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Roles of Protein Kinase C, Ca²⁺, Pyk2, and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK

¹From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; and ²Tufts University School of Dental Medicine, Tufts University, Boston, Massachusetts.

PURPOSE. Although p42/p44 mitogen-activated protein kinase (MAPK) negatively modulates protein secretion stimulated by cholinergic and _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 _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, _1D-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.