Moriah Hovde

Date of Award

January 2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

James D. Foster

Abstract

This dissertation focuses on the role of palmitoylation in regulating the sodium hydrogen exchanger isoform 1 (NHE1) and associated cellular functions as well as how various phosphorylation pathways alter NHE1 palmitoylation. Palmitoylation is the only known reversible lipid modification, allowing for the dynamic regulation of palmitoylated proteins not found with other lipid modifications. NHE1 regulates intracellular pH (pHi) by exchanging an extracellular sodium for an intracellular hydrogen. Additionally, NHE1 is involved in regulating cell volume, cytoskeletal organization, protein anchoring, cell migration, and cell proliferation. NHE1 is highly regulated by protein interactions and modifications including phosphorylation. To begin, the first study shows NHE1 is palmitoylated in cells and rat tissues and can be inhibited by 2-bromopalmitate (2BP), a global inhibitor of palmitoylation. Furthermore, inhibition of palmitoylation with 2BP affects multiple NHE1 associated cell functions including pHi, stress fiber formation, proliferation and migration.

Continuing to look NHE1 regulation, we used various agonists known to effect NHE1 phosphorylation followed by examination of NHE1 palmitoylation. Serum, lysophosphatidic acid (LPA) and phorbol 12-myristate (PMA) treatments all increased NHE1 palmitoylation while insulin decreased NHE1 palmitoylation. Multiple studies have shown that palmitoylation and phosphorylation within the same protein can impact each other in various ways and thereby work together to regulate protein function. As NHE1 is highly phosphorylated, we also utilized specific kinase inhibitors and measured the effect on NHE1 palmitoylation. We found inhibition of PI3K, AKT, Rsk, and ROCK increased NHE1 palmitoylation while MEK inhibition decreased NHE1 palmitoylation. Together these studies establish a role for palmitoylation in the regulation of NHE1 activity and associated cellular functions and demonstrate palmitoylation and phosphorylation events may work in a coordinated manner to regulate NHE1.

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