title: Activation of the Na+/H+exchanger in isolated cardiomyocytes through β-Raf dependent pathways. Role of Thr653of the cytosolic tail.
abstract: The mammalian Na+/H+exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that is a key regulator of intracellular pH in isolated cardiomyocytes. A 500 amino acid membrane domain removes protons and is regulated by a 315 amino acid cytosolic domain. In the myocardium, aberrant regulation of NHE1 contributes to ischemia reperfusion damage and to heart hypertrophy. We examined mechanisms of regulation of NHE1 in the myocardium by endothelin and β-Raf. Endothelin stimulated NHE1 activity and activated Erk-dependent pathways. Inhibition of β-Raf reduced NHE1 activity and Erk-pathway activation. We demonstrated that myocardial β-Raf binds to the C-terminal 182 amino acids of the NHE1 protein and that β-Raf is associated with NHE1 in intact cardiomyocytes. NHE1 was phosphorylated in vivo and the protein kinase inhibitor sorafenib reduced NHE1 phosphorylation levels. Immunoprecipitates of β-Raf from cardiomyocytes phosphorylated the C-terminal 182 amino acids of NHE1 and mass spectrometry analysis showed that amino acid Thr653was phosphorylated. Mutation of this amino acid to Ala resulted in defective activity while mutation to Asp restored the activity. The results demonstrate that Thr653is an important regulatory amino acid of NHE1 that is activated through β-Raf dependent pathways by phosphorylation either directly or indirectly by β-Raf, and this affects NHE1 activity.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada. Electronic address: xjli@ualberta.ca.
authors.forename: Xiuju
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada. Electronic address: aruna@ualberta.ca.
authors.forename: Aruna
authors.initials: A
authors.lastname: Augustine
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada. Electronic address: difei2@ualberta.ca.
authors.forename: Difei
authors.initials: D
authors.lastname: Sun
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada. Electronic address: liang.li@ualberta.ca.
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada. Electronic address: lfliegel@ualberta.ca.
authors.forename: Larry
authors.initials: L
authors.lastname: Fliegel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013970:Thyronines / Q000235:genetics / Q000378:metabolism
chemical_list: D017923:Sodium-Hydrogen Exchangers; D013970:Thyronines; C069139:growth factor-activatable Na-H exchanger NHE-1; D048493:Proto-Oncogene Proteins B-raf
keywords: Endothelin; Isolated cardiomyocytes; Na(+)/H(+) exchanger; pH regulation; β-Raf