily covalently modified. We identified similar to 40 PTM sites composed of 13 different types of modification on both HIF alpha isoforms, including multiple cysteine modifications and an unusual phosphocysteine. More than 80% of the PTMs identified were not previously known and about half exhibited oxygen dependency. We further characterized an evolutionarily conserved phosphorylation of Ser(31) in HIF-1 alpha as a regulator of its transcriptional function, and we propose functional roles for Thr(406), Thr(528), and Ser(581) in HIF-2 alpha. These data will help to delineate the different physiological roles of these closely related isoforms in fine-tuning the hypoxic response.