The hypoxia-inducible factor (HIF-1 alpha) functions as a master regulator of oxygen homeostasis. Oxygen dependent hydroxylation of HIF-1 alpha is tightly regulated by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). The prolyl hydroxylation facilitates the recruitment of the von HippelLindau (VHL) protein, leading to ubiquitination and degradation of HIF-1 alpha by the proteasomes. Besides prolyl hydroxylation, phosphorylation of HIF-1 alpha is another central post-translational modification, which regulates its stability under hypoxic conditions as well as normoxic conditions. By use of LC/MS/MSbased analysis, we were able to identify a specific serine residue (Ser451) of HIF-1 alpha phosphorylated under hypoxic conditions. Using plasmids expressing wild type (WT), non-phosphorylatable mutant HIF1 alpha (S451A), and phosphomimetic mutant HIF-1 alpha (S451E), we demonstrated that the phosphorylation at Ser451 is important in maintaining the HIF-1 alpha protein stability. Notably, phosphorylation at S451 interrupts the interaction of HIF-1 alpha with PHD and pVHL. A phosphomimetic construct of HIF-1 alpha at Ser451 (S451E) is significantly more stable than WT HIF-1 alpha under normoxic conditions. Cells transfected with unphosphorylatable HIF-1 alpha exhibited significantly lower HIF-1 transcriptional activity than WT cells and markedly reduced tumor cell migration. Further, tumors derived from the phosphomimetic mutant cells grew faster, whereas the tumors derived from non-phosphorylatable mutant cells grew slower than the control tumors, suggesting that the phosphorylation of HIF-1 alpha at the Ser451 site is critical to promote tumor growth in vivo. Taken together, our data suggest an alternative mechanism responsible for the regulation of HIF-1 alpha stability. (C) 2021 Elsevier Inc. All rights reserved.