title: Effects of heterologous expression of human cyclic nucleotide phosphodiesterase 3A (hPDE3A) on redox regulation in yeast.
abstract: Oxidative stress plays a pivotal role in pathogenesis of cardiovascular diseases and diabetes; however, the roles of protein kinase A (PKA) and human phosphodiesterase 3A (hPDE3A) remain unknown. Here, we show that yeast expressing wild-type (WT) hPDE3A or K13R hPDE3A (putative ubiquitinylation site mutant) exhibited resistance or sensitivity to exogenous hydrogen peroxide (H2O2), respectively. H2O2-stimulated ROS production was markedly increased in yeast expressing K13R hPDE3A (Oxidative stress Sensitive 1, OxiS1), compared with yeast expressing WT hPDE3A (Oxidative stress Resistant 1, OxiR1). In OxiR1, YAP1 and YAP1-dependent antioxidant genes were up-regulated, accompanied by a reduction in thioredoxin peroxidase. In OxiS1, expression of YAP1 and YAP1-dependent genes was impaired, and the thioredoxin system malfunctioned. H2O2increased cyclic adenosine monophosphate (cAMP)-hydrolyzing activity of WT hPDE3A, but not K13R hPDE3A, through PKA-dependent phosphorylation of hPDE3A, which was correlated with its ubiquitinylation. The changes in antioxidant gene expression did not directly correlate with differences in cAMP-PKA signaling. Despite differences in their capacities to hydrolyze cAMP, total cAMP levels among OxiR1, OxiS1, and mock were similar; PKA activity, however, was lower in OxiS1 than in OxiR1 or mock. During exposure to H2O2, however, Sch9p activity, a target of Rapamycin complex 1-regulated Rps6 kinase and negative-regulator of PKA, was rapidly reduced in OxiR1, and Tpk1p, a PKA catalytic subunit, was diffusely spread throughout the cytosol, with PKA activation. In OxiS1, Sch9p activity was unchanged during exposure to H2O2, consistent with reduced activation of PKA. These results suggest that, during oxidative stress, TOR-Sch9 signaling might regulate PKA activity, and that post-translational modifications of hPDE3A are critical in its regulation of cellular recovery from oxidative stress.
authors.affiliation: Laboratory of Biochemical Physiology, Cardiovascular and Pulmonary Branch, NHLBI/NIH, Room 5N-307, Building 10, Bethesda, MD 20892-1434, USA.
authors.forename: Dong Keun
authors.initials: DK
authors.lastname: Rhee
authors.affiliation: Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD, USA.
authors.forename: Jung Chae
authors.initials: JC
authors.lastname: Lim
authors.affiliation: Laboratory of Biochemical Physiology, Cardiovascular and Pulmonary Branch, NHLBI/NIH, Room 5N-307, Building 10, Bethesda, MD 20892-1434, USA.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Hockman
authors.affiliation: Laboratory of Biochemical Physiology, Cardiovascular and Pulmonary Branch, NHLBI/NIH, Room 5N-307, Building 10, Bethesda, MD 20892-1434, USA.
authors.forename: Faiyaz
authors.initials: F
authors.lastname: Ahmad
authors.affiliation: Nervous System Development and Plasticity Section, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Dong Ho
authors.initials: DH
authors.lastname: Woo
authors.affiliation: Laboratory of Biochemical Physiology, Cardiovascular and Pulmonary Branch, NHLBI/NIH, Room 5N-307, Building 10, Bethesda, MD 20892-1434, USA.
authors.forename: Youn Wook
authors.initials: YW
authors.lastname: Chung
authors.affiliation: Laboratory of Biochemical Physiology, Cardiovascular and Pulmonary Branch, NHLBI/NIH, Room 5N-307, Building 10, Bethesda, MD 20892-1434, USA.
authors.forename: Shiwei
authors.initials: S
authors.lastname: Liu
authors.affiliation: Laboratory of Biochemical Physiology, Cardiovascular and Pulmonary Branch, NHLBI/NIH, Room 5N-307, Building 10, Bethesda, MD 20892-1434, USA.
authors.forename: Allison L
authors.initials: AL
authors.lastname: Hockman
authors.affiliation: Laboratory of Biochemical Physiology, Cardiovascular and Pulmonary Branch, NHLBI/NIH, Room 5N-307, Building 10, Bethesda, MD 20892-1434, USA.
authors.forename: Vincent C
authors.initials: VC
authors.lastname: Manganiello
mesh_terms: D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D006861:Hydrogen Peroxide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human
keywords: PKA; Sch9; cAMP; oxidative stress; phosphodiesterase 3A; thioredoxin