title: Selective regulation of cyclic nucleotide phosphodiesterase PDE3A isoforms.
abstract: Inhibitors of cyclic nucleotide phosphodiesterase (PDE) PDE3A have inotropic actions in human myocardium, but their long-term use increases mortality in patients with heart failure. Two isoforms in cardiac myocytes, PDE3A1 and PDE3A2, have identical amino acid sequences except for a unique N-terminal extension in PDE3A1. We expressed FLAG-tagged PDE3A1 and PDE3A2 in HEK293 cells and examined their regulation by PKA- and PKC-mediated phosphorylation. PDE3A1, which is localized to intracellular membranes, and PDE3A2, which is cytosolic, were phosphorylated at different sites within their common sequence. Exposure to isoproterenol led to phosphorylation of PDE3A1 at the 14-3-3-binding site S312, whereas exposure to PMA led to phosphorylation of PDE3A2 at an alternative 14-3-3-binding site, S428. PDE3A2 activity was stimulated by phosphorylation at S428, whereas PDE3A1 activity was not affected by phosphorylation at either site. Phosphorylation of PDE3A1 by PKA and of PDE3A2 by PKC led to shifts in elution on gel-filtration chromatography consistent with increased interactions with other proteins, and 2D electrophoresis of coimmunoprecipitated proteins revealed that the two isoforms have distinct protein interactomes. A similar pattern of differential phosphorylation of endogenous PDE3A1 and PDE3A2 at S312 and S428 is observed in human myocardium. The selective phosphorylation of PDE3A1 and PDE3A2 at alternative sites through different signaling pathways, along with the different functional consequences of phosphorylation for each isoform, suggest they are likely to have distinct roles in cyclic nucleotide-mediated signaling in human myocardium, and raise the possibility that isoform-selective inhibition may allow inotropic responses without an increase in mortality.
authors.affiliation: Cardiology Section, VA Salt Lake City Health Care System, Salt Lake City, UT.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Vandeput
authors.forename: Nicolas
authors.initials: N
authors.lastname: Szabo-Fresnais
authors.forename: Faiyaz
authors.initials: F
authors.lastname: Ahmad
authors.forename: Changwon
authors.initials: C
authors.lastname: Kho
authors.forename: Ahyoung
authors.initials: A
authors.lastname: Lee
authors.forename: Judith
authors.initials: J
authors.lastname: Krall
authors.forename: Allan
authors.initials: A
authors.lastname: Dunlop
authors.forename: Mark W
authors.initials: MW
authors.lastname: Hazel
authors.forename: James A
authors.initials: JA
authors.lastname: Wohlschlegel
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Hajjar
authors.forename: Miles D
authors.initials: MD
authors.lastname: Houslay
authors.forename: Vincent C
authors.initials: VC
authors.lastname: Manganiello
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Movsesian
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002850:Chromatography, Gel; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D009200:Myocardial Contraction / Q000187:drug effects*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D058987:Phosphodiesterase 3 Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D007527:Isoenzymes; D058987:Phosphodiesterase 3 Inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; D007545:Isoproterenol
keywords: cAMP; protein–protein interactions