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Investigation of Post-Translational Modification on NHE-1 Transport Function

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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. 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 by several protein kinases. We recently identified that NHE-1 is reversibly palmitoylated (S-acetylation). 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 NHE-1 activity in a novel rheostat of the cell.

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Investigation of Post-Translational Modification on NHE-1 Transport Function

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. 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 by several protein kinases. We recently identified that NHE-1 is reversibly palmitoylated (S-acetylation). 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 NHE-1 activity in a novel rheostat of the cell.