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*

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.

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 also involve tyrosine kinases of the Src family (6,7). MAPK activation by PTX-sensitive G o proteins appears to be independent of G ␤␥ and Ras, but requires PKC (8). G s -coupled receptors such as the ␤-adrenergic receptor were found to exert a dual effect on MAPK involving G ␤␥ -mediated activation and cAMP-mediated inhibition (9). Alternatively, Ullrich and co-workers (10 -12) have suggested an epidermal growth factor receptor transactivation by both G iand G q/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)P 3 ), followed by rapid degradation to PtdIns(3,4)P 2 . Both molecules have been proposed to act as second messengers. Recent studies indicate that both PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 can directly activate certain PKC isoforms and the serine/threonine-protein kinase Akt/ PKB (for review, see Refs. 13 and 14). In terms of mode of regulation, class I members are subdivided into receptor tyrosine kinase-associated (class I A ) or G protein-coupled receptoractivated (class I B ) PI3Ks (for review, see Ref. 15). The class I A 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 I B member (termed PI3K␥) consists of a p110 catalytic subunit that lacks the binding site for p85, but is associated with a p101 non-catalytic subunit (16). The p110␥ catalytic subunit is directly stimulated by ␤␥-complexes of G proteins (17). G ␣ subunits of G i (but not G q or G 12 ) proteins only moderately activate p110␥ (17,18). The functional discrimination of class I A and I B members was questioned very recently since, in vitro, PI3K␤ has been shown to respond synergistically to both G ␤␥ and a synthetic phosphotyrosyl peptide that binds to the SH2 domain of p85 (19). These and other studies (20,21) suggest that also a p85/p110 PI3K may be regulated in the downstream region of pertussis toxin-sensitive G proteins.

Materials-[[3,4-
Cell Culture and Membrane Preparation-Human colon adenocarcinoma SW-480 cells (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, 10 g/ml streptomycin, and 0.25 g/ml amphotericin B in humidified air with 5% CO 2 at 37°C. For stimulation experiments, SW-480 cells were grown in 6-well dishes, and for [ 3 H]thymidine incorporation, they were grown in 24-well dishes and treated as indicated in the figure legends. An SW-480 particulate fraction (referred to as "membranes") was prepared by resuspending cells in 50 mM HEPES, pH 7.5, and centrifuging at 100,000 ϫ g for 20 min at 4°C. The pellets were resuspended in 50 mM HEPES, pH 7.5, containing bacitracin (100 g/ml), phenylmethylsulfonyl fluoride (0.1 mM), and leupeptin (2 g/ml) and were stored at -80°C. Protein concentration was determined according to Bradford (22).
[ 3 H]Thymidine Incorporation-Subconfluent cells were deprived of serum for 24 h and then treated with bradykinin (10 nM) with or without the respective inhibitors as indicated. The cells were incubated for another 24 h, followed by the addition of [ 3 H]thymidine (1 Ci/ml, 2 M) for 12 h. Finally, cells were filtered through Whatman GF/C glassfiber filters using a Brandel harvester and washed three times with 5 ml of 10 mM HEPES, pH 7.4. The filters were dried, and the cells were counted for incorporated radioactivity by liquid scintillation counting.
[ 3 H]Bradykinin Binding-The bradykinin receptor binding assay was performed as described previously (23)  Phosphatidylinositol Turnover-Determination of total inositol phosphates was performed as described previously (23). In brief, SW-480 cells in 24-well plates were prelabeled with 4 Ci/ml myo-[ 3 H]inositol for 24 h. At 2 h prior to stimulation, the cells were incubated in serum-free medium containing 20 mM HEPES, pH 7.4, and 1 M captopril. The cells were stimulated with increasing concentrations of bradykinin, as indicated, in the presence of LiCl for 10 min. For termination, the medium was replaced by 1 ml of 10% trichloroacetic acid. After 10 min, the extracts were collected, and the trichloroacetic acid was removed by washing four times with 2 volumes of water-saturated diethyl ether. After neutralization by adding Tris base, the samples were diluted to 4 ml with distilled water. The inositol phosphate fractions containing inositol mono-, bis-, and trisphosphates were obtained by eluting five times with 2 ml of 1.0 M ammonium formate and 0.1 M formic acid from AG 1-X8 columns (200 -400 mesh, formate form; Bio-Rad). Radioactivity of the inositol phosphate-containing fractions was determined by liquid scintillation counting.
Measurement of p44 MAPK (Erk1) Activity-SW-480 cells were preincubated in serum-free RPMI 1640 medium for 2 h and then treated with the different inhibitors and/or BK as indicated in the figure legends. After stimulation, cells were scraped off and centrifuged for 1 min at 5000 ϫ g. The medium was removed, and the pellets were lysed in 1 ml of lysis buffer (20 mM HEPES, pH 7.5, 10 mM EGTA, 40 mM ␤-glycerophosphate, 1% Triton X-100, 2.5 mM MgCl 2 , 2 mM orthovanadate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 g/ml aprotinin, and 20 g/ml leupeptin). After a 30-min incubation on ice, the lysates were centrifuged (10 min, 15,000 ϫ g, 4°C) to pellet insoluble material. The supernatants were transferred into new tubes, and Erk1 was immunoprecipitated using a rabbit polyclonal antibody (1 g/ml of lysate) from Santa Cruz Biotechnology. The immunoprecipitates were subsequently washed with phosphate-buffered saline containing 1% Triton X-100 and 2 mM orthovanadate; Tris-HCl, pH 7.5, containing 0.5 M LiCl; and kinase buffer (12.5 mM MOPS, pH 7.5, 12.5 mM ␤-glycerophosphate, 7.5 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM sodium fluoride, and 0.5 mM orthovanadate). Phosphorylation of immunoprecipitates was performed in 30 l of kinase buffer supplemented with 1 Ci of [␥-32 P]ATP, 20 M ATP, 1.5 mg/ml myelin basic protein, and 3.3 M dithiothreitol. After 20 min at 30°C, the reaction was terminated by the addition of 10 l of SDS-polyacrylamide gel electrophoresis buffer. The samples were boiled for 5 min and analyzed by SDS gel electrophoresis on 12% (w/v) gels. The dried gels were autoradiographed, and the radioactivity incorporated into myelin basic protein was quantified using a PhosphorImager (NIH Image Version 1.57).
PKC Translocation-For the measurement of PKC translocation, SW-480 cells were subjected to serum-free RPMI 1640 medium for 2 h before stimulation. The cells were then exposed to BK (100 nM) for 5 min at 37°C. For several experiments, cells were pretreated with the PI3K inhibitor wortmannin for 30 min. The incubation was terminated by removing the cells and centrifuging at 20,000 ϫ g for 1 min at 4°C. The pellets were resuspended in 50 mM HEPES, pH 7.4, containing bacitracin (100 g/ml), phenylmethylsulfonyl fluoride (0.1 mM), pepstatin A (1 g/ml), and leupeptin (2 g/ml) and were stored at -80°C. Protein concentration was determined according to Bradford (22) with BSA as a standard. For Western blot analysis, these membranes were separated on 7.5% gels by SDS-polyacrylamide gel electrophoresis and transferred to Hybond PVDF membranes. After blocking in 1% (w/v) BSA and 1% (w/v) nonfat dried milk powder overnight, the PVDF strips were incubated with the PKC antibodies as indicated (1 g/ml of the blocking solution). The strips were washed twice with Tris-buffered saline, pH 7.6, containing 0.05% (v/v) Tween 20; incubated for 45 min with goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology); and washed again four times as described above. Secondary antibodies were detected using the ECL Western blotting detection system by exposure to Biomax films.
Immunoprecipitation of PKC⑀ and Western Blot Analysis-Cell lysates prepared as described above were incubated with anti-PKC⑀ an-tibody (1 g/ml) at 4°C for 2 h on a rotating drive. Antigen-antibody complexes were recovered using protein A-Sepharose. The immunoprecipitates were washed three times with phosphate-buffered saline, pH 7.4, containing 1% Triton X-100 and 2 mM vanadate; resuspended in 50 l of electrophoresis sample buffer, boiled for 3 min; and subjected to SDS-polyacrylamide gel electrophoresis using a 7.5% gel, followed by transfer to PVDF membranes. After blocking overnight with 3% nonfat dried milk in Tris-buffered saline and 0.5 M NaCl, PVDF blots were incubated with the appropriate primary antibodies (Santa Cruz Biotechnology), and horseradish peroxidase-conjugated anti-rabbit IgG was used for detection with the ECL system. 32 P i Labeling of SW-480 Cells and Analysis of Phosphatidylinositol Phosphates-PI3K lipid kinase activity was determined using the method of Stephens et al. (20) with minor modifications. Briefly, SW-480 cells were freshly isolated; washed two times with phosphate-free RPMI 1640 medium; and incubated for 1 h in phosphate-free RPMI 1640 medium containing 25 mM HEPES, pH 7.5, 1 mg/ml fatty acid-free BSA, and 10% fetal calf serum. The SW-480 cells were then labeled overnight with 100 Ci of 32 P i /dish (6 ϫ 10 6 cells/2 ml). After labeling, cells were washed two times with 140 mM NaCl, 5 mM KCl, 2.8 mM NaHCO 3 , 1.5 mM CaCl 2 , 1 mM MgCl 2 , 0.06 mM MgSO 4 , 15 mM HEPES, 5.6 mM glucose, and 0.1% BSA, pH 7.2, at 37°C; centrifuged at 1200 rpm for 5 min; resuspended in 0.5 ml of the above buffer; and treated with BK as indicated. Reactions were terminated by the addition of 1 ml of ice-cold 2.4 N HCl. Then, 1 ml of chloroform/methanol/HCl (1:2:1), 0.75 ml of chloroform/phosphoinositide mixture (with 10 g of phosphoinositide mixture/point; Sigma), and 1 ml of chloroform were added subsequently. The mixture was thoroughly vortexed, and phase separation was performed by a short centrifugation (2500 rpm, 4 min). The lower chloroform phase was transferred to a new vial, and the upper phase was re-extracted twice with 1.5 ml of chloroform. Pooled chloroform phases were dried, and the lipids were deacylated by incubation for 1 h in methylamine (33% (v/v) in ethanol; Fluka) at 50°C. After removal of the methylamine, the samples were resuspended in 1 ml of water and extracted twice with 1 ml of 1-butanol. The aqueous phase containing the labeled lipid head group was analyzed by high pressure liquid chromatography as described (24).  (Fig. 1A). After prelabeling of SW-480 cells with myo-[ 3 H]inositol, BK induced a concentration-dependent increase in inositol phosphate formation with an EC 50 value of ϳ3 nM (Fig. 1B). The phosphatidylinositol system represents the main signaling pathway of bradykinin B 2 receptors in most tissues or cells (25). Both the binding parameter and the dose-response curve are in a good agreement with those for other B 2 receptors (25). As in small cell lung cancer cells (26), BK exerted a mitogenic effect in SW-480 cells as measured with the thymidine incorporation assay. This effect of BK was completely blocked in the presence of the nonpeptidic bradykinin B 2 receptor antagonist FR 173657 (27), suggesting the involvement of the B 2 receptor subtype in the mitogenic action of BK (Fig. 1C).

Bradykinin B 2 Receptor-mediated Mitogenic Effects in SW-
Bradykinin-induced Cell Proliferation Is Mediated via the Extracellular Signal-regulated Protein Kinase/MAPK Pathway-Treatment of SW-480 cells with BK led to the immediate activation of p44 MAPK as determined using the myelin basic protein assay (Fig. 2). To investigate whether activation of the MAPK pathway is required for the induction of cell division by BK, we measured the effect of BK on thymidine incorporation in the presence of PD 098059, which inhibits the activation of MAPK by blocking the activity of MAPK kinase (MEK) (28). Under the conditions used, both the BK-induced cell proliferation and the MAPK activation by BK were completely abolished in the presence of PD 098059 (Fig. 2). It may be concluded that the proliferation of SW-480 cells in response to BK is dependent on the activation of the MAPK pathway.

Effects of CTX or PTX on MAPK Activation in Response to
Bradykinin-In SW-480 cells, the BK-induced MAPK activation was insensitive to treatment with PTX (200 ng/ml) (Fig.  3A). The same PTX concentration was shown to effectively inhibit MAPK activation by lysophosphatidic acid in PC-12 cells (29). Besides G i and G q/11 proteins, immunoblotting experiments with specific antibodies (Santa Cruz Biotechnology) revealed the presence of G s , G 12 , and G 13 proteins, whereas G o and G z were not detected in SW-480 cells (data not shown). To investigate whether the PTX-insensitive G s protein might play a role in the mitogenic signaling pathway of BK, SW-480 cells were treated with CTX. The effect of BK on MAPK activity was clearly abolished by CTX (Fig. 3 ? B). In addition, treatment of SW-480 cells with forskolin also prevented the activation of MAPK by BK (Fig. 3C). Since cAMP has been reported to inhibit MAPK in smooth muscle cells and some fibroblast cell lines (30,31), we conclude that the permanent activated adenylate cyclase in the presence of CTX counteracts the stimulation of MAPK activity in response to BK.
Effects of BK on DNA Synthesis and MAPK Are Blocked by Both Inhibitors of PI3K and PKC-Next we tested two different inhibitors of PI3K, wortmannin and LY 294002, for their ability to affect the mitogenic action of BK in SW-480 cells. When PI3K was blocked, neither DNA synthesis (Fig. 4) nor MAPK activity (Fig. 5) was stimulated by BK, suggesting an involvement of a PI3K in the BK signaling pathway in SW-480 cells. Furthermore, two different inhibitors of PKC, bisindolylmale- imide and Ro 31-8220, were used to study the involvement of PKC in the mitogenic action of BK in SW-480 cells. As shown in Figs. 4 and 6, also in the presence of PKC inhibitors, BK failed to induce both stimulation of DNA synthesis and activation of MAPK, suggesting an involvement of protein kinase C in the mitogenic signaling pathway of BK in SW-480 cells as well. Taken together, these results obtained with different inhibitors and different experimental approaches indicate that a PI3K as well as a PKC are downstream mediators of the G q proteincoupled bradykinin receptor in SW-480 cells.

FIG. 1. Bradykinin binding, bradykinin-induced inositol phosphate formation, and effect of bradykinin on [ 3 H]thymidine incorporation in SW-480 cells. A, competition curve of unlabeled bradykinin for binding of [ 3 H]BK to
Bradykinin Stimulates Accumulation of PI3K Products in Intact SW-480 Cells-In SW-480 cells prelabeled with 32 P i , BK rapidly stimulated the accumulation of PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 (Fig. 7). , is comparable to their pattern of accumulation in human neutrophils after stimulation with fMet-Leu-Phe (20). In SW-480 cells pretreated with wortmannin, BK failed to stimulate lipid kinase activity. SW-480 Cells Contain p85/p110 PI3K␤-To investigate which subtype of class I PI3Ks may be activated by BK we analyzed SW-480 cell lysates by Western blotting using specific antibodies against the catalytic subunits p110␣, p110␤, and p110␥ and against the regulatory subunits p85␣ and p85␤. Fig.  8 shows that in SW-480 cells, only p110␤ and the p85␣ and p85␤ subunits exhibited significant expression, whereas p110␣ and p110␥ were not detectable by immunoblotting. Thus, heterodimeric PI3K␤, but not monomeric PI3K␥, appears to be the target of the bradykinin receptor-stimulated G q protein. PKC⑀ May Be a Mediator Connecting PI3K␤ with the MAPK Cascade-Among the different PKC isoforms, the novel PKC⑀, PKC␦, and PKC as well as the atypical PKC have been demonstrated to be activated by PtdIns(3,4,5)P 3 and/or PtdIns(3,4)P 2 in vitro (32,33). Western blotting of whole cell extracts established that SW-480 cells express the PKC isoforms ⑀, ␦, and . For activation studies, we measured the stimulus-induced translocation of PKC from the cytosol to the plasma membrane. Following the kinetics of BK-induced translocation of PKC isoforms in other cells (29,33) SW-480 cells were stimulated with 100 nM BK for 5 min. Throughout the repeated experiments, only PKC⑀ showed an increased membrane association when cells were triggered with BK. The BK-induced translocation of PKC⑀ was completely abolished in the presence of wortmannin (Fig. 9), suggesting that activation of PKC⑀ is a downstream event of the BK-induced activation of PI3K␤. The mechanism whereby PKC isoforms may be activated by PI3K in vivo is not yet clear. Recently, a specific association (co-immunoprecipitation) of PKC␦ with PI3K after cytokine stimulation in human erythroleukemia cells was reported (34). Therefore, we examined a possible association of PKC⑀ with p110␤. Cell lysates from SW-480 cells were immunoprecipitated with anti-PKC⑀ antibodies and analyzed with antibodies to p110␤. Indeed, PI3K␤ and PKC⑀ were found to co-immunoprecipitate in SW-480 cells in a specific manner as demonstrated by control experiments with non-immune serum (Fig. 10). There was no detectable increase in association of PKC⑀ and PI3K␤ in BK-treated cells (data not shown).

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/p110␤ PI3K downstream of the bradykinin B 2 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 toxininsensitive G protein of the G q/11 family.
Recent studies have suggested that G i -coupled receptor-and G ␤␥ -stimulated MAPK activation is attenuated by the PI3K inhibitors wortmannin and LY 294002 (21). Furthermore, the PI3K␥ isoform was identified as the target of G ␤␥ complexes from PTX-sensitive G proteins and was suggested to link G icoupled receptors to the MAPK pathway (3,17,18).
In SW-480 cells, bradykinin was found to activate phospholipase C␤, leading to production of inositol polyphosphates, and to exert a mitogenic action via the bradykinin B 2 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 BKinduced 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)P 3 and PtdIns(3,4)P 2 (14). Immunoblotting experiments revealed that SW-480 cells lack the p110␥ and p110␣ isoforms, but express the heterodimeric isoform p85/p110␤ (PI3K␤). Thus, PI3K␥ may be excluded from participating in the signaling pathway from the bradykinin receptor to MAPK in SW-480 cells. Recently, Kurosu et al. (19) reported that p85/p110␤ was stimulated by G ␤␥ subunits from rat liver in vitro. Quite recently, this group demonstrated a potentiation of insulin-induced PtdIns(3,4,5)P 3 accumulation by adenosine and prostaglandin E 2 in rat adipocytes (35). Our results suggest that a G proteincoupled receptor is also capable of activating PI3K␤ 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 ␤␥-complexes released from G i proteins, the G protein involved in SW-480 cells is PTX-insensitive. Among the PTX-insensitive G proteins expressed in SW-480 cells, G 12/13 do not stimulate phosphatidylinositol hydrolysis (36) and may be excluded from linking the bradykinin receptor to phospholipase C␤. The bradykinin receptor appears to be capable of interacting with multiple G proteins, including also G s (23,37). If the effect of bradykinin on MAPK is triggered by ␤␥-complexes released from a G s protein as demonstrated for the ␤-adrenergic receptor (9), it might be expected that permanent activation of G s 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 BKinduced activation of MAPK was abolished in the presence of forskolin, which activates adenylate cyclase independently of the G s 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 C␤ by BK and stimulation of MAPK in response to BK belongs to the G q/11 family.
Our results suggest the involvement of a PKC upstream or downstream of PI3K␤. One plausible candidate to play a role as a downstream effector of PI3K is PKC⑀ since PKC⑀ is activated by both lipid-derived second messengers of PI3K, PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 (33,38). Overexpression of PKC⑀, but not that of PKC␦, another target of PI3K, has been shown to induce cell transformation (39) as well as activation of Raf-1 kinase (40) and MAPK (5). Both PKC ␦ and PKC⑀ were found to associate with PI3K in TF-1 cells, a human erythroleukemia cell line (41). In addition, PKC⑀ was suggested to be a mediator connecting PI3K with the MAPK pathway in erythroid progenitor cells (42).
We obtained two lines of evidence indicating a link between PKC⑀ and PI3K in SW-480 cells. First, BK-induced translocation of PKC⑀ is sensitive to wortmannin, and second, PKC⑀ associates with p110␤ 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 PKC␦, but not that with PKC⑀, was found to be increased after cytokine stimulation (41). 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 (41). In our case, the inhibitory effect of wortmannin on the BK-induced translocation of PKC⑀ from the cytosol to the membrane favors an essential role of lipid kinasegenerated second messengers and suggests a downstream position of PKC⑀ related to PI3K.
In conclusion, we have shown that, in SW-480 cells, the mitogenic signaling of bradykinin involves the consecutive activation of a G q/11 protein, PI3K␤, PKC⑀, and MAPK (Fig. 11). Thus, this study defines a novel connection between a G q pro-tein-coupled receptor and the MAPK pathway with putative functional consequences for cell growth and carcinogenesis. Lysates from SW-480 cells were subjected to Western blot analysis using specific antibodies to p110␣, p110␤, p110␥, p85␣, and p85␤. No significant immunoreactivity was detected with antibodies to p110␣ and p110␥.
FIG. 9. Effect of wortmannin on bradykinin-stimulated translocation of PKC⑀. SW-480 cells were exposed to 100 nM BK for 10 min in the absence or presence of wortmannin (100 nM, 30-min preincubation). Membranes were prepared and analyzed by immunoblotting with antisera to the different PKC isoforms indicated (1 g/ml) as described under "Experimental Procedures." Representative immunoblots are shown after background smoothing and quantification with the program NIH Image Version 1.57 of experiments repeated three times with similar results. Activation of a G q/11 protein in response to BK leads to release of ␤␥-complexes, which probably mediate activation of PI3K␤ (19). By an unknown mechanism, p110␤ recruits and activates PKC⑀, which presumably precedes activation of Raf kinase (5) and, subsequently, MAPK. BKR, BK receptor.