Abstract only
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.
Determine the effect of palmitoylation on the sodium hydrogen exchanger isoform 1 (NHE1), a member of the SLC9 family.
NHE1 expressed in native rat tissues or in heterologous cells was assessed for ...palmitoylation by acyl-biotinyl exchange (ABE) and metabolic labeling with 3Hpalmitate. Cellular palmitoylation was inhibited using 2-bromopalmitate (2BP) followed by determination of NHE1 palmitoylation status, intracellular pH, stress fiber formation, and cell migration. In addition, NHE1 was activated with LPA treatment followed by determination of NHE1 palmitoylation status and LPA-induced change in intracellular pH was determined in the presence and absence of preincubation with 2BP.
In this study we demonstrate for the first time that NHE1 is palmitoylated in both cells and rat tissue, and that processes controlled by NHE1 including intracellular pH (pHi), stress fiber formation, and cell migration, are regulated in concert with NHE1 palmitoylation status. Importantly, LPA stimulates NHE1 palmitoylation, and 2BP pretreatment dampens LPA-induced increased pHi which is dependent on the presence of NHE1.
Palmitoylation is a reversible lipid modification that regulates an array of critical protein functions including activity, trafficking, membrane microlocalization and protein-protein interactions. Our results suggest that palmitoylation of NHE1 and other control/signaling proteins play a major role in NHE1 regulation that could significantly impact multiple critical cellular functions.
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