An Investigation of the Impact of NHE1 Phosphorylation on Steady State Intracellular pH

Abstract

The Sodium‐Hydrogen Exchanger 1 (NHE1) is involved in cell scaffolding, mobility and intracellular pH (pHi) homeostasis in most cells. The C‐terminus of NHE1 is phosphorylated by several protein kinases, each contributing to NHE1 function. While investigation of one or two sites have been conducted with various agonists, we do not have an understanding of how these sites coordinate or if there is a hierarchical phosphorylation regulation of NHE1. To determine the potential phosphorylation‐dependent regulation of NHE1 on pHi, we focus on four protein kinases with known sites on NHE1; ERK 1⁄2, protein kinase B (AKT), RhoA kinase (Rock) and ribosomal s6 kinase (RSK). To measure the impact of these four kinases we will treat cells with three different agonist; lysophophatidic acid (LPA), platelet‐derived growth factor (PDGF), and phenylephrine (PE). We first determined the dose response for each agonist in fibroblasts deficient in NHE1 expression (PS120 cells) and in stable expressing human NHE1 in PS120 cells (PSN cells). To determine the role of each kinase on NHE1 basal activity, cells were treated with each agonist and teady state pHi was determined 15‐20 min after addition of agonist. Increases in pHi of 0.18 to 0.2 were observed for agonists. The role of each kinase was determined by treating cells prior to agonist with a specific cell permeable kinase inhibitor (1uM MK‐2206, 10uM BI‐D1870, 10uM Y27632 and 0.5uM SCH772984). The impact of direct phosphorylation was determined for each agonist in cells expressing human NHE1 with Ser/Thr to Ala mutations for each kinase site. This work begins to identify the coordinated and role of multiple phosphorylation events on NHE1.