Phosphorylation of the Na + ‐H + Exchanger Isoform 1 Initiated by α 1 ‐Adrenergic Receptor Signaling Regulates Cell Growth and Movement

Abstract

α1-adrenergic receptor signaling has been implicated in cancer progression through the stimulation of cell proliferation, cell migration and cell invasion. Conversely, blocking α1-adrenergic receptor signaling decreases tumor formation in animal models. The α1-adrenergic receptor is a G protein-coupled receptor that initiates a complex series of intracellular signaling events. Receptor activation stimulates phospholipases, protein kinases, and intracellular calcium release. The α1-adrenergic receptor can activate three distinct protein kinases that phosphorylate NHE1 in four different locations. These kinases and the locations they phosphorylate are: 1) Rock which phosphorylates NHE1 at T653, 2) Rsk which phosphorylates NHE1 at S703, and 3) Erk which phosphorylates NHE1 at both S770 and S771. To evaluate the role of each of these phosphorylation sites in the regulation of cellular function we have created a series of cell lines each expressing human NHE1 with one of the phosphorylation sites mutated to an alanine, thus removing that ability for NHE1 to be phosphorylated at that location. We will evaluate each of these cell lines, PSN T653A, PSN S703A, PSN S770A, and PSN S771A to determine the role of each phosphorylation site. We will use an XTT assay to evaluate cell proliferation, AlexaFluor 488 phalloidin to evaluate stress fiber formation, and ibidi wound healing inserts to evaluate cell migration.