Sodium Hydrogen Exchanger Isoform 1 (NHE1) Palmitoylation and Phosphorylation Barcoding: Implications on Regulation and Function

Abstract

The sodium hydrogen exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by exchanging an intracellular proton for an extracellular sodium ion. Multiple cellular processes are associated with NHE1 activity including coordinated cell migration, cellular proliferation, and control of cell volume. NHE1 also functions as a membrane anchoring and scaffolding protein resulting in the organization of protein complexes, formation of stress fibers, and regulation of signaling pathways within the cell. The large C‐terminus (amino acids 503–815) of NHE1 extends intracellularly and functions as the major regulatory domain of the exchanger where binding partners and posttranslational modifications (PTM) influence the exchanger in both a short‐ and long‐term manner. Elucidating mechanisms of NHE1 regulation by posttranslational modifications has been focused on contributions from the many phosphorylation sites on functional cellular outcomes. Previously, we have shown NHE1 is also regulated by palmitoylation, a reversible lipid modification in which a 16‐carbon fatty acid is covalently linked to a cysteine residue via a thioester bond and have demonstrated that inhibition of palmitoylation in cells expressing NHE1 decreases NHE1 activity, as well as stress fiber formation and cell migration, two cellular functions that are regulated through NHE1 phosphorylation. Multiple studies demonstrating a relationship between phosphorylation and palmitoylation on protein regulation have been reported with our lab recently demonstrating a reciprocal relationship between phosphorylation and palmitoylation in regulation of the dopamine transporter. The goal of this study was to begin to understand how the presence or absence of palmitoylation and phosphorylation in a coordinated manner contributes to a barcode that dictates a regulatory outcome and cellular response involving NHE1. We utilized various stimuli including insulin, reduced serum, and lysophosphatidic acid (LPA), all of which are known to impact NHE1 phosphorylation and regulation through multiple kinase pathways. We show that increasing phosphorylation with phorbol 12‐myristate (PMA) and LPA results in increased palmitoylation of NHE1, while treatment with insulin which also increases NHE1 phosphorylation, results in decreased NHE1 palmitoylation. Using this information, we have begun studies to inhibit specific kinases involved in these pathways to further explore the mechanism through which palmitoylation is altered. Together, this data supports the idea of a barcode hypothesis where phosphorylation and palmitoylation and other PTMs work in concert to regulate protein function including NHE1. Future studies will identify the specific palmitoylation sites and their role in the barcode hypothesis pertaining to NHE1.Support or Funding InformationThis work was supported by the University of North Dakota School of Medicine and Health Sciences and P20 GM103442 (to U.N.D.) from the INBRE program of the National Institute of General Medical Sciences and the ND EPSCoR Doctoral Dissertation AssistantshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.