Proline‐Rich Tyrosine Kinase Phosphorylation's Effect On the Na+/H+ Exchanger Isoform 1

Abstract

The Na+‐H+ exchanger isoform 1 (NHE1) is a 12‐pass transmembrane protein that functions by exchanging one extracellular sodium ion for one intracellular proton. The transmembrane domain of NHE1 is involved in ion exchange, and the cytoplasmic tail is the regulatory domain which includes a series of phosphorylation sites as well as lipid and proteins binding sites that alter transport activity. NHE1 is a central driver of the hallmarks of cancer through its involvement in the regulation of cellular proliferation, migration, and invasion. While the exact number of phosphorylation sites on the cytoplasmic domain of NHE1 is still unclear, many studies have implicated 22 phosphorylation sites and 12 different kinases. One of the kinases believed to directly or indirectly lead to the phosphorylation of NHE1 at S602 and S605 is proline rich tyrosine kinase 2 (PyK2). To evaluate the role of PyK2 in NHE1 regulation we prepared three cell lines derived from Chinese hamster lung fibroblast lacking NHE1 expression (PS120 cells). These cell lines are PSN (PS120 cells expressing human NHE1), PSN S602A/S605A (PSN cells with serine to alanine mutations of the reported PyK2 phosphorylation sites) and PSN S602D/S605D (PSN cells with serine to aspartic acid mutations of the reported PyK2 phosphorylation sites to mimic phosphorylation). Some studies have found that PyK2 works inversely on NHE1 and causes inhibition upon phosphorylation, while other studies have shown PyK2 to enhance NHE1 activity. Here we evaluate the impact of PyK2 inhibition and mutation of the PyK2 phosphorylation sites on proliferation and migration. Western blot analysis is used to evaluate changes in Pyk2 expression and phosphorylation over time under low and normal serum conditions. Cell proliferation and cell migration are both evaluated. Our results suggest that serum stimulation increases Pyk2 phosphorylation and enhances both cell proliferation and cell migration. Inhibition of PyK2 by PF43I396 or the mutation of the Pyk2 phosphorylation sites on NHE1 decreases proliferation and migration rates in serum stimulated cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.