title: B-Raf associates with and activates the NHE1 isoform of the Na+/H+ exchanger.
abstract: The serine/threonine kinase B-Raf is the second most frequently occurring human oncogene after Ras. Mutations of B-Raf occur with the highest incidences in melanoma, and the most common mutant, V600E, renders B-Raf constitutively active. The sodium proton exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein responsible for regulating intracellular pH, cell volume, cell migration, and proliferation. A screen of protein kinases that bind to NHE1 revealed that B-Raf bound to the cytosolic regulatory tail of NHE1. Immunoprecipitation of NHE1 from HeLa and HEK cells confirmed the association of B-Raf with NHE1 in vivo. The expressed and purified C-terminal 182 amino acids of the NHE1 protein were also shown to associate with B-Raf protein in vitro. Because treatment with the kinase inhibitor sorafenib decreased NHE1 activity in HeLa and HEK cells, we examined the role of B-Raf in regulating NHE1 in malignant melanoma cells. Melanoma cells with the B-Raf(V600E) mutation demonstrated increased resting intracellular pH that was dependent on elevated NHE1 activity. NHE1 activity after an acute acid load was also elevated in these cell lines. Moreover, inhibition of B-Raf activity by either sorafenib, PLX4720, or siRNA reduction of B-Raf levels abolished ERK phosphorylation and decreased NHE1 activity. These results demonstrate that B-Raf associates with and stimulates NHE1 activity and that B-Raf(V600E) also increases NHE1 activity that raises intracellular pH.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Pratap
authors.initials: P
authors.lastname: Karki
authors.forename: Xiuju
authors.initials: X
authors.lastname: Li
authors.forename: David
authors.initials: D
authors.lastname: Schrama
authors.forename: Larry
authors.initials: L
authors.lastname: Fliegel
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001557:Benzenesulfonates / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D048429:Cell Size; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000494:pharmacology; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020125:Mutation, Missense; D009536:Niacinamide / Q000031:analogs & derivatives; D010671:Phenylurea Compounds; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D000077157:Sorafenib; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D001557:Benzenesulfonates; D027682:Cation Transport Proteins; D007211:Indoles; C528407:PLX 4720; D010671:Phenylurea Compounds; D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D013449:Sulfonamides; D009536:Niacinamide; D000077157:Sorafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D048049:Extracellular Signal-Regulated MAP Kinases