ACTIVATION OF MAP KINASE KINASE (MEK) AND RAS BY CHOLECYSTOKININ IN RAT PANCREATIC ACINI

To evaluate the mechanism of MAP kinase activation, we studied the eff ects of CCK on MAP kinase kinase (MEK) in rat pancreatic acini. Two fo rms of MEK were identified by immunoblotting, using antibodies specifi c to MEK1 and MEK2. MEK activity in acinar extracts and after immunopr ecipitation with anti-MEK was detected using a recombinant fusion prot ein, glutathione S-transferase-MAP kinase, as a substrate. MEK activit y rapidly increased after stimulation of acini by CCK, with significan t stimulation at 1 min and a maximal effect at 5 min, followed by a sl ow decline to slightly above control levels after 30 min. The threshol d concentration of CCK was similar to 10 pM, and the maximal effect wa s induced by 1 nM CCK, which increased MEK activity by 120%. In additi on to CCK, bombesin and carbachol, but not secretin or vasoactive inte stinal peptide, enhanced MEK activity. Phorbol ester mimicked the effe ct of CCK, whereas ionomycin and thapsigargin failed to activate MEK. We further studied the activation of Ras, an important component leadi ng to activation of MEK by growth factors. Ras in acini was immunoprec ipitated and identified by Western blotting. CCK and 12-O-tetradecanoy lphorbol-13-acetate stimulated the incorporation of GTP into Ras, a re quirement for its activation, reaching a maximum at 10 min of similar to 120% over control. In conclusion, the activation of MAP kinase by C CK can be explained by activation of MEK and may involve the activatio n of Ras by a protein kinase C-dependent mechanism.