CHOLECYSTOKININ-STIMULATED TYROSINE PHOSPHORYLATION OF P125(FAK) AND PAXILLIN IS MEDIATED BY PHOSPHOLIPASE C-DEPENDENT AND C-INDEPENDENT MECHANISMS AND REQUIRES THE INTEGRITY OF THE ACTIN CYTOSKELETON AND PARTICIPATION OF P21(RHO)

Recent studies show that the effects of some oncogenes, integrins, gro wth factors and neuropeptides are mediated by tyrosine phosphorylation of the cytosolic kinase p125 focal adhesion kinase (p125(FAK)) and th e cytoskeletal protein paxillin. Recently we demonstrated that cholecy stokinin (CCK) C-terminal octapeptide (CCK-8) causes tyrosine phosphor ylation of p125(FAK) and paxillin in rat pancreatic acini. The present study was aimed at examining whether protein kinase C (PKC) activatio n, calcium mobilization, cytoskeletal organization and small G-protein p21(rho) activation play a role in mediating the stimulation of tyros ine phosphorylation by CCK-8 in acini. CCK-8-stimulated phosphorylatio n of p125(FAK) and paxillin reached a maximum within 2.5 min. The CCK- 8 dose response for causing changes in the cytosolic calcium concentra tion ([Ca2+](i)) was similar to that for p125(FAK) and paxillin phosph orylation, and both were to the left of that for receptor occupation a nd inositol phosphate production. PMA increased tyrosine phosphorylati on of both proteins. The calcium ionophore A23187 caused only 25 % of the maximal stimulation caused by CCK-8. GF109203X, a PKC inhibitor, c ompletely inhibited phosphorylation with PMA but had no effect on the response to CCK-g. Depletion of [Ca2+](i) by thapsigargin had no effec t on CCK-8-stimulated phosphorylation. Pretreatment with both GF109203 X and thapsigargin decreased CCK-8-stimulated phosphorylation of both proteins by 50 %. Cytochalasin D, but not colchicine, completely inhib ited CCK-8- and PMA-induced p125(FAK) and paxillin phosphorylation. Tr eatment with Clostridium botulinum C3 transferase, which inactivates p 21(rho), caused significant inhibition of CCK-8-stimulated p125(FAK) a nd paxillin phosphorylation. These results demonstrate that, in pancre atic acini, CCK-8 causes rapid p125(FAK) and paxillin phosphorylation that is mediated by bath phospholipase C-dependent and -independent me chanisms. For this tyrosine phosphorylation to occur, the integrity of the actin, but not the microtubule, cytoskeleton is essential as well as the activation of p21(rho).