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-1α and HIF-2α under normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. Hypoxia substantially altered the complexity and composition of the HIFα protein interaction networks, particularly for HIF-2α, with the hypoxic networks of both isoforms being enriched for mitochondrial proteins. Moreover, both HIFα isoforms were heavily covalently modified. We identified ~40 PTM sites composed of 13 different types of modification on both HIFα 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 Ser31 in HIF-1α as a regulator of its transcriptional function, and we propose functional roles for Thr406, Thr528, and Ser581 in HIF-2α. These data will help to delineate the different physiological roles of these closely related isoforms in fine-tuning the hypoxic response.
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