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.
Pertussis toxin (PTx), which inactivates G(i/o) type G proteins, is widely used to investigate the involvement of G(i/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 G(i)betagamma-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 G(i) proteins in addition to G(q/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.
Piiper
A
A
Department of Internal Medicine, Johann Wolfgang Goethe-University, Frankfurt, Germany. piiper@em.uni-frankfurt.de
Gebhardt
R
R
Kronenberger
B
B
Giannini
C D
CD
Elez
R
R
Zeuzem
S
S
D037361:Adenylate Cyclase Toxin; D000818:Animals; D002766:Cholecystokinin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D037342:Pertussis Toxin; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors*; D051381:Rats; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D010566:Virulence Factors, Bordetella; D004815:Epidermal Growth Factor; D002766:Cholecystokinin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases