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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*

Open AccessPublished:November 27, 1998DOI:https://doi.org/10.1074/jbc.273.48.32016
      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 Gq/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 PKCε isoform from the cytosol to the membrane. Both effects of BK were inhibited by wortmannin, too. Using subtype-specific antibodies, only the PI3K subunits p110β and p85, but not p110α and p110γ, were detected in SW-480 cells. Finally, p110β was found to be co-immunoprecipitated with PKCε. Our data suggest that in SW-480 cells, (i) dimeric PI3Kβ is activated via a Gq/11 protein; (ii) PKCε is a downstream target of PI3Kβ mediating the mitogenic signal to the MAPK pathway; and (iii) PKCε associates with the p110 subunit of PI3Kβ. Thus, these results add a novel possibility to the emerging picture of multiple pathways linking G protein-coupled receptors to MAPK.
      MAPK
      mitogen-activated protein kinase
      PTX
      pertussis toxin
      PI3K
      phosphatidylinositol 3-kinase
      PKC
      protein kinase C
      PtdIns(3
      4)P2, phosphatidylinositol 3,4-bisphosphate
      PtdIns(3
      4,5)P3, phosphatidylinositol 3,4,5-trisphosphate
      BK
      bradykinin
      PVDF
      polyvinylidene difluoride
      CTX
      cholera toxin
      BSA
      bovine serum albumin
      TES
      2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] amino}ethanesulfonic acid
      MOPS
      4-morpholinepropanesulfonic acid.
      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.
      • van Biesen T.
      • Luttrell L.M.
      • Hawes B.E.
      • Lefkowitz R.J.
      and
      • Gutkind J.S.
      ). Thus, MAPK activation via pertussis toxin (PTX)-sensitive Giprotein-coupled receptor, such as the m2 muscarinic receptor, was found to be mediated by Gβγ subunits, phosphatidylinositol 3-kinase γ (PI3Kγ), and Ras (
      • Lopez-Illasaca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ). In contrast, receptors coupled to G proteins of the PTX-insensitive Gq/11 family, such as the m1 muscarinic receptor, mediate MAPK activation via a Gα subunit that is Ras-independent and may involve PKC (
      • Hawes B.E.
      • van Biesen T.
      • Koch W.J.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ). Once activated, the different PKC isoforms, with the exception of PKCζ, activate the MAPK cascade at the level of Raf (
      • Schönwasser D.C.
      • Marais R.M.
      • Marshall C.J.
      • Parker P.J.
      ), but may also involve tyrosine kinases of the Src family (
      • Rodriguez-Fernandez J.L.
      • Rozengurt E.
      ,
      • Wan Y.
      • Kurosaki T.
      • Huang X.-Y.
      ). MAPK activation by PTX-sensitive Go proteins appears to be independent of Gβγ and Ras, but requires PKC (
      • van Biesen T.
      • Hawes B.E.
      • Raymund J.R.
      • Luttrell L.M.
      • Koch W.J.
      • Lefkowitz R.J.
      ). Gs-coupled receptors such as the β-adrenergic receptor were found to exert a dual effect on MAPK involving Gβγ-mediated activation and cAMP-mediated inhibition (
      • Crespo P.
      • Cachero T.G.
      • Xu N.
      • Gutkind J.S.
      ). Alternatively, Ullrich and co-workers (
      • Daub H.
      • Weiss F.U.
      • Wallasch C.
      • Ullrich A.
      ,
      • Daub H.
      • Wallasch C.
      • Lankenau A.
      • Herrlich A.
      • Ullrich A.
      ,
      • Zwick E.
      • Daub H.
      • Aoki N.
      • Yamaguchi-Aoki Y.
      • Tinhofer I.
      • Maly K.
      • Ullrich A.
      ) have suggested an epidermal growth factor receptor transactivation by both Gi- and Gq/11-coupled receptors as an essential prerequisite for MAPK activation. They propose an epidermal growth factor receptor tyrosine phosphorylation by G protein-coupled receptors as the key event, which might be mediated by cytosolic tyrosine kinases such as Src and PYK2.
      In addition to receptor tyrosine kinases and PKC, PI3Ks appear to be key signaling enzymes implicated in the regulation of receptor-stimulated mitogenesis. After activation, they preferentially utilize phosphatidylinositol 4,5-bisphosphate as substrate, which is phosphorylated to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), followed by rapid degradation to PtdIns(3,4)P2. Both molecules have been proposed to act as second messengers. Recent studies indicate that both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 can directly activate certain PKC isoforms and the serine/threonine-protein kinase Akt/PKB (for review, see Refs.
      • Carpenter C.L.
      • Cantley L.C.
      and
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ). In terms of mode of regulation, class I members are subdivided into receptor tyrosine kinase-associated (class IA) or G protein-coupled receptor-activated (class IB) PI3Ks (for review, see Ref.
      • Domin J.
      • Waterfield M.D.
      ). The class IA types have been structurally characterized as a heterodimer consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). They are stimulated through receptors with intrinsic or associated tyrosine kinase activity that bind to the p85 subunit, thereby inducing PI3K activity. The only known class IB member (termed PI3Kγ) consists of a p110 catalytic subunit that lacks the binding site for p85, but is associated with a p101 non-catalytic subunit (
      • Stephens L.R.
      • Eguinoa A.
      • Erdjument-Bromage M.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.S.
      • Thelen M.
      • Cadwallader T.
      • Tempot P.
      • Hawkins P.T.
      ). The p110γ catalytic subunit is directly stimulated by βγ-complexes of G proteins (
      • Leopold D.
      • Hanck T.
      • Exner T.
      • Maier U.
      • Wetzker R.
      • Nürnberg B.
      ). Gα subunits of Gi (but not Gq or G12) proteins only moderately activate p110γ (
      • Leopold D.
      • Hanck T.
      • Exner T.
      • Maier U.
      • Wetzker R.
      • Nürnberg B.
      ,
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck M.
      • Dhand R.
      • Nürnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.D.
      • Wetzker R.
      ). The functional discrimination of class IAand IB members was questioned very recently since, in vitro, PI3Kβ has been shown to respond synergistically to both Gβγ and a synthetic phosphotyrosyl peptide that binds to the SH2 domain of p85 (
      • Kurosu H.
      • Maehama T.
      • Okada T.
      • Yamamoto T.
      • Hoshino S.
      • Fukui Y.
      • Ui M.
      • Hazeki O.
      • Katada T.
      ). These and other studies (
      • Stephens L.
      • Eguinoa A.
      • Corey S.
      • Jackson T.
      • Hawkins P.T.
      ,
      • Hawes B.E.
      • Luttrell L.M.
      • van Biesen T.
      • Lefkowitz R.J.
      ) suggest that also a p85/p110 PI3K may be regulated in the downstream region of pertussis toxin-sensitive G proteins.
      In this report, we present evidence for the activation of p85/p110β by the Gq protein-coupled bradykinin receptor in intact human colon carcinoma SW-480 cells. In addition, we obtained results showing that protein kinase Cε is a mediator connecting PI3Kβ with the MAPK signaling cascade in this endothelial cell line.

      DISCUSSION

      In this study, we investigated the signaling pathway linking the endogenously expressed bradykinin receptor to MAPK in the human colon carcinoma cell line SW-480. We present evidence for the activation of p85/p110β PI3K downstream of the bradykinin B2 receptor, which couples to a PTX-insensitive G protein. To our knowledge, this is the first demonstration that (i) a tyrosine kinase-associated PI3K is activated by a G protein-coupled receptor solely in an intact cell system and that (ii) the activation of a PI3K is mediated via a pertussis toxin-insensitive G protein of the Gq/11family.
      Recent studies have suggested that Gi-coupled receptor- and Gβγ-stimulated MAPK activation is attenuated by the PI3K inhibitors wortmannin and LY 294002 (
      • Hawes B.E.
      • Luttrell L.M.
      • van Biesen T.
      • Lefkowitz R.J.
      ). Furthermore, the PI3Kγ isoform was identified as the target of Gβγ complexes from PTX-sensitive G proteins and was suggested to link Gi-coupled receptors to the MAPK pathway (
      • Lopez-Illasaca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ,
      • Leopold D.
      • Hanck T.
      • Exner T.
      • Maier U.
      • Wetzker R.
      • Nürnberg B.
      ,
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck M.
      • Dhand R.
      • Nürnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.D.
      • Wetzker R.
      ).
      In SW-480 cells, bradykinin was found to activate phospholipase Cβ, leading to production of inositol polyphosphates, and to exert a mitogenic action via the bradykinin B2 receptor subtype. In addition, using two different experimental approaches, we obtained results indicating the involvement of a PI3K in the mitogenic bradykinin signaling. First, both BK-induced stimulation of DNA synthesis and activation of MAPK are inhibited by wortmannin or LY 294002. Activation of MAPK represents an essential step in the mitogenic action of BK in SW-480 cells because the effect of bradykinin on DNA synthesis was completely blocked by the MAPK inhibitor PD 098059. Second, bradykinin is capable of stimulating the lipid kinase activity of PI3K in SW-480 cells, resulting in the formation of the putative second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2 (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ). Immunoblotting experiments revealed that SW-480 cells lack the p110γ and p110α isoforms, but express the heterodimeric isoform p85/p110β (PI3Kβ). Thus, PI3Kγ may be excluded from participating in the signaling pathway from the bradykinin receptor to MAPK in SW-480 cells. Recently, Kurosu et al. (
      • Kurosu H.
      • Maehama T.
      • Okada T.
      • Yamamoto T.
      • Hoshino S.
      • Fukui Y.
      • Ui M.
      • Hazeki O.
      • Katada T.
      ) reported that p85/p110β was stimulated by Gβγ subunits from rat liver in vitro. Quite recently, this group demonstrated a potentiation of insulin-induced PtdIns(3,4,5)P3 accumulation by adenosine and prostaglandin E2 in rat adipocytes (
      • Hazeki O.
      • Okada T.
      • Kurosu H.
      • Takasuga S.
      • Suzuki T.
      • Katada T
      ). Our results suggest that a G protein-coupled receptor is also capable of activating PI3Kβ in an intact cell system independently of simultaneous activation of a receptor tyrosine kinase.
      In contrast to the hitherto existing idea that PI3K exclusively mediates the effect of βγ-complexes released from Giproteins, the G protein involved in SW-480 cells is PTX-insensitive. Among the PTX-insensitive G proteins expressed in SW-480 cells, G12/13 do not stimulate phosphatidylinositol hydrolysis (
      • Xu N.
      • Bradley L.
      • Ambudakar I.
      • Gutkind S.
      ) and may be excluded from linking the bradykinin receptor to phospholipase Cβ. The bradykinin receptor appears to be capable of interacting with multiple G proteins, including also Gs(
      • Liebmann C.
      • Graneß A.
      • Ludwig B.
      • Adomeit A.
      • Boehmer A.
      • Boehmer F.-D.
      • Nürnberg B.
      • Wetzker R.
      ,
      • Liebmann C.
      • Graneß A.
      • Böhmer A.
      • Kovalenko M.
      • Adomeit A.
      • Steinmetzer T.
      • Nürnberg B.
      • Wetzker R.
      • Böhmer F.-D.
      ). If the effect of bradykinin on MAPK is triggered by βγ-complexes released from a Gs protein as demonstrated for the β-adrenergic receptor (
      • Crespo P.
      • Cachero T.G.
      • Xu N.
      • Gutkind J.S.
      ), it might be expected that permanent activation of Gs in the presence of CTX simulates or potentiates the BK action on MAPK. Surprisingly, treatment of SW-480 cells with CTX completely prevented the activation of MAPK induced by BK. Furthermore, the BK-induced activation of MAPK was abolished in the presence of forskolin, which activates adenylate cyclase independently of the Gs protein. It may therefore be assumed that the inhibitory effect of CTX on the BK-induced stimulation of MAPK activity is due to cAMP triggered by CTX. We conclude that the G protein involved in both stimulation of phospholipase Cβ by BK and stimulation of MAPK in response to BK belongs to the Gq/11family.
      Our results suggest the involvement of a PKC upstream or downstream of PI3Kβ. One plausible candidate to play a role as a downstream effector of PI3K is PKCε since PKCε is activated by both lipid-derived second messengers of PI3K, PtdIns(3,4,5)P3and PtdIns(3,4)P2 (
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ,
      • Moriya S.
      • Kazlauskas A.
      • Akimoto K.
      • Hirai S.
      • Mizuno K.
      • Takenawa T.
      • Fukui Y.
      • Watanabe Y.
      • Ozaki S.
      • Ohno S.
      ). Overexpression of PKCε, but not that of PKCδ, another target of PI3K, has been shown to induce cell transformation (
      • Mischak H.
      • Goodnight J.A.
      • Kolch W.
      • Martiny-Baron G.
      • Schaechtle C.
      • Kazanietz M.G.
      • Blumberg P.M.
      • Pierce J.H.
      • Mushinski J.F.
      ) as well as activation of Raf-1 kinase (
      • Cai H.
      • Smola U.
      • Wixler V.
      • Eisenmann-Trappe I.
      • Diaz-Meco M.T.
      • Mosat J.
      • Rapp U.
      • Cooper G.M.
      ) and MAPK (
      • Schönwasser D.C.
      • Marais R.M.
      • Marshall C.J.
      • Parker P.J.
      ). Both PKC δ and PKCε were found to associate with PI3K in TF-1 cells, a human erythroleukemia cell line (
      • Ettinger S.L.
      • Lauener R.W.
      • Duronio V.
      ). In addition, PKCε was suggested to be a mediator connecting PI3K with the MAPK pathway in erythroid progenitor cells (
      • Klingmüller U.
      • Wu H.
      • Hsiao J.G.
      • Toker A.
      • Duckworth B.C.
      • Cantley L.C.
      • Lodish H.F.
      ).
      We obtained two lines of evidence indicating a link between PKCε and PI3K in SW-480 cells. First, BK-induced translocation of PKCε is sensitive to wortmannin, and second, PKCε associates with p110β as demonstrated by co-immunoprecipitation. This association was not enhanced after stimulation of SW-480 cells with bradykinin. Similarly, in TF-1 cells, only the association of PI3K with PKCδ, but not that with PKCε, was found to be increased after cytokine stimulation (
      • Ettinger S.L.
      • Lauener R.W.
      • Duronio V.
      ). There are also contradictory results whether or not PI3K lipid products may be a prerequisite for the PI3K/PKC association. In TF-1 cells, wortmannin inhibited this association, whereas LY 294002 did not (
      • Ettinger S.L.
      • Lauener R.W.
      • Duronio V.
      ). In our case, the inhibitory effect of wortmannin on the BK-induced translocation of PKCε from the cytosol to the membrane favors an essential role of lipid kinase-generated second messengers and suggests a downstream position of PKCε related to PI3K.
      In conclusion, we have shown that, in SW-480 cells, the mitogenic signaling of bradykinin involves the consecutive activation of a Gq/11 protein, PI3Kβ, PKCε, and MAPK (Fig. 11). Thus, this study defines a novel connection between a Gq protein-coupled receptor and the MAPK pathway with putative functional consequences for cell growth and carcinogenesis.
      Figure thumbnail gr11
      Figure 11Model of bradykinin receptor-mediated PI3Kβ- and PKCεdependent MAPK activation in SW-480 cells. Activation of a Gq/11 protein in response to BK leads to release of βγ-complexes, which probably mediate activation of PI3Kβ (
      • Kurosu H.
      • Maehama T.
      • Okada T.
      • Yamamoto T.
      • Hoshino S.
      • Fukui Y.
      • Ui M.
      • Hazeki O.
      • Katada T.
      ). By an unknown mechanism, p110β recruits and activates PKCε, which presumably precedes activation of Raf kinase (
      • Schönwasser D.C.
      • Marais R.M.
      • Marshall C.J.
      • Parker P.J.
      ) and, subsequently, MAPK. BKR, BK receptor.

      ACKNOWLEDGEMENT

      We thank Carmen Mertens for excellent technical assistance.

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