Phosphorylation and regulation of the Na+/H+ exchanger through mitogen-activated protein kinase.
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.
Wang
H
H
Departments of Biochemistry and Pediatrics, University of Alberta, Edmonton, Canada.
Silva
N L
NL
Lucchesi
P A
PA
Haworth
R
R
Wang
K
K
Michalak
M
M
Pelech
S
S
Fliegel
L
L
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*
D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D017871:Calcium-Calmodulin-Dependent Protein Kinases