PKA phosphorylates Thr63 and Ser692 to increase HIF-1α stability.

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PKA phosphorylates Thr63 and Ser692 to increase HIF-1α stability. PKA phosphorylates Thr63 and Ser692 to increase HIF-1α stability. (A) GST fusion proteins encompassing the indicated HIF-1α residues were incubated with the rCa subunit of PKA and analyzed for phospho–total protein (p-TP) by Pro-Q Diamond staining (top) or total protein by Coomassie Blue staining (bottom). Phosphorylated proteins are indicated (arrows) (representative of n = 2 independent experiments). (B) The schematic shows HIF-1α residues that were phosphorylated (P) by rCa in vitro as determined by LC-MS/MS analysis of LysC- or trypsin-digested peptides (n = 1 experiment per digestion). The location of the bHLH, Per-Arnt-Sim homology (PAS-A and PAS-B), O2-dependent degradation (ODDD), ID, and TAD-N and TAD-C domains and sites of hydroxylation (OH) are shown. (C) Immunoblot assays were performed using lysates of HeLa cells expressing FLAG-tagged HIF-1α–DM or a deletion mutant containing the indicated HIF-1α residues and exposed to vehicle or H89. The bar graph shows the H89/vehicle ratio of FLAG/actin densitometry ratios for each fusion protein (means ± SD, n = 3 independent experiments). *P < 0.05 compared to vehicle. (D to F) HeLa cells expressing FLAG-tagged HIF-1α–DM (DM) or S692A-, T700A-, or S727A-mutant DM (D); FLAG-tagged HIF-1α1–200, which was either wild-type (WT) or contained an S31A or T63A mutation (E); or FLAG-tagged HIF-1α531–826, which was WT or contained an S692A mutation (F), were treated with vehicle or H89 and subjected to immunoblot assays. (G) HeLa cells expressing DM or T63A- + S692A-mutant DM and exposed to vehicle, forskolin + IBMX, or H89 were subjected to immunoblot assays. *P < 0.05 compared to vehicle; #P < 0.05 compared to DM under same condition. FLAG/actin ratios displayed as a number below each blot (D to F) or as a bar graph are means ± SD from three independent experiments normalized to vehicle (D) or respective WT (E and F) or DM (G) vehicle-treated controls. (H) MS/MS spectra of the PKA-phosphorylated HIF-1α tryptic peptides ASVMRLpTISYLRVRK (left) and SPNVLpSVALSQR (right) demonstrating phospho-Thr63 (pThr63) and phospho-Ser692 (pSer692), respectively. Monoisotopic b′- and y′-type fragment ion masses are displayed above the respective annotated ion peaks (n = 1 experiment per digestion). The Mann-Whitney U test was used to analyze statistical significance for all data in this figure. John W. Bullen et al., Sci. Signal. 2016;9:ra56 ©2016 by American Association for the Advancement of Science