title: Regulation of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) activity by phosphodiesterase 3A (PDE3A) in human myocardium: phosphorylation-dependent interaction of PDE3A1 with SERCA2.
abstract: Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.
authors.affiliation: From the Cardiovascular Pulmonary Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, Ahmadf@nhlbi.nih.gov.
authors.forename: Faiyaz
authors.initials: F
authors.lastname: Ahmad
authors.affiliation: From the Cardiovascular Pulmonary Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Weixing
authors.initials: W
authors.lastname: Shen
authors.affiliation: VA Salt Lake City Health Care System and University of Utah, Salt Lake City, Utah.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Vandeput
authors.affiliation: VA Salt Lake City Health Care System and University of Utah, Salt Lake City, Utah.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Szabo-Fresnais
authors.affiliation: VA Salt Lake City Health Care System and University of Utah, Salt Lake City, Utah.
authors.forename: Judith
authors.initials: J
authors.lastname: Krall
authors.affiliation: Department of Experimental Medical Science, Division for Diabetes, Metabolism, and Endocrinology, Lund University, Lund, Sweden.
authors.forename: Eva
authors.initials: E
authors.lastname: Degerman
authors.affiliation: Max Delbrueck Center for Molecular Medicine Berlin-Buch (MDC), 13125 Germany, and.
authors.forename: Frank
authors.initials: F
authors.lastname: Goetz
authors.affiliation: Max Delbrueck Center for Molecular Medicine Berlin-Buch (MDC), 13125 Germany, and DZHK, German Centre for Cardiovascular Research, 13347 Berlin, Germany.
authors.forename: Enno
authors.initials: E
authors.lastname: Klussmann
authors.affiliation: VA Salt Lake City Health Care System and University of Utah, Salt Lake City, Utah.
authors.forename: Matthew
authors.initials: M
authors.lastname: Movsesian
authors.affiliation: From the Cardiovascular Pulmonary Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Vincent
authors.initials: V
authors.lastname: Manganiello
mesh_terms: D054758:A Kinase Anchor Proteins / Q000032:analysis / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D060066:Protein Interaction Maps; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000032:analysis / Q000378:metabolism*
chemical_list: D054758:A Kinase Anchor Proteins; D020033:Protein Isoforms; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; D002118:Calcium
keywords: A-kinase Anchoring Protein (AKAP); Cyclic AMP (cAMP); Cyclic Nucleotide Phosphodiesterase; Immunohistochemistry; PDE3A; Phospholamban; Protein Kinase A (PKA); SERCA2; Subcellular Fractionation