Bradykinin B₂ Receptor-Mediated Mitogen-Activated Protein Kinase Activation in COS-7 Cells Requires Dual Signaling via Both Protein Kinase C Pathway and Epidermal Growth Factor Receptor Transactivation

Abstract

The signaling routes linking G-protein-coupled receptors to mitogen-activated protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-kinase gamma (PI3Kgamma), and protein kinase C (PKC). To characterize the mitogenic pathway of bradykinin (BK), COS-7 cells were transiently cotransfected with the human bradykinin B(2) receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK is mediated via the alpha subunits of a G(q/11) protein. Both activation of Raf-1 and activation of MAPK in response to BK were blocked by inhibitors of PKC as well as of the epidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 cells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-Kgamma or Src kinase failed to diminish MAPK activation by BK. BK-induced translocation and overexpression of PKC isoforms as well as coexpression of inactive or constitutively active mutants of different PKC isozymes provided evidence for a role of the diacylglycerol-sensitive PKCs alpha and epsilon in BK signaling toward MAPK. In addition to PKC activation, BK also induced tyrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. Inhibition 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 activation and EGF receptor transactivation in response to BK also suggests simultaneous rather than consecutive signaling. We conclude that in COS-7 cells, BK activates MAPK via a permanent dual signaling pathway involving the independent activation of the PKC isoforms alpha and epsilon and transactivation of the EGF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.

Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation

“…HEK cells (36,37). We incubated purified FLAG-KLHL3 with ATP and 0.3 units of PKC-α, PKC-βI, or PKC-e in vitro.…”

Membrane-Type 1 Matrix Metalloproteinase Stimulates Cell Migration through Epidermal Growth Factor Receptor Transactivation

“…As a result of the poor transfection efficiency of BAEC, we further investigated the mechanisms involved in the MT1-MMP -dependent transactivation of EGFR using COS-7 cells. This cell type has been used in number of studies to determine the signaling mechanisms involved in GPCR-mediated transactivation of EGFR (32,(40)(41)(42)(43)(44). These cells contain detectable amounts of the S1P receptor S1P 1 (45), but no detectable MT1-MMP, and thus constitute an interesting and appropriate cell model to study the mechanisms involved in S1P-stimulated MT1-MMP -dependent EGFR transactivation.…”

Metalloproteinase-dependent Transforming Growth Factor-α Release Mediates Neurotensin-stimulated MAP Kinase Activation in Human Colonic Epithelial Cells

“…In addition to protein kinases C, which can activate MAP kinase by directly phosphorylating its upstream molecule Raf-1 (27), epidermal growth factor (EGF) receptor transactivation has been shown to mediate MAP kinase activation by numerous G protein-coupled receptors such as endothelin-1, lysophosphatic acid, thrombin (28), substance P (29), and bradykinin (30), as well as by exposure of target cells to the pathogenic bacterium Helicobacter pylori (31) and to dermatonecrotic toxin produced by Pasteurella multocida (32). Transactivation of EGF receptors by several GPCRs involves metalloproteinasedependent cleavage of either proHB-EGF (33)(34)(35), TGF␣ (36), or amphiregulin (35,37).…”