title: Protein kinase-mediated regulation of the Na(+)/H(+) exchanger in the rat myocardium by mitogen-activated protein kinase-dependent pathways.
abstract: We examined regulation of the Na(+)/H(+) exchanger isoform 1 by phosphorylation in the rat myocardium. We utilized cell extracts from adult rat hearts, adult rat extracts fractionated by fast performance liquid chromatography, and extracts from cultured neonatal cardiac myocytes. The carboxyl-terminal 178 amino acids of the Na(+)/H(+) exchanger were expressed in Escherichia coli fused with glutathione S-transferase. The purified protein was used as a substrate for in vitro phosphorylation and in-gel kinase assays. Unfractionated extracts from neonatal myocytes or adult hearts phosphorylated the COOH-terminal domain of the antiporter. Western blot analysis revealed that mitogen-activated protein (MAP) kinase (44 and 42 kDa) and p90(rsk) (90 kDa) were present in specific fractions of cardiac extracts that phosphorylated the COOH-terminal protein. In-gel kinase assays confirmed that protein kinases of approximately 44 and 90 kDa could phosphorylate this domain. MAP kinase and p90(rsk)-dependent phosphorylation of the antiporter could be demonstrated by immunoprecipitation of these kinases from extracts of neonatal cardiac myocytes. PD98059, a mitogen-activated protein kinase kinase inhibitor, decreased MAP kinase and p90(rsk) phosphorylation of the antiporter and abolished serum and endothelin 1-stimulated increases in steady-state pH(i). These results confirm the presence of MAP kinase-dependent phosphorylation in the regulation of the Na(+)/H(+) exchanger in the rat myocardium and suggest an important role for p90(rsk) phosphorylation in regulation of the protein by endothelin-mediated stimulation of the antiporter.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: A N
authors.initials: AN
authors.lastname: Moor
authors.forename: L
authors.initials: L
authors.lastname: Fliegel
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D007700:Kinetics; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
chemical_list: D017923:Sodium-Hydrogen Exchangers; D017871:Calcium-Calmodulin-Dependent Protein Kinases