Characterizing Palmitoylation of the Sodium Hydrogen Exchanger Isoform 1 (NHE1)

Abstract

The sodium hydrogen exchanger isoform 1 (NHE1) is an integral membrane protein that has the primary action of regulating intracellular pH through exchanging an intracellular proton for an extracellular sodium ion. Regulation of pH plays a role in many cellular processes including coordination of cell migration, control of cell volume, and anchoring protein interactions at the cell membrane. The involvement of NHE1 in these processes plays a role in neoplastic transformation of cells and tumorgenesis. The large C‐terminus of NHE1 functions as the regulatory domain of the exchanger. Within this regulatory domain many binding partners including ERM, PIP2, and CHP bind NHE1 and influence both the exchanger and the cell as a whole. Multiple phosphorylation sites are also present on the C‐terminus which have varying effects on NHE1 and cell physiology. Multiple Cysteine residues are present on the C‐terminus as well, which are potential sites at which S‐palmitoylation may occur. S‐palmitoylation is a posttranslational modification in which a 16 carbon palmitic acid is added to a cysteine residue via a thioester linkage. Palmitoylation is a dynamic and reversible post‐translational modification that is regulated by palmitoylating enzymes, palmitoyl acyl transferases (PATs) and depalmitoylating enzymes that include protein palmitoyl thioesterases (PPTs), acyl protein thioesterases (APTs) and the a/b hydrolases (ABHD) family of proteins. Palmitoylation is involved in many cellular processes including protein trafficking, membrane microdomain localization, and protein‐protein interactions. These findings prompted us to investigate the presence of this modification on NHE1 using acyl‐biotinyl exchange (ABE), an in vitro assay for post‐hoc detection of endogenous palmitoylation, which revealed that NHE1 is palmitoylated. We then treated Chinese hamster lung fibroblasts expressing human NHE1 (PSN) with the irreversible PAT inhibitor 2‐bromopalmitate (2BP) and found that 2BP suppresses NHE1 palmitoylation in both a time and dose dependent manner. Additionally, PSN cell migration assessed using electric cell‐substrate impedance sensing was inhibited by 2BP in a manner consistent with the degree of inhibition of NHE1 palmitoylation. To further evaluate the nature of NHE1 palmitoylation we have utilized site directed mutagenesis of various cysteine residues paired with the ABE to assess the sites that contribute to NHE1 palmitoylation. Interplay between palmitoylation and phosphorylation has been demonstrated on various other proteins. To determine if this is the case with NHE1 we have utilized various NHE1 phosphomutatant cell lines paired with the ABE which has allowed us to analyze the effect removing known phosphorylation sites has on NHE1 palmitoylation levels. Together this information is providing the necessary stepping stones to understanding how palmitoylation regulates NHE1.Support or Funding InformationSupport: National Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.