No 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.

The Na+‐H+ exchanger isoform 1 (NHE1) is a transmembrane protein that regulates a range of cellular functions essential for cancer progression including cell adhesion, proliferation and migration. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors to support NHE1 function. The CHP1 and CHP 2 binding domain on NHE1 is the same, amino acids 515 to 530. CHP2 is expressed primarily in tumor cells where it binds to NHE1 with a 5‐10 fold higher affinity than CHP1. CHP2 expression in tumor cells supports increased invasion and migration. Here we investigate the ability of mutations to a key amino acid in the binding domain (N519) to alter CHP2 binding to NHE1 in vivo. Two distinct site directed mutations to NHE1, N519A and N519D, have been constructed along with the accompanying stable cell lines expressing NHE1 with these mutations. We will present data evaluating CHP2 binding to NHE1 in these cells using a GFP‐CHP construct. We will also evaluate how a change in CHP2 binding alters adhesion, proliferation, and migration in mutant expressing cells as compared to cells expressing wild‐type NHE1.

The Na+‐H+ exchanger isoform 1 (NHE1) is a transmembrane protein that regulates a spectrum of function in cells from solid tumors including cell proliferation, adhesion, migration and invasion. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors for NHE1 function. Both CHP1 and CHP 2 bind to the same domain on NHE1, amino acids 515 to 530. CHP2 is expressed primarily in tumor cells where it binds to NHE1 with a 5‐10 fold higher affinity than CHP1. CHP2 expression in tumor cells activation of NHE1, leading to enhances tumorigenic behavior. We will investigate the ability of mutations to a key amino acid in the binding domain (H523) to alter CHP2 binding to NHE1 in vivo. Two distinct site directed mutations to NHE1, H523I and H523G, have been constructed along with the accompanying stable cell lines expressing NHE1 with these mutations. We will present data evaluating CHP2 binding to NHE1 in these cells using a GFP‐CHP construct as well as how changes in CHP2 binding alters adhesion, proliferation, and migration in cells expressing the NHE1 mutations relative to cells expressing wild‐type NHE1.