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Robin R. Hodges, Jose D. Rios, Joanna Vrouvlianis, Isao Ota, Driss Zoukhri, Darlene A. Dartt; Roles of Protein Kinase C, Ca2+, Pyk2, and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK. Invest. Ophthalmol. Vis. Sci. 2006;47(8):3352-3359. doi: 10.1167/iovs.06-0028.
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purpose. Although p42/p44 mitogen-activated protein kinase (MAPK) negatively modulates protein secretion stimulated by cholinergic and α1D-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 α1D-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, α1D-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.
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