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Cholecystokinin and EGF activate a MAPK cascade by different mechanisms in rat pancreatic acinar cells
A Dabrowski1, G E Groblewski, C Schäfer
1Department of Physiology, University of Michigan, Ann Arbor 48109-0622, USA.
Abstract:
The effects of activating the Gq protein-coupled cholecystokinin (CCK) receptor on different proteins/signaling molecules in the mitogen-activated protein kinase (MAPK) cascade in pancreatic acinar cells were analyzed and compared with the effects of activating the tyrosine kinase-coupled epidermal growth factor (EGF) receptor. Both EGF and CCK octapeptide rapidly increased the activity of the MAPKs [extracellular signal-regulated kinase (ERK) 1 and ERK2], reaching a maximum within 2.5 min when 3.9- and 8.5-fold increases, respectively, were observed. The EGF-induced increase of MAPK activity was transient, with only a slight elevation after 30 min, whereas CCK-stimulated MAPK remained at a high level of activation to 60 min. The protein kinase C inhibitor GF-109203X abolished the activation by phorbol ester and inhibited the effect of CCK by 78% but had no effect on EGF-activated MAPK activity. EGF and CCK activated both forms of MAPK kinase (MEK), with CCK having a much larger effect, activating MEK1 by 6-fold and MEK2 by 10-fold, whereas EGF activated both MEKs by only 2-fold. Immunoblotting revealed three different forms of Raf in pancreatic acinar cells. Of the total basal Raf kinase activity, 3.7% was Raf-A, 89.0% was Raf-B, and 7.3% was c-Raf-1. All three forms of Raf were stimulated to a greater extent by CCK than by EGF, which was especially evident for Raf-A and c-Raf-1. The effect of CCK in activating Rafs was at least partially mimicked by stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. EGF significantly increased GTP-bound Ras by 183 and 164% at 2.5 and 10 min, respectively; CCK and TPA had no measurable effect. Our study suggests that CCK and EGF activate the MAPK cascade by distinct mechanisms in pancreatic acinar cells.
Insights
Cholecystokinin (CCK) and epidermal growth factor (EGF) activate mitogen-activated protein kinase (MAPK) pathways in pancreatic cells. CCK and EGF utilize distinct signaling mechanisms to activate MAPK cascade proteins, influencing cellular responses.
Area of Science:
- Cellular signaling and molecular biology
- Gastrointestinal physiology and endocrinology
Background:
- Pancreatic acinar cells respond to various extracellular signals.
- Mitogen-activated protein kinase (MAPK) cascades are crucial for cellular processes.
- Cholecystokinin (CCK) and epidermal growth factor (EGF) receptors initiate distinct signaling pathways.
Purpose of the Study:
- To compare the effects of CCK receptor activation versus EGF receptor activation on the MAPK cascade in pancreatic acinar cells.
- To elucidate the distinct mechanisms by which CCK and EGF activate MAPK signaling.
Main Methods:
- Activation of CCK and EGF receptors in pancreatic acinar cells.
- Measurement of MAPK, MAPK kinase (MEK), and Raf kinase activities.
- Analysis of Ras GTP-binding protein activation.
- Use of specific inhibitors, including GF-109203X (protein kinase C inhibitor).
Main Results:
- Both CCK and EGF rapidly increased extracellular signal-regulated kinase (ERK) 1/2 activity, with CCK showing sustained activation.
- CCK significantly activated MEK1 and MEK2 more potently than EGF.
- CCK stimulated Raf kinase activity (Raf-A, Raf-B, c-Raf-1) more effectively than EGF, while EGF specifically increased GTP-bound Ras.
- Protein kinase C inhibition affected CCK-induced, but not EGF-induced, MAPK activation.
Conclusions:
- CCK and EGF activate the MAPK cascade through distinct molecular mechanisms in pancreatic acinar cells.
- CCK signaling involves protein kinase C and leads to sustained MAPK activation.
- EGF signaling primarily activates Ras and results in transient MAPK activation.