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

• Published in: Cellular signalling Vol. 27; no. 3; pp. 498 - 509
• Main Authors: Wallert, Mark A., Hammes, Daniel, Nguyen, Tony, Kiefer, Lea, Berthelsen, Nick, Kern, Andrew, Anderson-Tiege, Kristina, Shabb, John B., Muhonen, Wallace W., Grove, Bryon D., Provost, Joseph J.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.03.2015
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Animals; Cell Line; Cell Movement - drug effects; Cell Proliferation; Cricetinae; Cytoskeleton; Cytoskeleton - drug effects; Humans; Hydrogen-Ion Concentration; Lysophosphatidic acid; Lysophospholipids - pharmacology; Molecular Sequence Data; NHE1; Phosphopeptides - analysis; Phosphorylation - drug effects; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; rhoA GTP-Binding Protein - genetics; rhoA GTP-Binding Protein - metabolism; Ribosomal Protein S6 Kinases, 90-kDa - genetics; Ribosomal Protein S6 Kinases, 90-kDa - metabolism; Rock kinase; Rsk; Sequence Alignment; Sodium hydrogen exchanger; Sodium-Hydrogen Exchangers - genetics; Sodium-Hydrogen Exchangers - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; NHE1; ctNHE; Rock; Lysophosphatidic acid; Rock kinase; Cytoskeleton; RSK; Sodium hydrogen exchanger; LPA; Rsk
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2015.01.002

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. [Display omitted] •Directed cell migration involves the sodium hydrogen exchanger 1 (NHE1).•NHE1 is phosphorylated by Rock at serine 653.•Both Rsk and Rock cooperatively influence NHE1 transport and are critical for cell motility events.•Rock directly regulates both functions of NHE1 impacting lysophosphatidic acid (LPA) mediated cell motility.•Both LPA signaling pathways equally contribute to NHE1 function.