Abstract
Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. CCK exerts its growth modulatory effects through G(q)-coupled receptors (CCK(A) and CCK(B)) and activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). In the present study, we investigated the different mechanisms participating in CCK-induced activation of ERK1/2 in pancreatic AR42J cells expressing both CCK(A) and CCK(B). CCK activated ERK1/2 and Raf-1 to a similar extent as epidermal growth factor (EGF). Inhibition of EGF receptor (EGFR) tyrosine kinase or expression of dominant-negative Ras reduced CCK-induced ERK1/2 activation, indicating participation of the EGFR and Ras in CCK-induced ERK1/2 activation. However, compared with EGF, CCK caused only small increases in tyrosine phosphorylation of the EGFR and Shc, Shc-Grb2 complex formation, and Ras activation. Signal amplification between Ras and Raf in a CCK-induced ERK cascade appears to be mediated by activation of protein kinase Cepsilon (PKCepsilon), because 1) down-modulation of phorbol ester-sensitive PKCs inhibited CCK-induced activation of Ras, Raf, and ERK1/2 without influencing Shc-Grb2 complex formation; 2) PKCepsilon, but not PKCalpha or PKCdelta, was detectable in Raf-1 immunoprecipitates, although CCK activated all three PKC isoenzymes. In addition, the present study provides evidence that the Src family tyrosine kinase Yes is activated by CCK and mediates CCK-induced tyrosine phosphorylation of Shc. Furthermore, we show that CCK-induced activation of the EGFR and Yes is achieved through the CCK(B) receptor. Together, our data show that different signals emanating from the CCK receptors mediate ERK1/2 activation; activation of Yes and the EGFR mediate Shc-Grb2 recruitment, and activation of PKC, most likely PKCepsilon, augments CCK-stimulated ERK1/2 activation at the Ras/Raf level.
Highlights
Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer
To determine whether the EGFR is involved in CCKinduced activation of extracellular signal-regulated kinase 1/2 (ERK1/2), we tested the effect of AG1478, a well established inhibitor of the EGFR tyrosine kinase, on CCK-induced ERK1/2 activation
Since protein kinase C (PKC) has been proposed as a modulator of Ras-Raf activation and activation of PKC is clearly involved in CCKinduced ERK1/2 activation [25, 26, 60], we studied the effect of prolonged treatment of the cells with TPA, which leads to down-modulation of diacylglycerol-sensitive classical and novel PKC isoenzymes, on CCK-induced tyrosine phosphorylation of Shc, Shc-Grb2 complex formation, and activation of Ras, Raf, and ERK1/2
Summary
Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. Since cleavage of pro-HB-EGF by metalloproteinases has been shown to mediate EGFR transactivation by Gi- and Gq-coupled receptors in several different cell lines [41], we investigated the effect of neutralizing anti-HB-EGF antibody and of the HBEGF inhibitor [Glu52]diphtheria toxin (CRM197) on CCK-induced tyrosine phosphorylation of the EGFR and ERK1/2 activation.
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