Research ArticleCell Biology

Protein kinase A–dependent phosphorylation stimulates the transcriptional activity of hypoxia-inducible factor 1

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Science Signaling  31 May 2016:
Vol. 9, Issue 430, pp. ra56
DOI: 10.1126/scisignal.aaf0583
  • Fig. 1 PKA interacts with HIF-1α.

    (A) Immunoblot assays of lysates (left) and proteins pulled down by GST or GST–HIF-1α531–826 from the same lysates (right), which were prepared from H9c2 cells subjected to no treatment (NT); treated with vehicle (V), isoproterenol (ISO), phenylephrine (PE), or isoproterenol/phenylephrine; or exposed to 1% O2 (Hyp) (n ≥ 3 independent experiments). (B) Immunoprecipitation (IP) of R1a from NRCMs (left panel; n = 2 biological replicates) or H9c2 cells (middle panel; n = 2 biological replicates) and immunoprecipitation of HIF-1α from HeLa cells (right panel; n = 2 biological replicates) were performed, followed by immunoblot assays using antibodies against HIF-1α, R1a, or Ca. Primary antibody (Ab) alone and lysate incubated with nonspecific immunoglobulin G (IgG) were included as negative controls. (C) Pull-down assays were performed using GST or GST–HIF-1α531–826 and recombinant His-tagged Ca or R1a, followed by immunoblot assays using the indicated antibodies (n = 2 independent experiments).

  • Fig. 2 PKA increases HIF-1α protein abundance and transcriptional activity.

    (A and B) Immunoblot assays were performed using lysates from primary human left ventricular cardiomyocytes (CMs) exposed to vehicle, isoproterenol, or isoproterenol + phenylephrine (A) (n = 2 biological replicates), or HeLa cells exposed to vehicle, forskolin (F), H89 (H), or forskolin + H89 at 20 or 1% O2 (B) (representative of n = 3 independent experiments). (C) HeLa cells were cotransfected with p2.1 and pSV-Renilla and exposed to vehicle, forskolin, H89, forskolin + H89, or PKI at 20 or 1% O2, and the ratio of firefly/Renilla luciferase was determined (HIF luciferase activity; means ± SD, n = 9 biological replicates from three independent experiments). *P < 0.05 compared to vehicle at same % O2; #P < 0.05 compared to forskolin at same % O2. a.u., arbitrary units. (D) HeLa cells were cotransfected with p2.1, pSV-Renilla, and vector encoding red fluorescent protein (RFP), RFP-Ca fusion protein, or hemagglutinin-tagged Cb and exposed to 20 or 1% O2, and HIF luciferase activity was determined (means ± SD, n = 9 biological replicates from three independent experiments). *P < 0.05 compared to RFP at the same % O2. (E) Immunoblot assays were performed using lysates of HeLa cells expressing RFP or RFP-Ca and exposed to 20 or 1% O2 (n = 3 independent experiments). (F and G) HIF-dependent luciferase reporter assays (F) [means ± SD, n = 9 biological replicates from three independent experiments; *P < 0.05 compared to scrambled control (SC) at same % O2] and immunoblot assays (G) (n = 3 independent experiments) were performed using lysates of HeLa cells expressing a scrambled control short hairpin RNA (shRNA) or either of two shRNAs targeting Ca (shCa-1 or shCa-5) and were exposed to 20 or 1% O2. (H) HIF-1α mRNA abundance was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in parental HeLa cells (left panel), scrambled control, shCa-1 and shCa-5 HeLa subclones (middle panel), or NRCMs (right panel), which were exposed to 20 or 1% O2 (means ± SD, n = 6 biological replicates from three independent experiments; none of the differences were statistically significant). Data in (C), (D), (F), and (H) are graphed as fold change from respective controls at 20% O2. The Mann-Whitney U test was used to assess statistical significance for all data in this figure.

  • Fig. 3 PKA inhibits the proteasomal degradation of HIF-1α independently of prolyl hydroxylation.

    (A and B) HeLa cells were exposed to vehicle, forskolin, H89, forskolin + H89, or PKI at 20 or 1% O2 (A) or to vehicle, H89, MG132 (M), or MG132 + H89 (M/H) at 1% O2 (B). (C) HEK293T cells expressing FLAG-tagged P402A/P564A HIF-1α–DM were exposed to vehicle or forskolin. For each condition, two biological replicates are shown. (D) HeLa cells expressing FLAG-tagged HIF-1α–DM were exposed to vehicle, forskolin, H89, forskolin + H89, forskolin + IBMX (F/I) or forskolin + IBMX + H89 (F/I/H). (E) HeLa cells transfected with empty vectors (EVs) or FLAG-tagged HIF-1α–DM and RFP or RFP-Ca. Data in all panels are representative of at least three independent experiments unless otherwise indicated.

  • 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 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.

  • Fig. 5 PKA stimulates the TAD function of HIF-1α.

    (A) The schematic illustrates the HIF-1α TAD function reporter assay (G, Gal4-binding sites). (B and C) Luciferase assays (means ± SD, n = 9 biological replicates from three independent experiments) were performed using lysates from HeLa cells treated as indicated (B) or HeLa cells expressing RFP or RFP-Ca and exposed to 20 or 1% O2 (C). *P < 0.05 compared to vehicle or RFP at the same % O2; #P < 0.05 compared to forskolin + IBMX at the same % O2. (D) HeLa cells expressing GalA or GalA with an N803A mutation were exposed to 20 or 1% O2 and subjected to immunoblot (top) or luciferase (bottom) assays (means ± SD, n = 9 biological replicates from three independent experiments). *P < 0.05 compared to vehicle at the same % O2. (E) HeLa cells expressing empty vector or FLAG-tagged HIF-1α–DM were exposed to vehicle, MG132, MG132 + forskolin + IBMX, or MG132 + H89 and subjected to immunoblot assays (left), or p300 (top right) or FLAG (bottom right) immunoprecipitation was performed (n = 3 independent experiments). (F) Immunoblot assays were performed using lysates and p300 immunoprecipitated from HeLa cells expressing FLAG-tagged P402A/P564A/N803A HIF-1α–TM and treated as in (E) (n = 3 independent experiments). (G) The schematic shows Gal4–HIF-1α fusion proteins and HIF-1α residues identified as phosphorylated by the rCa subunit of PKA in vitro using LC-MS/MS on Glu-C–digested peptides (n = 1 experiment). Locations of ID, TAD-N and TAD-C, and the Asn hydroxylation site are shown. Fusion proteins with TAD function that was not inhibited by H89 are highlighted in red. (H) Luciferase assays were performed using lysates from HeLa cells, which were transfected with Gal4-DBD fused to HIF-1α residues 531–575 (GalL), 577–826 (GalB), 757–826 (GalG), or 786–826 (GalH), and incubated at 20 or 1% O2 (left) (means ± SD, n = 6 biological replicates from two independent experiments). *P < 0.05 compared to vehicle at the same % O2. Endogenous HIF-1α (right, top panel), GalG and GalH (right, middle panel), and actin (right, bottom panel) were detected by immunoblot assays. (I) GST–HIF-1α fusion proteins were tested for their ability to capture the Ca or R1a subunits of PKA from HeLa cell lysates (representative of n = 3 independent experiments). (J and K) Luciferase assays were performed using lysates from HeLa cells expressing unmutated WT GalG, or GalG with S760A + S761A, or S809A mutations and exposed to vehicle or H89 at 20 or 1% O2 (means ± SD, n = 6 biological replicates from two independent experiments); *P < 0.05 compared to vehicle at same % O2. Data in (B) to (D), (G), (J), and (K) are graphed as fold change from respective controls at 20% O2. The Mann-Whitney U test was used to analyze statistical significance for all data in this figure.

  • Fig. 6 PKA stimulates HIF-1 target gene expression.

    (A) CA9 and PDK1 mRNA expression was determined by RT-qPCR in parental HeLa cells; scrambled shRNA control, shCa-1, or shCa-5 HeLa subclones; and NRCMs exposed to vehicle, forskolin, H89, or forskolin + H89 at 20 or 1% O2 (means ± SD, n = 6 biological replicates from two independent experiments). *P < 0.05 compared to vehicle; #P < 0.05 compared to scrambled control; ^P < 0.05 compared to H89. (B) CD39 and CD73 mRNA expression and the abundance of the corresponding proteins in HeLa cells or NRCMs exposed to vehicle, forskolin, H89, or forskolin + H89 at 20 or 1% O2 were determined by RT-qPCR (means ± SD, n = 6 biological replicates from two independent experiments) and immunoblot assays (n = 3 independent experiments). ^P < 0.05 compared to 20% O2; *P < 0.05 compared to vehicle; #P < 0.05 compared to H89. Data in (A) and (B) are graphed as fold change from respective controls at 20% O2. The Mann-Whitney U test was used to assess statistical significance for all data in (A) and (B). (C) PKA regulates HIF-1α protein stability and TAD-C function. Phosphorylation of Thr63 and Ser692 by PKA inhibits proteasomal degradation of HIF-1α. PKA also stimulates p300 binding to overcome the inhibitory effect of FIH-1–mediated Asn803 hydroxylation, thereby increasing transactivation by HIF-1α.

Supplementary Materials

  • Supplementary Materials for:

    Protein kinase A–dependent phosphorylation stimulates the transcriptional activity of hypoxia-inducible factor 1

    John W. Bullen, Irina Tchernyshyov, Ronald J. Holewinski, Lauren DeVine, Fan Wu, Vidya Venkatraman, David L. Kass, Robert N. Cole, Jennifer Van Eyk, Gregg L. Semenza*

    *Corresponding author. Email: gsemenza{at}jhmi.edu

    This PDF file includes:

    • Table S1. List of primer and shRNA nucleotide sequences.
    • Table S2. Antibodies used for immunoprecipitation and immunoblot assays.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Summary of MS/MS data for R1a peptide and protein group identification.

    Citation: J. W. Bullen, I. Tchernyshyov, R. J. Holewinski, L. DeVine, F. Wu, V. Venkatraman, D. L. Kass, R. N. Cole, J. Van Eyk, G. L. Semenza, Protein kinase A–dependent phosphorylation stimulates the transcriptional activity of hypoxia-inducible factor 1. Sci. Signal. 9, ra56 (2016).

    © 2016 American Association for the Advancement of Science

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