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...

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The correct functioning of Ras proteins requires post-translational modification of the GTP hydrolases (GTPases). These modifications provide hydrophobic moieties that lead to the attachment of Ras to the inner side of the plasma membrane. In this study we investigated the role of Ras processing in the interaction with various putative Ras-effector proteins. We describe a specific, GTP-independent interaction between post-translationally modified Ha-and Ki-Ras4B and the G-protein responsive phosphoinositide 3-kinase p110g. Our data demonstrate that post-translational processing increases markedly the binding of Ras to p110g in vitro and in Sf9 cells, whereas the interaction with p110a is unaffected under the same conditions. Using in vitro farnesylated Ras, we show that farnesylation of Ras is sufficient to produce this effect. The complex of p110g and farnesylated RasGTP exhibits a reduced dissociation rate leading to the efficient shielding of the GTPase from GTPase activating protein (GAP) action. Moreover, Ras processing affects the dissociation rate of the RasGTP complex with the Ras binding domain (RBD) of Raf-1, indicating that processing induces alterations in the conformation of RasGTP. The results suggest a direct interaction between a moiety present only on fully processed or farnesylated Ras and the putative target protein p110g.Keywords: Ras; post-translational modifications; phosphoinositide 3-kinase; farnesylation.The Ras GTP hydrolases (GTPases) are a family of related proteins involved in the regulation of numerous cellular processes such as mitogen-induced G0/G1 phase transition, apoptosis, cytoskeletal rearrangements and regular cell-cycle progression. Ras proteins cycle between a GDP-bound and a GTP-bound state, the latter being the active, signal transducing version of the GTPase.The correct subcellular localization of Ras is critical for the activation of its downstream targets. Immunocytochemical studies have shown, that the majority of Ras is localized to the inner side of the plasma membrane. This association with the plasma membrane has been causally connected to a series of post-translational modifications of Ras. Inhibition of the posttranslational modification reactions abolishes the plasma membrane localization and leads to the loss of the biological activity of Ras [1,2]. The transforming potential of oncogenic Ras variants is severely affected by interfering with their post-translational modifications [2±4]. The Raf kinase isoform Raf-1 binds to, and is activated by, Ras in insect Sf9 cells [5] and vertebrate cell lines [6,7] and this activation, although not the association, is lost if post-translational processing of Ras is prevented [7]. A farnesylation-deficient point mutant of oncogenic Ha-Ras has been identified that can no longer activate the phosphoinositide 3-kinase (PI3K) isoform p110a in COS7 cells [8]. These and other findings have been interpreted in such a way that the post-translational processing of Ras functions to localize both Ras and, eventually, its target pro...