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 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.
Rho associated kinase p160 ROCK (Rock) and Ribosome S‐6 Kinase (RSK) both phosphorylate the Na+/H+ exchanger isoform 1 (NHE1) regulating ion transport, cell migration and binding to cytoskeletal linker proteins, ezrin, radixin, and moesin (ERM). NHE1 is an integral membrane protein involved in the regulation of intracellular pH, extracellular acidification, cellular migration, and cytoskeletal organization. Phosphorylation of NHE1 induces an intracellular alkalynization which promotes cellular proliferation and coordinates cellular migration through ERM binding and the accompanying organization of actin filaments. Using the Rock consensus sequence of RXXS/T and RXS/T and MS analysis of Rock phosphorylated NHE1, we have identified potential phosphorylation sites and created a series of site directed mutants. With the phosphorylation sites identified we have confirmed phosphorylation using in vitro labeling and generated stable cell lines expressing single and double Ser‐Ala mutations of the Rock and RSK sites to study changes in NHE regulation of ion translocation and cell motility events. This study intends to identify the Rock phosphorylation site on NHE1 and provide a new understanding for how transporters function to direct cell motility. This work was supported with funds from NSF‐MCB‐081778, NSF‐RUI‐MCB 0930432, and NIH‐1‐R15‐CA135616‐01
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