title: Phosphorylation and regulation of the Na+/H+ exchanger through mitogen-activated protein kinase.
abstract: We examined mitogen-activated protein kinase-mediated phosphorylation and activation of the Na+/H+ exchanger isoform type 1. A rabbit skeletal muscle extract was fractionated by FPLC chromatography. Four main fractions had the ability to phosphorylate the carboxyl-terminal region of NHE1. Western blot analysis and immunoprecipitation showed that three of these were associated with MAP kinase-dependent phosphorylation. Phosphorylation studies using purified MAP kinase showed that the region involved was the carboxyl-terminal 178 amino acids of the protein and that the stoichiometry was 1 phosphate/mol of protein. In-gel kinase assays showed that cytosolic extracts from smooth muscle cells also phosphorylate the carboxyl-terminal of NHE1 and that the MAP kinase-dependent phosphorylation could be activated by PDGF and AngII. Mutant cell lines with an inducible dominant negative MAP kinase showed decreased serum activation of Na+/H+ exchange but normal hypertonic activation of the protein. The results show that MAP kinase is intimately involved in regulation of the Na+/H+ exchanger, possibly through phosphorylation of one amino acid of the carboxyl-terminal cytosolic domain.
authors.affiliation: Departments of Biochemistry and Pediatrics, University of Alberta, Edmonton, Canada.
authors.forename: H
authors.initials: H
authors.lastname: Wang
authors.forename: N L
authors.initials: NL
authors.lastname: Silva
authors.forename: P A
authors.initials: PA
authors.lastname: Lucchesi
authors.forename: R
authors.initials: R
authors.lastname: Haworth
authors.forename: K
authors.initials: K
authors.lastname: Wang
authors.forename: M
authors.initials: M
authors.lastname: Michalak
authors.forename: S
authors.initials: S
authors.lastname: Pelech
authors.forename: L
authors.initials: L
authors.lastname: Fliegel
mesh_terms: D000818:Animals; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000502:physiology*; D006224:Cricetinae; D003593:Cytoplasm / Q000201:enzymology; D004790:Enzyme Induction; D007536:Isomerism; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011233:Precipitin Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D017871:Calcium-Calmodulin-Dependent Protein Kinases