Alternative regulation of HIF-1? stability through Phosphorylation on Ser451.
The hypoxia-inducible factor (HIF-1?) functions as a master regulator of oxygen homeostasis. Oxygen-dependent hydroxylation of HIF-1? is tightly regulated by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). The prolyl hydroxylation facilitates the recruitment of the von Hippel-Lindau (VHL) protein, leading to ubiquitination and degradation of HIF-1? by the ... proteasomes. Besides prolyl hydroxylation, phosphorylation of HIF-1? is another central post-translational modification, which regulates its stability under hypoxic conditions as well as normoxic conditions. By use of LC/MS/MS-based analysis, we were able to identify a specific serine residue (Ser451) of HIF-1? phosphorylated under hypoxic conditions. Using plasmids expressing wild type (WT), non-phosphorylatable mutant HIF-1? (S451A), and phosphomimetic mutant HIF-1? (S451E), we demonstrated that the phosphorylation at Ser451 is important in maintaining the HIF-1? protein stability. Notably, phosphorylation at S451 interrupts the interaction of HIF-1? with PHD and pVHL. A phosphomimetic construct of HIF-1? at Ser451 (S451E) is significantly more stable than WT HIF-1? under normoxic conditions. Cells transfected with unphosphorylatable HIF-1? 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? 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? stability.
Mesh Terms:
Amino Acid Substitution, Biomimetic Materials, Cell Hypoxia, HCT116 Cells, HEK293 Cells, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Models, Biological, Mutagenesis, Site-Directed, NIMA-Interacting Peptidylprolyl Isomerase, Phosphorylation, Prolyl Hydroxylases, Protein Interaction Domains and Motifs, Protein Processing, Post-Translational, Protein Stability, Serine, Von Hippel-Lindau Tumor Suppressor Protein
Amino Acid Substitution, Biomimetic Materials, Cell Hypoxia, HCT116 Cells, HEK293 Cells, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Models, Biological, Mutagenesis, Site-Directed, NIMA-Interacting Peptidylprolyl Isomerase, Phosphorylation, Prolyl Hydroxylases, Protein Interaction Domains and Motifs, Protein Processing, Post-Translational, Protein Stability, Serine, Von Hippel-Lindau Tumor Suppressor Protein
Biochem Biophys Res Commun
Date: Dec. 19, 2020
PubMed ID: 33550096
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