Abstract
Early adaptive responses to hypoxia are essential for cell survival, but their nature and underlying mechanisms are poorly known. We have studied the post-transcriptional changes in the proteome of mammalian cells elicited by acute hypoxia and found that phosphorylation of eukaryotic elongation factor 2 (eEF2), a ribosomal translocase whose phosphorylation inhibits protein synthesis, is under the precise and reversible control of O(2) tension. Upon exposure to hypoxia, phosphorylation of eEF2 at Thr(56) occurred rapidly (<15 min) and resulted in modest translational arrest, a fundamental homeostatic response to hypoxia that spares ATP and thus facilitates cell survival. Acute inhibitory eEF2 phosphorylation occurred without ATP depletion or AMP kinase activation. Furthermore, eEF2 phosphorylation was mimicked by prolyl hydroxylase (PHD) inhibition with dimethyloxalylglycine or by selective PHD2 siRNA silencing but was independent of hypoxia-inducible factor α stabilization. Moreover, overexpression of PHD2 blocked hypoxic accumulation of phosphorylated eEF2. Therefore, our findings suggest that eEF2 phosphorylation status (and, as a consequence, translation rate) is controlled by PHD2 activity. They unravel a novel pathway for cell adaptation to hypoxia that could have pathophysiologic relevance in tissue ischemia and cancer.
Highlights
Translational arrest is a classical cellular response to hypoxia, the underlying mechanisms of which are unknown
We focused on eukaryotic elongation factor 2 (eEF2), a translocase necessary for protein synthesis, we identified two other proteins altered by lowering O2 tension
We have shown that hypoxic inhibitory eEF2 phosphorylation is independent of AMPK activity and is mimicked by DMOG treatment, suggesting that eEF2 activity and protein synthesis are modulated by prolyl hydroxylases (PHDs)
Summary
Translational arrest is a classical cellular response to hypoxia, the underlying mechanisms of which are unknown. Contribute to HIF-independent inhibition of protein translation under low O2 conditions [7, 9, 11,12,13] These phosphorylation/dephosphorylation cascades regulate protein synthesis by precisely modulating the activity of ribosomal initiation and elongation factors [10, 14]. Using a proteomic approach designed to detect early adaptive changes during short-term (5–30 min) exposure to hypoxia, we have identified several proteins acutely regulated by O2 availability Among these proteins, eEF2 was modulated by dimethyloxalylglycine (DMOG), an inhibitor of the O2-sensing PHDs. we show that eEF2 activity is rapidly and reversibly regulated by O2 tension in a PHD2-dependent manner. These observations, which unveil unexpected actions of the O2-sensing PHD2, could have profound implications for the pathophysiology and pharmacology of cell adaptation to hypoxia
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