title: Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A.
abstract: The cardiac Na(+)/Ca(2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-(32)P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.
authors.affiliation: Institute for Experimental Medical Research, Oslo Univ. Hospital, Ullevaal, Oslo, Norway. pimthanya.wanichawan@medisin.uio.no
authors.forename: Pimthanya
authors.initials: P
authors.lastname: Wanichawan
authors.forename: William E
authors.initials: WE
authors.lastname: Louch
authors.forename: Kristin H
authors.initials: KH
authors.lastname: Hortemo
authors.forename: Bjørg
authors.initials: B
authors.lastname: Austbø
authors.forename: Per Kristian
authors.initials: PK
authors.lastname: Lunde
authors.forename: John D
authors.initials: JD
authors.lastname: Scott
authors.forename: Ole M
authors.initials: OM
authors.lastname: Sejersted
authors.forename: Cathrine R
authors.initials: CR
authors.lastname: Carlson
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D019295:Computational Biology / Q000379:methods; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D057809:HEK293 Cells; D006321:Heart / Q000187:drug effects; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism*
chemical_list: D000318:Adrenergic beta-Agonists; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol