Role of Phosphorylation and Palmitoylation Post‐Translational Modification of NHE1 on Fibroblast Motility

Abstract

The sodium hydrogen exchanger protein isoform 1 (NHE1) is a trans‐membrane protein expressed in nearly all cells and plays an essential role in regulating intracellular pH as well as cell motility. In actively motile cells, NHE1 localizes to the leading edge of migrating cells and regulates directional cell movement. These mechanisms include the formation of intra‐ and extracellular pH‐gradients through Na+/H+ ion exchange, and likely also direct protein‐protein interactions and local cell volume changes. The purpose of this study was to understand the impact of palmitoylation on the NHE1 driven migration of lung cells. Palmitoylation of NHE1 involves the reversible covalent attachment of a palmitic acid to an amino acid found in NHE (cysteine): proteins are modified by palmitate via enzyme palmitoyl acyl‐transferases (PATs). To study the impact of palmitoylation on NHE1 and cell motility, an inhibitor of palmitoylation known as 2‐bromo‐palmitate (2BP) was incubated during a wound‐healing cell migration assay. Treatment with 2‐BP resulted in a significant and dose‐responsive reduction in cell motility, indicating a positive correlation between palmitoylation and NHE1 driven migration. Previous research indicates that phosphorylation also plays an important role in NHE1 activity and potentially palmitoylation. Therefore we investigated the impact of inhibition of both posttranslational modifications on cell motility. Specifically, we focused on agonists and stimulation factors which lead to increases in both NHE1 phosphorylation and palmitoylation: LPA, insulin, serum and PMA. The interplay between kinases and PAT regulation of NHE1 will provide a novel and underappreciated level of regulation of the transporter that may be a model for other transport proteins.