Palmitoylation Regulates the Functions of NHE1

Abstract

The sodium hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane protein that regulates intercellular pH (pHi) and interacts with proteins involved in cell migration and adhesion. Within the extended cytosolic tail of NHE1, there are multiple sites of post‐translational modification including phosphorylation and lipidation. Our understanding of impact of reversible palmitoylation on membrane transporters is growing. 2‐bromopalmitate (2BP) irreversibly inhibits the enzyme, palmitoyl acyltransferase, that transfer palmitate to cysteines. We found NHE1 is reversibly palmitoylated and here, demonstrate the impact of the lipidation on NHE1 function. Lung fibroblasts expressing NHE1 (PSN) showed minimal decrease (8.5 ± 2.5% SEM) in cell viability after treatment with 50 μM 2BP after 72 h. Palmitoylation plays a critical role in cell adhesion and motility. Cells that were incubated in 15 μM 2BP for 18 hrs displayed a significant decrease cell adhesion in low serum conditions and cells treated with 2BP did not fully migrate into vacated space after 24 hrs in a wounding assay. Impact on cytoskeletal structure by 2BP treatment was also investigated. Lysophosphatidic acid (LPA) increased actin stress fiber formation 40% over control cells. This LPA‐induced stress fiber formation was inhibited more than two‐fold in the presence of 2BP. This is important as LPA‐induced stress fiber formation requires NHE1; therefore, palmitoylation is required for NHE1‐dependent actin stress fiber formation. To determine the effect of palmitoylation on NHE1 transport activity, PSN cells were treated with 2BP and the pHi was measured. Addition of increasing concentrations of 2BP inhibited NHE1 transport in cells with and without serum, but the effect was enhanced in low serum conditions. Interestingly, LPA stimulation of NHE1 activity was inhibited by 60%, but not to baseline. This indicates that in addition to cell adherence, motility, and cytoskeletal formation, palmitoylation is critical for NHE1 transport.Support or Funding InformationThis research was partially funded by the Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.