A Novel Mitogenic Signaling Pathway of Bradykinin in the Human Colon Carcinoma Cell Line SW-480 Involves Sequential Activation of a Gq/11 Protein, Phosphatidylinositol 3-Kinase β, and Protein Kinase Cε

Abstract

The signaling routes connecting G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) pathway reveal a high degree of complexity and cell specificity. In the human colon carcinoma cell line SW-480, we detected a mitogenic effect of bradykinin (BK) that is mediated via a pertussis toxin-insensitive G protein of the G q/11 family and that involves activation of MAPK. Both BK-induced stimulation of DNA synthesis and activation of MAPK in response to BK were abolished by two different inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY 294002, as well as by two different inhibitors of protein kinase C (PKC), bisindolylmaleimide and Ro 31-8220. Stimulation of SW-480 cells by BK led to increased formation of PI3K lipid products (phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate) and to enhanced translocation of the PKC⑀ isoform from the cytosol to the membrane. Both effects of BK were inhibited by wortmannin, too. Using subtype-specific antibodies, only the PI3K subunits p110␤ and p85, but not p110␣ and p110␥, were detected in SW-480 cells. Finally, p110␤ was found to be co-immunoprecipitated with PKC⑀. Our data suggest that in SW-480 cells, (i) dimeric PI3K␤ is activated via a G q/11 protein; (ii) PKC⑀ is a downstream target of PI3K␤ mediating the mitogenic signal to the MAPK pathway; and (iii) PKC⑀ associates with the p110 subunit of PI3K␤. Thus, these results add a novel possibility to the emerging picture of multiple pathways linking G protein-coupled receptors to MAPK.G protein-coupled receptors mediate effects of peptide hormones and neurotransmitters on intermediary metabolism as well as play an important role in the regulation of cell growth and differentiation. Similar to receptor tyrosine kinases, they initiate signaling pathways that finally activate members of the mitogen-activated protein kinase (MAPK) 1 family. One MAPK subfamily, which includes the extracellular signal-regulated kinases Erk1 and Erk2, is stimulated via a consecutive activation of the protein kinases Raf and MEK. The MAPK cascade is initially switched on via activation of the low molecular mass GTP-binding protein Ras. GTP-bound Ras associates the proximal kinase Raf to the plasma membrane, resulting in its activation. Several signal transduction pathways from G protein-coupled receptors to MAPK have been proposed that may be classified according to the type of G protein involved (for review, see Refs. 1 and 2). Thus, MAPK activation via pertussis toxin (PTX)-sensitive G i protein-coupled receptor, such as the m 2 muscarinic receptor, was found to be mediated by G ␤␥ subunits, phosphatidylinositol 3-kinase ␥ (PI3K␥), and Ras (3). In contrast, receptors coupled to G proteins of the PTX-insensitive G q/11 family, such as the m 1 muscarinic receptor, mediate MAPK activation via a G ␣ subunit that is Ras-independent and may involve PKC (4). Once activated, the different PKC isoforms, with the exception of PKC, activate the MAPK cascade at the level of Raf (5), but may al...

Src-family tyrosine kinases, phosphoinositide 3-kinase and Gab1 regulate extracellular signal-regulated kinase 1 activation induced by the type A endothelin-1 G-protein-coupled receptor

“…However, the mechanism remains poorly defined. More recently, following GPCR activation, PI 3-kinase has been shown to induce the activation of members of the PKC family, which subsequently induce MAPK activation [27,28]. Our data reported in this study now suggest that another function of PI 3-kinase in GPCR-induced MAPK activation is to induce tyrosine phosphorylation of the multisubstrate docking protein, Gab1, which subsequently regulates signalling pathways leading to MAPK activation.…”

“…Other PI 3-kinase-dependent signalling pathways also regulate GPCR-induced activation of MAPK. Activation of MAPK by the G q -coupled bradykinin receptor is dependent upon PI 3-kinaseβ and protein kinase C (PKC) ε [27]. Activation of MAPK by the G i -coupled lysophosphatidic acid (LPA) receptor is regulated by PI 3-kinaseγ-induced activation of PKCζ [28].…”

Bradykinin reduces growth factor-induced glomerular ERK1/2 phosphorylation

“…Moreover, the activation of ERK1/2 by BK has been demonstrated in various cells lines: A431, mesangial, and vascular smooth muscle cells (20,25,50). It is noteworthy that the proliferative effect of BK has been essentially demonstrated in quiescent mesangial cells with high concentrations of BK.…”

“…The profibrogenic effects of BK are associated with the phosphorylation of ERK1/2, which is a prerequisite for the activation of this MAPK (18). Finally, the activation of ERK1/2 by BK has been demonstrated in various cells lines: A431, mesangial, and vascular smooth muscle cells (20,25,50).…”

A specific function for phosphatidylinositol 3-kinase α (p85α-p110α) in cell survival and for phosphatidylinositol 3-kinase β (p85α-p110β) in de novo DNA synthesis of human colon carcinoma cells

“…p110a and p110b peptidic sequence only share 50% of identity and speci®c interactors of p110b have been recently identi®ed including the bg subunits of Gi protein (Kurosu et al, 1997;Maier et al, 1999). Therefore, important mitogens for these cancer cells may utilize seven trans-membrane receptors coupled to G protein for PI3Kb recruitment as recently demonstrated in non transformed ®broblasts (Roche et al, 1998) and suggested in SW480 colon cancer cells (Graness et al, 1998). On the other hand Ras was also described as an important regulator of cell survival in epithelial cells which involves its e ector PI3Ka (Khwaja et al, 1997).…”