%0 Journal Article %1 Iwam_1996_13609 %A Iwamoto, T. %A Pan, Y. %A Wakabayashi, S. %A Imagawa, T. %A Yamanaka, H. I. %A Shigekawa, M. %D 1996 %J J. Biol. Chem. %K Acetate, Acid Adenosine Amino Animals; Antibodies; Base C, Calcium, Carrier Cell Chickens; DNA Data; Dogs; Exchanger; Expression; Fusion Gene Ion Kinase Line; Molecular Myocardium, Phosphorylation; Primers, Protein Proteins, Rats; Recombinant Sequence Sequence; Sodium, Sodium-Calcium Tetradecanoylphorbol Transfection Transport, Triphosphate, drug effects; enzymology/metabolism; genetics/immunology/metabolism; genetics; metabolism; pharmacology; %N 23 %P 13609--13615 %T Phosphorylation-dependent regulation of cardiac Na$^+$/Ca$^2+$ exchanger via protein kinase C. %U http://www.jbc.org/cgi/content/full/271/23/13609 %V 271 %X The cardiac Na$^+$/Ca$^2+$ exchanger (NCX1) plays a major role in the extrusion of Ca$^2+$ from cardiomyocytes. We studied the role of protein phosphorylation in the regulation of cardiac NCX1 using CCL39 stably overexpressing the canine cardiac NCX1 and rat neonatal cardiomyocytes. In both cell types, the NCX1 protein immunoprecipitated with a chicken anti-NCX1 antibody exhibited a significant basal phosphorylation that was further enhanced by treatment with endothelin-1, acidic fibroblast growth factor, phorbol 12-myristate 13-acetate, or okadaic acid. In contrast, calphostin C, K252a, or EGTA inhibited the phosphorylation. The phosphorylation occurred on two major tryptic phosphopeptides (P1 and P2) exclusively on serine residues. Evidence is presented suggesting that P2 was derived from an N-terminal half (amino acids 240-475) of the central cytoplasmic domain of NCX1 and was phosphorylated directly by protein kinase C (PKC). The agents that increased NCX1 phosphorylation significantly enhanced both the forward and reverse modes of Na$^+$/Ca$^2+$ exchange. This exchange activation exhibited a very good correlation with the NCX1 phosphorylation. In NCX1-transfected cells, PKC down-regulation following prolonged exposure to phorbol 12-myristate 13-acetate abolished the acidic fibroblast growth factor-induced activation of exchange activity. On the other hand, cell ATP depletion reduced the exchange activity and abolished the effects of the above agents on exchange activity. These results indicate that the cardiac NCX1 is up-regulated by PKC-catalyzed phosphorylation. The cardiac NCX1 thus could play an important role in the previously reported negative inotropic actions of phorbol esters and other PKC-activating agents.

@article{Iwam_1996_13609, abstract = {The cardiac {N}a$^{+}$/{C}a$^{2+}$ exchanger (NCX1) plays a major role in the extrusion of {C}a$^{2+}$ from cardiomyocytes. We studied the role of protein phosphorylation in the regulation of cardiac NCX1 using CCL39 stably overexpressing the canine cardiac NCX1 and rat neonatal cardiomyocytes. In both cell types, the NCX1 protein immunoprecipitated with a chicken anti-NCX1 antibody exhibited a significant basal phosphorylation that was further enhanced by treatment with endothelin-1, acidic fibroblast growth factor, phorbol 12-myristate 13-acetate, or okadaic acid. In contrast, calphostin C, K252a, or EGTA inhibited the phosphorylation. The phosphorylation occurred on two major tryptic phosphopeptides (P1 and P2) exclusively on serine residues. Evidence is presented suggesting that P2 was derived from an N-terminal half (amino acids 240-475) of the central cytoplasmic domain of NCX1 and was phosphorylated directly by protein kinase C (PKC). The agents that increased NCX1 phosphorylation significantly enhanced both the forward and reverse modes of {N}a$^{+}$/{C}a$^{2+}$ exchange. This exchange activation exhibited a very good correlation with the NCX1 phosphorylation. In NCX1-transfected cells, PKC down-regulation following prolonged exposure to phorbol 12-myristate 13-acetate abolished the acidic fibroblast growth factor-induced activation of exchange activity. On the other hand, cell ATP depletion reduced the exchange activity and abolished the effects of the above agents on exchange activity. These results indicate that the cardiac NCX1 is up-regulated by PKC-catalyzed phosphorylation. The cardiac NCX1 thus could play an important role in the previously reported negative inotropic actions of phorbol esters and other PKC-activating agents.}, added-at = {2009-06-03T11:20:58.000+0200}, author = {Iwamoto, T. and Pan, Y. and Wakabayashi, S. and Imagawa, T. and Yamanaka, H. I. and Shigekawa, M.}, biburl = {https://www.bibsonomy.org/bibtex/25d0b8cbeebe8f59270434716beabc5ab/hake}, description = {The whole bibliography file I use.}, file = {Iwam_1996_13609.pdf:Iwam_1996_13609.pdf:PDF}, institution = {Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565, Japan.}, interhash = {acc6427e27bdd2e8a06aaef35b627dbc}, intrahash = {5d0b8cbeebe8f59270434716beabc5ab}, journal = {J. Biol. Chem.}, keywords = {Acetate, Acid Adenosine Amino Animals; Antibodies; Base C, Calcium, Carrier Cell Chickens; DNA Data; Dogs; Exchanger; Expression; Fusion Gene Ion Kinase Line; Molecular Myocardium, Phosphorylation; Primers, Protein Proteins, Rats; Recombinant Sequence Sequence; Sodium, Sodium-Calcium Tetradecanoylphorbol Transfection Transport, Triphosphate, drug effects; enzymology/metabolism; genetics/immunology/metabolism; genetics; metabolism; pharmacology;}, month = Jun, number = 23, pages = {13609--13615}, pdf = {Iwam_1996_13609.pdf}, pmid = {8662755}, timestamp = {2009-06-03T11:21:16.000+0200}, title = {Phosphorylation-dependent regulation of cardiac {N}a$^{+}$/{C}a$^{2+}$ exchanger via protein kinase C.}, url = {http://www.jbc.org/cgi/content/full/271/23/13609}, volume = 271, year = 1996 }