Phosphorylation at S345 Converts HIF-2α from a Transcription Factor to an RNA binding protein

Oxygen sensing and adaptation are vital for metazoan survival. At the cellular level the response to hypoxia is characterised by a switch in transcriptional programming, primarily mediated by hypoxia-inducible factors (HIFs). HIFs comprise an obligatory heterodimer between an oxygen-sensitive HIF- subunit and an oxygen-insensitive HIF-1{beta} subunit, which together regulate gene expression in response to hypoxia. Among the three HIF- proteins in vertebrates (HIF-1, HIF-2, and HIF-3), post-translational modifications (PTMs) play a crucial role in modulating their stability, localisation, and activity. While many modifications have been investigated on HIF-1, our knowledge of HIF-2 regulation is very limited. Here, we investigated the function of the recently identified HIF-2 S345 phosphorylation site and demonstrate that this disrupts the interaction between HIF-2 and HIF-1{beta}, thereby silencing the HIF-2 transcriptional response. Additionally, our findings suggest that this modification redirects HIF-2 from its normal transcription factor function towards a role in mRNA fate control, as indicated by mass spectrometry interactome analysis and RNA-immunoprecipitation. Our discoveries highlight a new regulatory mechanism of HIF-2 activity, where S345 phosphorylation impedes HIF-2/HIF-1{beta} heterodimer formation, creating a switch in HIF-2 function. As HIF-2 is the only HIF- isoform with a clinically approved inhibitor, these insights are crucial for advancing therapeutic strategies targeting hypoxia signalling.