Carbachol-stimulated transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells is mediated by intracellular Ca2+, PYK-2, and p60(src)

Abstract

Ca2+-dependent agonists, such as carbachol (CCh), stimulate epidermal growth factor receptor (EGFR) transactivation and mitogen-activated protein kinase activation in Ts4 intestinal epithelial cells. This pathway constitutes an antisecretory mechanism by which CCh-stimulated chloride secretion is limited. Here, we investigated mechanisms underlying CCh- stimulated epidermal growth factor receptor (EGFR) transactivation. Thapsigargin (TG, 2 μM) stimulated EGFR and extracellular signal-regulated kinase (ERK) phosphorylation in T84 cells. Inhibition of either EGFR or ERK activation, with tyrphostin AG1478 (1 μM) and PD 98059 (20 μM), respectively, potentiated chloride secretory responses to TG, as measured by changes in short-circuit current (I(sc)) across T84 cells. CCh (100 μM) stimulated tyrosine phosphorylation and association of the Ca2+-dependent tyrosine kinase, PYK-2, with the EGFR, which was inhibited by the Ca2+ chelator, BAPTA (20 μM). The calmodulin inhibitor, fluphenazine (50 μM) inhibited CCh-stimulated PYK-2 association with the EGFR and phosphorylation of EGFR and ERK. CCh also induced tyrosine phosphorylation of p60(src) and association of p60(src) with both PYK-2 and the EGFR. The Src family kinase inhibitor, PP2 (20 nM-20 μM) attenuated CCh-stimulated EGFR and ERK phosphorylation and potentiated chloride secretory responses to CCh. We conclude that CCh-stimulated transactivation of the EGFR is mediated by a pathway involving elevations in intracellular Ca2+, calmodulin, PYK-2, and p60(src). This pathway represents a mechanism that limits CCh-stimulated chloride secretion across intestinal epithelia.