title: Protein kinase A-dependent phosphorylation stimulates the transcriptional activity of hypoxia-inducible factor 1.
abstract: Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes encoding proteins that enable cells to adapt to reduced O2 availability. Proteins encoded by HIF-1 target genes play a central role in mediating physiological processes that are dysregulated in cancer and heart disease. These diseases are also characterized by increased production of cyclic adenosine monophosphate (cAMP), the allosteric activator of cAMP-dependent protein kinase A (PKA). Using glutathione S-transferase pull-down, coimmunoprecipitation, and mass spectrometry analyses, we demonstrated that PKA interacts with HIF-1α in HeLa cervical carcinoma cells and rat cardiomyocytes. PKA phosphorylated Thr(63) and Ser(692) on HIF-1α in vitro and enhanced HIF transcriptional activity and target gene expression in HeLa cells and rat cardiomyocytes. PKA inhibited the proteasomal degradation of HIF-1α in an O2-independent manner that required the phosphorylation of Thr(63) and Ser(692) and was not affected by prolyl hydroxylation. PKA also stimulated the binding of the coactivator p300 to HIF-1α to enhance its transcriptional activity and counteracted the inhibitory effect of asparaginyl hydroxylation on the association of p300 with HIF-1α. Furthermore, increased cAMP concentrations enhanced the expression of HIF target genes encoding CD39 and CD73, which are enzymes that convert extracellular adenosine 5'-triphosphate to adenosine, a molecule that enhances tumor immunosuppression and reduces heart rate and contractility. These data link stimuli that promote cAMP signaling, HIF-1α-dependent changes in gene expression, and increased adenosine, all of which contribute to the pathophysiology of cancer and heart disease.
authors.affiliation: Vascular Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Bullen
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Tchernyshyov
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Holewinski
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: DeVine
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Fan
authors.initials: F
authors.lastname: Wu
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Vidya
authors.initials: V
authors.lastname: Venkatraman
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: Kass
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Robert N
authors.initials: RN
authors.lastname: Cole
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Van Eyk
authors.affiliation: Vascular Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Radiation Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. gsemenza@jhmi.edu.
authors.forename: Gregg L
authors.initials: GL
authors.lastname: Semenza
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001081:Apyrase / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D018450:Disease Progression; D058851:GPI-Linked Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007165:Immunosuppression Therapy; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D014158:Transcription, Genetic
chemical_list: D015703:Antigens, CD; D058851:GPI-Linked Proteins; C497442:HIF1A protein, human; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D000242:Cyclic AMP; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D015720:5'-Nucleotidase; C548241:NT5E protein, human; D001081:Apyrase; C067869:CD39 antigen