title: RhoA Kinase (Rock) and p90 Ribosomal S6 Kinase (p90Rsk) phosphorylation of the sodium hydrogen exchanger (NHE1) is required for lysophosphatidic acid-induced transport, cytoskeletal organization and migration.
abstract: The sodium hydrogen exchanger isoform one (NHE1) plays a critical role coordinating asymmetric events at the leading edge of migrating cells and is regulated by a number of phosphorylation events influencing both the ion transport and cytoskeletal anchoring required for directed migration. Lysophosphatidic acid (LPA) activation of RhoA kinase (Rock) and the Ras-ERK growth factor pathway induces cytoskeletal reorganization, activates NHE1 and induces an increase in cell motility. We report that both Rock I and II stoichiometrically phosphorylate NHE1 at threonine 653 in vitro using mass spectrometry and reconstituted kinase assays. In fibroblasts expressing NHE1 alanine mutants for either Rock (T653A) or ribosomal S6 kinase (Rsk; S703A) we show that each site is partially responsible for the LPA-induced increase in transport activity while NHE1 phosphorylation by either Rock or Rsk at their respective site is sufficient for LPA stimulated stress fiber formation and migration. Furthermore, mutation of either T653 or S703 leads to a higher basal pH level and a significantly higher proliferation rate. Our results identify the direct phosphorylation of NHE1 by Rock and suggest that both RhoA and Ras pathways mediate NHE1-dependent ion transport and migration in fibroblasts.
authors.affiliation: Minnesota State University Moorhead, Department of Biosciences, Moorhead, MN, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Wallert
authors.affiliation: Minnesota State University Moorhead, Department of Biosciences, Moorhead, MN, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hammes
authors.affiliation: Minnesota State University Moorhead, Department of Biosciences, Moorhead, MN, USA.
authors.forename: Tony
authors.initials: T
authors.lastname: Nguyen
authors.affiliation: University of San Diego, Department of Chemistry and Biochemistry, San Diego, CA, USA.
authors.forename: Lea
authors.initials: L
authors.lastname: Kiefer
authors.affiliation: Minnesota State University Moorhead, Department of Biosciences, Moorhead, MN, USA.
authors.forename: Nick
authors.initials: N
authors.lastname: Berthelsen
authors.affiliation: Minnesota State University Moorhead, Department of Biosciences, Moorhead, MN, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Kern
authors.affiliation: Minnesota State University Moorhead, Department of Biosciences, Moorhead, MN, USA.
authors.forename: Kristina
authors.initials: K
authors.lastname: Anderson-Tiege
authors.affiliation: Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, USA.
authors.forename: John B
authors.initials: JB
authors.lastname: Shabb
authors.affiliation: Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, USA.
authors.forename: Wallace W
authors.initials: WW
authors.lastname: Muhonen
authors.affiliation: Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, USA.
authors.forename: Bryon D
authors.initials: BD
authors.lastname: Grove
authors.affiliation: University of San Diego, Department of Chemistry and Biochemistry, San Diego, CA, USA. Electronic address: josephprovost@sandiego.edu.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Provost
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation; D006224:Cricetinae; D003599:Cytoskeleton / Q000187:drug effects*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008246:Lysophospholipids / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D008246:Lysophospholipids; D010748:Phosphopeptides; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D020742:rhoA GTP-Binding Protein; C032881:lysophosphatidic acid
keywords: Cytoskeleton; Lysophosphatidic acid; NHE1; Rock kinase; Rsk; Sodium hydrogen exchanger