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 transducti on. Activation of extracellular-regulated kinases 1 and 2 (ERK1/2) by G pro tein-coupled receptors has been described to occur either through a PTx-ins ensitive pathway involving activation of phospholipase C and protein kinase C (PKC), or through a PTx-sensitive pathway involving G(i)beta gamma-media ted activation of Src. Cholecystokinin (CCK) activates ERK1/2 by a PKC-depe ndent, and thus presumably PTx-insensitive, pathway. However, CCK has recen tly 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 activati on in pancreatic AR42J cells, although activation of phospholipase C was no t reduced. PTx also inhibited ERK1/2 activation in response to the PKC acti vator 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth facto r (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 act ivation of c-Raf-1 and ERK1/2 in a manner similar to that of PTx. In PTx-tr eated cells, the cAMP content was increased and forskolin did not further i nhibit 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 ac tivation could be a consequence of disinhibition of the adenylyl cyclase si gnaling pathway, which in turn causes inhibition of c-Raf-1 activation rath er than indicating involvement of a PTx-sensitive G protein in this signali ng pathway.