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Leu‐574 of human HIF‐1α is a molecular determinant of prolyl hydroxylation

Hypoxia‐inducible factor (HIF)‐1α, a master regulator of oxygen homeostasis, regulates genes crucial for cell growth and survival. In normoxia, HIF‐1α is constantly degraded via the ubiquitin‐proteasome pathway. The von Hippel‐Lindau (VHL) E3 ubiquitin ligase binds HIF‐1α through specific recognition of hydroxylated Pro‐402 or Pro‐564, both of which are modified by the oxygen‐dependent HIF prolyl hydroxylases (PHDs/HPHs). Despite the identification of a conserved Leu‐X‐X‐Leu‐Ala‐Pro motif, the molecular requirement of HIF‐1α for PHDs/HPHs binding remains elusive. Recently, we demonstrated that Leu‐574 of human HIF‐1α—10 residues downstream of Pro‐564—is essential for VHL recognition. We show here that the role of Leu‐574 is to recruit PHD2/HPH2 for Pro‐564 hydroxylation. An antibody specific for hydroxylated Pro‐564 has been used to determine the hydroxylation status; mutation or deletion of Leu‐574 results in a significant decrease in the ratio of the hydroxylated HIF‐1α to the total amount. The nine‐residue spacing between Pro‐564 and Leu‐574 is not obligatory for prolyl hydroxylation. Furthermore, mutation of Leu‐574 disrupts the binding of PHD2/HPH2, a key prolyl hydroxylase for oxygen‐dependent proteolysis of HIF‐1α. Hence, our findings indicate that Leu‐574 is essential for recruiting PHD2/HPH2, thereby providing a molecular basis for modulating HIF‐1α activity.