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Palmitoylation Impact on the Sodium Hydrogen Exchanger Isoform 1 Function
Author(s) -
Pritsch Emily,
Holland Alexander,
Kooiker Amanda J,
Rastedt Danielle E,
Vaughan Roxanne A,
Foster James D,
Wallert Mark Anthony,
Provost Joseph John
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.949.4
Subject(s) - palmitoylation , intracellular , microbiology and biotechnology , sodium–hydrogen antiporter , chemistry , biochemistry , gene isoform , motility , cysteine , biology , sodium , enzyme , organic chemistry , gene
The sodium hydrogen exchanger isoform 1 (NHE1) regulates intracellular pH and directs protein motility as an anchor for proteins at the leading edge of migrating cells. NHE1 has three putative palmitoylation cysteine targets in its cytoplasmic tail. Palmitoylation is a reversible, post‐translational fatty acid modification (S‐acetylation) of cysteine residues altering cellular location and function of many proteins. 2‐bromopalmitate (2BP) is a non‐metabolizable palmitate analog that irreversibly inhibits the protein acyltransferases responsible for palmitoylation. Using a live‐dead assay we found that up to 50 μM 2BP had a minimal (8.5 −/+ 2.5 % SEM) effect on cell viability and was not significantly different than the control palmitate at all concentrations. A 25% reduction in NHE1 palmitoylation was observed after the addition of 15 μM 2BP over 18 hours of incubation. Incubation of 15 μM 2BP in lung fibroblasts expressing human NHE1 (PSN) abrogated cell proliferation in low serum (0.5% FBS) conditions and reduced proliferation four fold in the presence of 10% FBS. Interestingly cells incubated with 2BP show a diminished adhesive ability on quartz coverslips. LPA induced stress fiber formation was decreased in cells treated with 2BP compared to vehicle control. Transiently transfected RFP tagged NHE1 cellular location and migration was determined in the absence and presence 2BP. Finally, we show the impact of 2BP and its control, palmitate, on intracellular pH indicating a role of palmitoylation on NHE1 function. Together this work shows a novel and yet undiscovered regulation of an important protein involved in intracellular pH homeostasis and directed cell motility. Support or Funding Information This work was funded in part by the Beckman Scholars Program, National Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)