Phosphorylation and Regulation of the Na+/H+ Exchanger through Mitogen-Activated Protein Kinase

• Published in: Biochemistry (Easton) 1997-07, Vol.36 (30), p.9151-9158
• Main Authors: Wang, H, Silva, N. L. C. L, Lucchesi, P. A, Haworth, R, Wang, K, Michalak, M, Pelech, S, Fliegel, L
• Format: Article
• Language: English
• Subjects: Animals; Calcium-Calmodulin-Dependent Protein Kinases - genetics; Calcium-Calmodulin-Dependent Protein Kinases - physiology; CHO Cells; Cricetinae; Cytoplasm - enzymology; Enzyme Induction; Isomerism; Muscle, Skeletal - enzymology; Mutagenesis, Site-Directed; Phosphorylation; Precipitin Tests; Rabbits; Recombinant Fusion Proteins - metabolism; Sodium-Hydrogen Exchangers - genetics; Sodium-Hydrogen Exchangers - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi970802f

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.