Investigation of Rho associated kinase phosphorylation of carboxyl terminus of the Sodium Hydrogen Exchanger Isoform 1

Abstract

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