Activation of MAP kinase kinase (MEK) and Ras by cholecystokinin in rat pancreatic acini.
Cholecystokinin (CCK) has recently been shown to activate mitogen-activated protein (MAP) kinase in rat pancreatic acini [Duan and Williams, Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G401-G408, 1994]. To evaluate the mechanism of MAP kinase activation, we studied the effects of CCK on MAP kinase kinase (MEK) in rat pancreatic acini. Two forms of MEK were identified by immunoblotting, using antibodies specific to MEK1 and MEK2. MEK activity in acinar extracts and after immunoprecipitation with anti-MEK was detected using a recombinant fusion protein, glutathione S-transferase-MAP kinase, as a substrate. MEK activity rapidly increased after stimulation of acini by CCK, with significant stimulation at 1 min and a maximal effect at 5 min, followed by a slow decline to slightly above control levels after 30 min. The threshold concentration of CCK was approximately 10 pM, and the maximal effect was induced by 1 nM CCK, which increased MEK activity by 120%. In addition to CCK, bombesin and carbachol, but not secretin or vasoactive intestinal peptide, enhanced MEK activity. Phorbol ester mimicked the effect of CCK, whereas ionomycin and thapsigargin failed to activate MEK. We further studied the activation of Ras, an important component leading to activation of MEK by growth factors. Ras in acini was immunoprecipitated and identified by Western blotting. CCK and 12-O-tetradecanoylphorbol-13-acetate stimulated the incorporation of GTP into Ras, a requirement for its activation, reaching a maximum at 10 min of approximately 120% over control. In conclusion, the activation of MAP kinase by CCK can be explained by activation of MEK and may involve the activation of Ras by a protein kinase C-dependent mechanism.
Duan
R D
RD
Department of Physiology, University of Michigan, Ann Arbor 48109, USA.
Zheng
C F
CF
Guan
K L
KL
Williams
J A
JA
D000818:Animals; D001839:Bombesin / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002766:Cholecystokinin / Q000494:pharmacology*; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000096:biosynthesis; D015151:Immunoblotting; D066298:In Vitro Techniques; D007700:Kinetics; D020929:Mitogen-Activated Protein Kinase Kinases; D016716:PC12 Cells; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012633:Secretin / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology; D018631:ras Proteins / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D012633:Secretin; D014660:Vasoactive Intestinal Peptide; D002217:Carbachol; D002766:Cholecystokinin; D005982:Glutathione Transferase; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate; D001839:Bombesin