Roles of protein kinase C, Ca2+, Pyk2, and c-Src in agonist activation of rat lacrimal gland p42/p44 MAPK

PURPOSE. Although p42/p44 mitogen-activated protein kinase ( MAPK) negatively modulates protein secretion stimulated by cholinergic and alpha(1D)-adrenergic agonists, it does not play a role in epidermal growth factor (EGF)-stimulated protein secretion. Therefore, this study was conducted to determine the roles that protein kinase C (PKC), intracellular Ca2+([Ca2+](i)), and nonreceptor tyrosine kinases Pyk2 and Src play in the activation of agonist- and EGF-stimulated MAPK activation. METHODS. Lacrimal gland acini were isolated by collagenase digestion and incubated with phorbol 12-myristate 13-acetate (PMA) to activate PKC or ionomycin, a Ca2+ ionophore. Acini were preincubated with the PKC inhibitors calphostin C or Ro-31-8220, EGTA to chelate Ca2+, or the c-Src inhibitor PP1 before stimulation with the cholinergic agonist carbachol, the alpha(1D)-adrenergic agonist phenylephrine, or EGF. Activated MAPK, Pyk2, and c-Src amounts were measured by Western blot analysis. RESULTS. PMA and ionomycin significantly increased the activation of MAPK in a time- and concentration-dependent manner. Inhibition of PKC partially inhibited carbachol-stimulated MAPK activation while completely inhibiting phenylephrine- and EGF-stimulated MAPK activation. Chelation of Ca2+ also partially inhibited carbachol-stimulated MAPK with no effect on phenylephrine- and EGF-stimulated MAPK activation. Carbachol increased the phosphorylation of Pyk2 on tyrosine 402 and c-src on tyrosine 416 in a time- dependent manner. The c-src inhibitor PP1 inhibited carbachol-stimulated phosphorylation of Pyk2. CONCLUSIONS. It was concluded that cholinergic agonists use Ca2+ and PKC to phosphorylate Pyk2 and c-Src, which subsequently stimulate MAPK activity. In contrast, alpha(1D)-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.