Impact of NHE1 Phosphorylation by Ribosomal S‐6 kinase and RhoA kinase on ERM binding and cell motility

Abstract

Cellular migration involves complex signaling and intricate reorganization of the cytoskeleton. Ezrin/Radixin/Moesin (ERM) proteins play a crucial role in the reorganization of the cytoskeleton by binding to membrane bound proteins including the Na+‐H+ exchanger (NHE) and anchoring stress fibers to the plasma membrane. During cellular migration, there is a reorganization of the membrane to localize NHE to the leading edge of the cell. Localization of NHE helps to give the cell polarity, which is important in directing stress fiber formation to assist the cell to move forward. The focus of this study was to determine the conditions by which ERM translocates to the membrane to bind to NHE and examine the role of the ERM:NHE binding complex in proper formation of unidirectional cell migration. We have prepared a series of Ser or Thr – Ala mutants at putative RhoA Kinase (Rock) and Ribosomal S‐6 kinase (RSK) phosphorylation sites and expressed these in NHE1‐null PS120 cells. Co‐immunoprecipitation of NHE1 identified ERM‐NHE1 interactions for each mutant following activation with lysophopsphatidic acid (LPA). Using wild‐type and non‐phosphorylatable EFGP‐ERM, we investigated the localization of ERM proteins and the impact of NHE1 phosphorylation. Finally we show the effect of NHE1 and ERM phosphorylation on stress fiber formation and cell motility.. This work was supported with funds from NSF‐MCB‐081778, NSF‐RUI‐MCB 0930432, and NIH‐1‐R15‐CA135616‐01.