Bradykinin B-2 receptor-mediated mitogen-activated protein kinase activation in COS-7 cells requires dual signaling via both protein kinase C pathwayand epidermal growth factor receptor transactivation

The signaling routes linking G-protein-coupled receptors to mitogen-activat ed protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-k inase gamma (PI3K gamma), and protein kinase C (PKC). To characterize the m itogenic path way of bradykinin (BR), COS-7 cells were transiently cotransf ected with the human bradykinin B-2 receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK. is mediated via the cu subunits of a G(q/11) protein. Both activation of Raf-l and activation of M APK in response to BK were blocked by inhibitors of PKC as well as of the e pidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 c ells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-K gam ma or Src kinase failed to diminish MAPK activation by BK. BK-induced trans location and overexpression of PKC isoforms as well as coexpression of inac tive or constitutively active mutants of different PKC isozymes provided ev idence for a role of the diacylglycerol sensitive PKCs alpha and epsilon in BK signaling toward MAPK. In addition to PRC activation, BK also induced t yrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. I nhibition of PKC did not alter BK-induced transactivation, and blockade of EGF receptor did not affect BK-stimulated phosphatidylinositol turnover or BK-induced PKC translocation, suggesting that PKC acts neither upstream nor downstream of the EGF receptor. Comparison of the kinetics of PKC activati on and EGF receptor transactivation in response to BK also suggests simulta neous rather than consecutive signaling. We conclude that in COS-7 cells, B K activates MAPK via a permanent dual signaling pathway involving the indep endent activation of the PKC isoforms ct and e and transactivation of the E GF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.