Pertussis Toxin Inhibits Cholecystokinin- and Epidermal Growth Factor-Induced Mitogen-Activated Protein Kinase Activation by Disinhibition of the cAMP Signaling Pathway and Inhibition of c-Raf-1
- Albrecht Piiper,
- Ralph Gebhardt,
- Bernd Kronenberger,
- Claudio D. Giannini,
- Robert Elez and
- Stefan Zeuzem
Abstract
Pertussis toxin (PTx), which inactivates Gi/o type G proteins, is widely used to investigate the involvement of Gi/o proteins in signal transduction. Activation of extracellular-regulated kinases 1 and 2 (ERK1/2) by G protein-coupled receptors has been described to occur either through a PTx-insensitive pathway involving activation of phospholipase C and protein kinase C (PKC), or through a PTx-sensitive pathway involving Giβγ-mediated activation of Src. Cholecystokinin (CCK) activates ERK1/2 by a PKC-dependent, and thus presumably PTx-insensitive, pathway. However, CCK has recently been shown to induce activation of Gi proteins in addition to Gq/11. In the present study, PTx partially inhibited CCK-induced ERK1/2 activation in pancreatic AR42J cells, although activation of phospholipase C was not reduced. PTx also inhibited ERK1/2 activation in response to the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) as well as activation of c-Raf-1 by EGF and CCK. In contrast, PTx, CCK, and EGF had only minor effects on A-Raf and B-Raf activity. Forskolin, a direct activator of adenylyl cyclase, inhibited CCK- and EGF-induced activation of c-Raf-1 and ERK1/2 in a manner similar to that of PTx. In PTx-treated cells, the cAMP content was increased and forskolin did not further inhibit CCK- and EGF-induced activation of c-Raf-1 or ERK1/2. In conclusion, the present study shows that PTx-sensitivity of receptor-induced ERK1/2 activation could be a consequence of disinhibition of the adenylyl cyclase signaling pathway, which in turn causes inhibition of c-Raf-1 activation rather than indicating involvement of a PTx-sensitive G protein in this signaling pathway.
Footnotes
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Send reprint requests to: Albrecht Piiper, M.D., Ph.D., or Stefan Zeuzem, M.D., University of Frankfurt, Department of Medicine II, Theodor-Stern-Kai 7, D-60590 Frankfurt A.M., Germany. E-mail:piiper{at}em.uni-frankfurt.de orzeuzem{at}em.uni-frankfurt.de
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This work was partly supported by grants from the Deutsche Forschungsgemeinschaft (Ze 237/4–5) and Nachlaβ Held/Hecker.
- Abbreviations:
- MAPK
- mitogen-activated protein kinase
- ERK
- extracellular signal-regulated kinase
- GPCR
- G protein-coupled receptor
- MEK
- mitogen-activated protein kinase kinase
- PTx
- pertussis toxin
- PKC
- protein kinase C
- CCK
- cholecystokinin
- TPA
- 12-O-tetradecanoylphorbol-13-acetate
- EGF
- epidermal growth factor
- DMEM
- Dulbecco's modified Eagle's medium
- IP3
- inositol trisphosphate
- [Ca2+]i
- intracellular calcium concentration
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- Received February 29, 2000.
- Accepted May 25, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
