Abstract

The sodium hydrogen exchanger isoform 1 (NHE‐1) is a sodium hydrogen antiporter that regulates intracellular pH (pHi), cell volume, dynamic actin remodeling processes, and coordination of cell migration in mammalian tissues. NHE‐1 is post‐translationally modified by phosphorylation and lipid modification (palmitoylation). The intracellular C‐terminal domain of NHE‐1 is reversibly phosphorylated at multiple sites byseveral kinases including: extracellular signal‐regulated kinase (ERK), protein kinase B (AKT), B‐Raf, p90 Ribosomal S6 Kinase (p90rsk), RhoA Kinase (ROCK), and other protein kinases. We recently identified that NHE‐1 is reversibly palmitoylated (S‐acetylation), in which a 16‐carbon palmitic acid is covalently attached to a cysteine residue on the C‐terminal domain via a thioester linkage. Such modifications are reported to regulate protein trafficking, membrane micro localization, and protein‐protein interactions. Steady‐state pHi assays incubated with pH‐sensitive dye were conducted to measure the change in pHi of Chinese Hamster Lung Fibroblast Cells due to NHE‐1. Investigating agonists and stimulating factors that increase NHE‐1 palmitoylation allows us to determine the impact of palmitoylation on NHE‐1 transport. Palmitoyl‐transferases (PATs) catalyze the lipidation of proteins and are inhibited by 2‐bromo‐palmitate (2BP). The presence of 2BP causes a loss of palmitoylation to occur, effectively inhibiting LPA‐induced NHE‐1 activity by 0.1 pH units. Fetal Bovine Serum (FBS), Lysophosphatidic acid (LPA), Phorbol 12‐myristate 13‐acetate (PMA), and Insulin effectively stimulate NHE‐1 by increasing pHi by 0.1 pH units. In the presence of kinase inhibitors, NHE‐1 activity significantly decreases compared to agonist or serum treatment alone. The impact of the combination of kinase inhibitors and palmitoylation inhibitors on NHE‐1 mediated pHi change was determined. The data supports the hypothesis that palmitoylation and phosphorylation coordinate to subtly modify NHE1 activity in a novel rheostat of the cell.

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