Fig. 4. PKA phosphorylates Thr63 and Ser692 to increase HIF-1α stability.
(A) GST fusion proteins encompassing the indicated HIF-1α residues were incubated with the recombinant Ca subunit of PKA (rCa) and analyzed for phospho-total protein (p-TP) by ProQ Diamond staining (top) or total protein (TP) 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 basic helix-loop-helix (bHLH), Per-Arnt-Sim homology (PAS-A and PAS-B), O2-dependent degradation (ODDD), inhibitory (ID), and amino- and carboxy-terminal transactivation (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 (V) or H89 (H). The bar graph shows the H/V ratio of FLAG:actin densitometry ratios for each fusion protein (mean ± SD, n = 3 independent experiments). *p < 0.05 compared to vehicle. (D-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 V or H and subjected to immunoblot assays. (G) HeLa cells expressing DM or T63A+S692A-mutant DM, and exposed to V, forskolin + IBMX (F/I) or H were subjected to immunoblot assays. *p < 0.05 compared to V, #p < 0.05 compared to DM under same condition. FLAG:actin ratios displayed as a number below each blot (D-F) or as a bar graph (G) are the mean ± SD from 3 independent experiments normalized to V (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.