A comparative survey of the impact of NHE1 phosphorylation on pHi and cell motility (994.1)

The sodium hydrogen exchanger isoform one (NHE1) exchanges an intracellular proton for an extracellular sodium ion playing a key role in the regulation of pH and directed cell motility. The regulation of NHE1 function is complex, involving multiple protein‐protein interactions and several phosphorylation sites. While most of the kinase sites have been identified the role of each phosphorylation event has not been defined in the context of the other phosphorylation events. There are many challenges involved in gaining a comprehensive understanding of the role of the different phosphorylation events. In part, these challenges come from the fact that data has been collected from multiple cell types being stimulated by a wide array of agonists. The key phosphorylation sites on NHE1 include: RhoA associated kinase (Rock), ribosomal S‐6 kinase (Rsk), protein kinase B (AKT) and extracellular‐signal regulated kinase (Erk). We have created cell lines expressing two distinct mutations at each Ser/Thr phosphorylation site. The first mutation changes the Ser/Thr to Ala removing the ability of a kinase to phosphorylate that site. The second changes a Ser/Thr to Asp to mimic the phosphorylation event. To screen the roles of each of these phosphorylation sites, changes in intracellular pHi and cell motility will be determined in cell lines stably expressing each NHE1 mutant. Our goal is to evaluate the relative impact of each phosphorylation site on NHE1 function in a comparative comprehensive manner