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Involvement of Protein Kinase C and Src Family Tyrosine Kinase in Gαq/11-induced Activation of c-Jun N-terminal Kinase and p38 Mitogen-activated Protein Kinase

1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês

10.1074/jbc.273.36.22892

ISSN

1083-351X

Autores

Motoshi Nagao, Junji Yamauchi, Yoshito Kaziro, Hiroshi Itoh,

Tópico(s)

Cell death mechanisms and regulation

Resumo

Mitogen-activated protein kinases (MAPKs) are activated by various extracellular stimuli. The signaling pathways from G protein-coupled receptors to extracellular signal-regulated kinase have been partially elucidated, whereas the mechanisms by which G protein-coupled receptors stimulate c-Jun N-terminal kinase (JNK) and p38 MAPK activities remain largely unknown. We have recently demonstrated that the signal from Gq/11-coupled m1 muscarinic acetylcholine receptor to p38 MAPK is mediated by both Gαq/11 and Gβγ in HEK-293 cells (Yamauchi, J., Nagao, M., Kaziro, Y., and Itoh, H. (1997) J. Biol. Chem.272, 27771–27777). In the present study, we report that a constitutively activated mutant of Gα11(Gα11 Q209L) activated not only p38 MAPK, but also JNK, and the activation of JNK and p38 MAPK by Gα11 Q209L was partially inhibited by prolonged treatment with phorbol 12-myristate 13-acetate and calphostin C. In addition, the Gα11Q209L-stimulated activation of both kinases was blocked by a specific inhibitor of protein tyrosine kinases (PP2) and Csk (C-terminal Src kinase). Furthermore, we demonstrated that Gα11 Q209L stimulated Src family kinase activity and induced tyrosine phosphorylation of several proteins in HEK-293 cells. These results suggest that Gαq/11 stimulates JNK and p38 MAPK activities through protein kinase C- and Src family kinase-dependent signaling pathways. Mitogen-activated protein kinases (MAPKs) are activated by various extracellular stimuli. The signaling pathways from G protein-coupled receptors to extracellular signal-regulated kinase have been partially elucidated, whereas the mechanisms by which G protein-coupled receptors stimulate c-Jun N-terminal kinase (JNK) and p38 MAPK activities remain largely unknown. We have recently demonstrated that the signal from Gq/11-coupled m1 muscarinic acetylcholine receptor to p38 MAPK is mediated by both Gαq/11 and Gβγ in HEK-293 cells (Yamauchi, J., Nagao, M., Kaziro, Y., and Itoh, H. (1997) J. Biol. Chem.272, 27771–27777). In the present study, we report that a constitutively activated mutant of Gα11(Gα11 Q209L) activated not only p38 MAPK, but also JNK, and the activation of JNK and p38 MAPK by Gα11 Q209L was partially inhibited by prolonged treatment with phorbol 12-myristate 13-acetate and calphostin C. In addition, the Gα11Q209L-stimulated activation of both kinases was blocked by a specific inhibitor of protein tyrosine kinases (PP2) and Csk (C-terminal Src kinase). Furthermore, we demonstrated that Gα11 Q209L stimulated Src family kinase activity and induced tyrosine phosphorylation of several proteins in HEK-293 cells. These results suggest that Gαq/11 stimulates JNK and p38 MAPK activities through protein kinase C- and Src family kinase-dependent signaling pathways. mitogen-activated protein kinases extracellular signal-regulated kinase c-Jun N-terminal kinase activating transcription factor 2 heterotrimeric guanine nucleotide-binding regulatory protein protein kinase C hemagglutinin phorbol 12-myristate 13-acetate glutathioneS-transferase polyacrylamide gel electrophoresis. Mitogen-activated protein kinases (MAPKs)1 are important mediators of signal transduction from the cell surface to the nucleus. Regulation by MAPKs has been implicated in many cellular processes such as proliferation, differentiation, and apoptotic death. In mammals, MAPKs are divided into at least three subfamilies: ERK, JNK/stress-activated protein kinase, and p38 MAPK (1Davis R.J. Trends Biochem. Sci. 1994; 19: 470-473Abstract Full Text PDF PubMed Scopus (918) Google Scholar). ERK is activated in response to a variety of growth factors, cytokines, and mitogens. The ERK signaling pathways stimulated by receptor tyrosine kinases are well understood (2Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar). JNK/stress-activated protein kinase and p38 MAPK are stimulated by cellular stresses such as heat shock, osmotic shock, and UV irradiation and by inflammatory cytokines such as tumor necrosis factor-α and interleukin-1 (3Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1026) Google Scholar). JNK/stress-activated protein kinase phosphorylates and activates transcription factors that include c-Jun and ATF2 (3Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1026) Google Scholar). p38 MAPK phosphorylates and activates transcription factors such as ATF2, CHOP, and MEF2C (3Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1026) Google Scholar, 4Wang X.-Z. Ron D. Science. 1996; 272: 1347-1349Crossref PubMed Scopus (743) Google Scholar, 5Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Nature. 1997; 386: 296-299Crossref PubMed Scopus (688) Google Scholar). It also activates MAPK-activated protein kinase-2 and -3 (3Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1026) Google Scholar, 6McLaughlin M.M. Kumar S. McDonnell P.C. Van Horn S. Lee J.C. Livi G.P. Young P.R. J. Biol. Chem. 1996; 271: 8488-8492Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are composed of α-, β-, and γ-subunits and transduce external signals from seven transmembrane receptors to intracellular effectors. Mammalian G protein α-subunits are grouped into four subfamilies: Gs, Gi, Gq, and G12 (7Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4728) Google Scholar, 8Kaziro Y. Itoh H. Kozasa T. Nakafuku M. Satoh T. Annu. Rev. Biochem. 1991; 60: 349-400Crossref PubMed Scopus (548) Google Scholar, 9Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1289) Google Scholar). The α-subunit of Gq/11(Gαq/11) activates phospholipase Cβ, which catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate and diacylglycerol. The inositol 1,4,5-trisphosphate released into the cytoplasm mobilizes Ca2+ from internal stores, whereas diacylglycerol activates protein kinase C (PKC) (10Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar). A constitutively activated mutant of Gαq induces the neoplastic transformation of NIH 3T3 cells (11Kalinec G. Nazarali A.J. Hermouet S. Xu N. Gutkind J.S. Mol. Cell. Biol. 1992; 12: 4687-4693Crossref PubMed Google Scholar, 12De Vivo M. Chen J. Codina J. Iyengar R. J. Biol. Chem. 1992; 267: 18263-18266Abstract Full Text PDF PubMed Google Scholar) and the differentiation of PC12 cells (13Heasley L.E. Storey B. Fanger G.R. Butterfield L. Zamarripa J. Blumberg D. Maue R.A. Mol. Cell. Biol. 1996; 16: 648-656Crossref PubMed Google Scholar). In COS-7 and Chinese hamster ovary cells, the activated mutant of Gαqcauses apoptosis (14Althoefer H. Eversole-Cire P. Simon M.I. J. Biol. Chem. 1997; 272: 24380-24386Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). It has been reported that the stimulation of G protein-coupled receptors activates ERK (15Post G.R. Brown J.H. FASEB J. 1996; 10: 741-749Crossref PubMed Scopus (199) Google Scholar). Recent studies have demonstrated that G protein-coupled receptor agonists such as carbachol (16Coso O.A. Chiariello M. Kalinec G. Kyriakis J.M. Woodgett J. Gutkind J.S. J. Biol. Chem. 1995; 270: 5620-5624Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 17Mitchell F.M. Russell M. Johnson G.L. Biochem. J. 1995; 309: 381-384Crossref PubMed Scopus (60) Google Scholar, 18Yamauchi J. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 27771-27777Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 19Bence K. Ma W. Kozasa T. Huang X.-Y. Nature. 1997; 389: 296-299Crossref PubMed Scopus (169) Google Scholar), angiotensin II (20Zohn I.E. Yu H. Li X. Cox A.D. Earp H.S. Mol. Cell. Biol. 1995; 15: 6160-6168Crossref PubMed Scopus (140) Google Scholar), and thrombin (21Shapiro P.S. Evans J.N. Davis R.J. Posada J.A. J. Biol. Chem. 1996; 271: 5750-5754Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 22Kramer R.M. Roberts E.F. Strifler B.A. Johnstone E.M. J. Biol. Chem. 1995; 270: 27395-27398Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) activate JNK or p38 MAPK in several kinds of cells. In addition, we and others have shown that constitutively activated Gαq/11 can stimulate JNK (13Heasley L.E. Storey B. Fanger G.R. Butterfield L. Zamarripa J. Blumberg D. Maue R.A. Mol. Cell. Biol. 1996; 16: 648-656Crossref PubMed Google Scholar,23Prasad M.V.V.S.V. Dermott J.M. Heasley L.E. Johnson G.L. Dhanasekaran N. J. Biol. Chem. 1995; 270: 18655-18659Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) and p38 MAPK (18Yamauchi J. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 27771-27777Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) activities. However, the signaling pathways from Gαq/11 to JNK and p38 MAPK activation remain to be determined. In this study, we investigated the mechanisms of JNK and p38 MAPK activation by Gαq/11 using the transient expression of an activated Gα11 mutant in HEK-293 cells. Our data indicate that PKC and Src family kinase are involved in the Gαq/11-stimulated activation of JNK and p38 MAPK. Rabbit polyclonal antibodies against Gα11 (D-17), Csk (C-terminal Srckinase) (C-20), Src (N-16), Src family kinases (Src, Yes, and Fyn) (SRC2), and Ha-Ras (C-20) were purchased from Santa Cruz Biotechnology, Inc. Mouse monoclonal antibodies (12CA5 and M2) against the hemagglutinin (HA) epitope and the FLAG epitope were obtained from Boehringer Mannheim and Eastman Kodak Co., respectively. Mouse monoclonal anti-phosphotyrosine antibody (PY20) was from Transduction Laboratories. Rabbit anti-mouse Ig antibody (55480) was obtained from Cappel. Anti-mouse (NA931) and anti-rabbit (NA934) Ig antibodies conjugated with horseradish peroxidase were from Amersham Pharmacia Biotech. PP1/AG1872 and PP2/AG1879 (24Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar) 2J. Waltenberger, A. Uecker, J. Kroll, H. Frank, U. Mayr, D. Fujita, A. Gazit, V. Hombach, A. Levitzki, and F.-D. Böhmer, submitted for publication. were kindly provided by A. Levitzki (Hebrew University of Jerusalem, Jerusalem, Israel). PMA was purchased from Sigma. Calphostin C, A23187, and thapsigargin were from Calbiochem. Complementary DNAs coding for mouse Gα11, CSBP1 (a human homologue of p38 MAPK), and the N terminus (amino acids 1–96) of ATF2 were isolated as described previously (18Yamauchi J. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 27771-27777Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 25Umemori H. Inoue T. Kume S. Sekiyama N. Nagao M. Itoh H. Nakanishi S. Mikoshiba K. Yamamoto T. Science. 1997; 276: 1878-1881Crossref PubMed Scopus (128) Google Scholar). A constitutively activated GTPase-deficient mutant of Gα11 (Gα11 Q209L) was made by primer-mediated mutagenesis as described previously (25Umemori H. Inoue T. Kume S. Sekiyama N. Nagao M. Itoh H. Nakanishi S. Mikoshiba K. Yamamoto T. Science. 1997; 276: 1878-1881Crossref PubMed Scopus (128) Google Scholar). Wild-type Gα11, Gα11 Q209L, and FLAG epitope-tagged p38 MAPK cDNAs were subcloned into mammalian expression vector pCMV5 (26Andersson S. Davis D.L. Dahlbäck H. Jörnvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar). cDNA for the N terminus of ATF2 was subcloned intoEscherichia coli expression vector pGEX-2T. The mammalian expression plasmid of HA-tagged JNK1 (pSRα-HA-JNK1) and the E. coli expression plasmid of glutathione S-transferase (GST)-c-Jun (amino acids 1–79) (27Minden A. Lin A. Claret F.-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar) were kindly provided by M. Karin (University of California at San Diego, La Jolla, CA). Csk (28Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Crossref PubMed Scopus (511) Google Scholar) and dominant-negative Ha-Ras(S17N) cDNAs, which were generous gifts from M. Okada (Institute for Protein Research, Osaka University, Osaka, Japan) and G. M. Cooper (Dana-Farber Cancer Institute, Boston, MA), respectively, were subcloned into pCMV5. The mammalian expression plasmid of human m1 muscarinic acetylcholine receptor was generously provided by E. M. Ross (University of Texas Southwestern Medical Center, Dallas, TX). The plasmids of v-Src and TAK1 were kindly provided by Y. Fukami (Kobe University, Kobe, Japan) and K. Matsumoto (Nagoya University, Nagoya, Japan), respectively. The isolated cDNAs and the mutations were confirmed by dideoxynucleotide sequencing. Human embryonic kidney (HEK) 293 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum, 50 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Inc.). The cells were cultured at 37 °C in a humidified 10% CO2 environment. HEK-293 cells on 60-mm dishes were transfected with each plasmid DNA using the calcium phosphate precipitation method. The total amount of plasmid DNA was adjusted to 30 μg/60-mm dish with empty vector (pCMV5). The medium was replaced 24 h after transfection, and the cells were starved in the serum-free medium containing 1 mg/ml bovine serum albumin (Sigma) for 24 h and were harvested. To measure JNK and p38 MAPK activities, the cells were transfected with pSRα-HA-JNK1 (5 μg) or pCMV5-FLAG-p38 MAPK (3 μg) together with pCMV5-Gα11 (10 μg) and the indicated expression plasmid. The transfected cells were lysed in 600 μl of lysis buffer A (20 mm HEPES-NaOH (pH 7.5), 3 mm MgCl2, 100 mm NaCl, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 mm EGTA, 1 mmNa3VO4, 10 mm NaF, 20 mm β-glycerophosphate, and 0.5% Nonidet P-40) 48 h after transfection. The cell lysates were centrifuged at 15,000 rpm for 10 min at 4 °C. The supernatants were incubated for 1 h at 4 °C with mouse anti-HA antibody (12CA5; 0.2 μg) or mouse anti-FLAG antibody (M2; 0.2 μg) and mixed for 1 h at 4 °C with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) preabsorbed with rabbit anti-mouse Ig antibody (1 μg). The immunoprecipitates were washed twice with lysis buffer A and twice with reaction buffer A (20 mm HEPES-NaOH (pH 7.5), 10 mmMgCl2, 0.5 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 0.1 μg/ml leupeptin, 0.1 mm EGTA, 0.1 mmNa3VO4, 1 mm NaF, and 2 mm β-glycerophosphate). The washed precipitates were incubated in 30 μl of reaction buffer A containing 1.5 μg of affinity-purified GST-c-Jun (amino acids 1–79) for JNK assay or 1 μg of affinity-purified GST-ATF2 (amino acids 1–96) for p38 MAPK assay, 20 μm ATP, and 5 μCi of [γ-32P]ATP at 30 °C for 20 min (for JNK assay) or 15 min (for p38 MAPK assay). The reactions were terminated by adding 10 μl of 4× Laemmli sample buffer (50 mm Tris-HCl (pH 6.8), 2% SDS, 30 mmdithiothreitol, and 10% glycerol). The boiled samples were subjected to SDS-PAGE, and the radioactivity incorporated into GST-c-Jun or GST-ATF2 was measured by an imaging analyzer (Fuji BAS2000) and detected by autoradiography. Src family kinase assay was performed as described previously (29Osherov N. Levitzki A. Eur. J. Biochem. 1994; 225: 1047-1053Crossref PubMed Scopus (267) Google Scholar) with some modifications. Cells were transfected with pCMV5-Gα11 (10 μg). The transfected cells were harvested and lysed in 600 μl of lysis buffer A after 48 h. The cell lysates were centrifuged at 15,000 rpm for 10 min at 4 °C. Aliquots of the supernatants containing 250 μg of protein were incubated for 1 h at 4 °C with 0.2 μg of anti-Src family kinase antibody (SRC2), which recognizes the C-terminal sequence conserved among Src, Yes, and Fyn, and mixed for 2 h at 4 °C with protein A-Sepharose CL-4B. The immunoprecipitates were washed twice with lysis buffer A and twice with reaction buffer B (40 mm HEPES-NaOH (pH 7.5), 10 mmMgCl2, 3 mm MnCl2, 0.5 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 0.1 μg/ml leupeptin, 0.1 mmNa3VO4, 1 mm NaF, and 2 mm β-glycerophosphate). Kinase reactions were carried out in 30 μl of reaction buffer B containing 4 μg of acid-denatured enolase (Boehringer Mannheim), 10 μm ATP, and 10 μCi of [γ-32P]ATP at 22 °C for 5 min. The reactions were stopped by adding 10 μl of 4× Laemmli sample buffer. The boiled samples were separated by 10% SDS-PAGE, and the radioactivity incorporated into the enolase was measured by an imaging analyzer (Fuji BAS2000) and detected by autoradiography. The transfected cells were lysed in 600 μl of lysis buffer A, and the cell lysates were centrifuged at 15,000 rpm for 10 min at 4 °C. The supernatants containing 350 μg of protein were incubated for 1 h at 4 °C with mouse monoclonal anti-phosphotyrosine antibody (PY20) (10 μg) and mixed for 2 h at 4 °C with protein A-Sepharose CL-4B preabsorbed with rabbit anti-mouse Ig antibody (20 μg). The immunoprecipitates were washed twice with lysis buffer A and twice with reaction buffer A. The precipitated proteins were boiled in Laemmli sample buffer. The boiled samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked using 1% bovine serum albumin in phosphate-buffered saline containing 0.1% Tween 20 for PY20 or using blocking solution (50 mmTris-HCl (pH 8.0), 2 mm CaCl2, 80 mm NaCl, 0.2% Nonidet P-40, 0.02% NaN3, and 5% nonfat dried milk) for other specific antibodies. Immunoreactive bands were visualized by using secondary horseradish peroxidase-conjugated antibodies and chemiluminescence (NEN Life Science Products). We previously reported that a constitutively activated mutant of Gα11(Gα11 Q209L) stimulates p38 MAPK activity in HEK-293 cells (18Yamauchi J. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 27771-27777Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In this study, we examined whether Gα11 Q209L can also stimulate JNK activity in the same cells. The cells were transfected with wild-type Gα11 or Gα11Q209L together with HA epitope-tagged JNK1. HA-JNK1 was immunoprecipitated from the transfected cell lysate with anti-HA antibody, and the JNK1 activity was assessed by phosphorylation of GST-c-Jun (amino acids 1–79). Wild-type Gα11 did not significantly enhance the JNK1 activity, whereas Gα11Q209L increased the JNK1 activity by 7–8-fold in HEK-293 cells (Fig. 1 A). Using FLAG epitope-tagged p38 MAPK and GST-ATF2, an ∼4-fold stimulation of p38 MAPK by Gα11 Q209L was also observed (Fig. 1 B). Immunoblot analysis indicated that HA-JNK1 and FLAG-p38 MAPK were expressed at similar levels in the transfected cells. We next examined the signaling pathways from Gαq/11 to JNK and p38 MAPK activation in HEK-293 cells. It is known that Gαq/11 activates phospholipase Cβ to hydrolyze phosphatidylinositol 4,5-bisphosphate and thereby generates inositol 1,4,5-trisphosphate and diacylglycerol. The inositol 1,4,5-trisphosphate released into the cytoplasm mobilizes Ca2+ from internal stores, whereas diacylglycerol activates some PKC isoforms (10Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar). We therefore investigated whether PKC participates in the JNK1 and p38 MAPK activation by Gα11Q209L. In HEK-293 cells, PMA caused an ∼2-fold increase in JNK1 and p38 MAPK activities (Fig. 2 A). The PMA-stimulated JNK1 and p38 MAPK activation was prevented by a 24-h pretreatment with PMA. In contrast, the prolonged PMA treatment did not diminish the anisomycin-induced activation of both kinases (data not shown). The cells transfected with Gα11 Q209L were treated with PMA for 24 h, and the effect of PKC inactivation on JNK1 and p38 MAPK activation was examined. The Gα11Q209L-stimulated activation of JNK1 and p38 MAPK was partially inhibited by the prolonged PMA treatment (Fig. 2 B). In addition, a PKC inhibitor (calphostin C) also blocked the JNK1 and p38 MAPK activation in a dose-dependent manner (Fig. 2 C). These results indicate that PKC may be involved in part in the activation of JNK1 and p38 MAPK by Gα11 Q209L. The expression of Gα11, HA-JNK1, or FLAG-p38 MAPK was not affected by treatment with PMA or calphostin C. Carbachol increased JNK1 and p38 MAPK activities by 3-fold in HEK-293 cells expressing Gq/11-coupled m1 muscarinic acetylcholine receptor, and calphostin C also reduced the activation by 50%. To explore the role of Ca2+ in JNK and p38 MAPK activation in HEK-293 cells, the cells were stimulated with A23187 or thapsigargin, which increases the intracellular Ca2+concentration. Activation of JNK1 and p38 MAPK by A23187 or thapsigargin was observed to a small extent (2-fold) (data not shown). Recently, it has been shown that Src family kinase and Ras contribute to ERK1/2 activation by Gq/11-coupled α1B-adrenergic receptor in HEK-293 cells (30Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). To determine whether protein tyrosine kinase and Ras are implicated in the Gαq/11-mediated signaling pathways leading to JNK1 and p38 MAPK activation, we examined the effects of a protein tyrosine kinase inhibitor (PP2) (24Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar), Csk, and dominant-negative Ras(S17N) on the Gα11 Q209L-stimulated JNK1 and p38 MAPK activation. The activation of both kinases by Gα11 Q209L was markedly attenuated by PP2 in a dose-dependent manner (Fig. 3, A and B). Similar inhibitory effects of another tyrosine kinase inhibitor (PP1) (24Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar) and PP2 were observed in the JNK1 and p38 MAPK activation by m1 muscarinic acetylcholine receptor (Fig. 3, C and D). Western blots analysis indicated that the PP1 and PP2 treatment did not affect the expression of Gα11, HA-JNK1, and FLAG-p38 MAPK. Csk is a cytoplasmic protein tyrosine kinase that inactivates Src family kinases (28Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Crossref PubMed Scopus (511) Google Scholar). The overexpression of Csk significantly blocked the Gα11 Q209L-stimulated JNK1 and p38 MAPK activation (Fig. 3 E). Furthermore, the expression of v-Src effectively stimulated the JNK1 and p38 MAPK activities in HEK-293 cells (Fig. 3 F). In contrast, Ras(S17N) failed to inhibit the Gα11 Q209L-induced JNK1 and p38 MAPK activation in the cells (Fig. 4). These data suggest that Src family kinases, but not Ras, are involved in the JNK1 and p38 MAPK activation by Gαq/11. Therefore, we tested whether Gα11 stimulates Src family kinase activity in HEK-293 cells. The cells were transfected with wild-type Gα11 or Gα11 Q209L, and endogenous Src family kinases were immunoprecipitated with anti-Src family kinase antibody (SRC2), which recognizes the C-terminal sequence conserved among Src, Yes, and Fyn. Src family kinase activity was assessed by phosphorylation of acid-denatured enolase. As shown in Fig. 5, Gα11 Q209L stimulated an ∼2-fold increase in Src family kinase activity over the basal level. Next, we investigated whether the tyrosine phosphorylation of the intracellular proteins was induced by the expression of the activated mutant of Gα11 in HEK-293 cells. Lysates of the cells transfected with wild-type Gα11 or Gα11Q209L were immunoprecipitated with anti-phosphotyrosine antibody (PY20). The immunoprecipitated proteins were separated by SDS-PAGE and subjected to immunoblot analysis with PY20. The expression of Gα11 Q209L promoted the tyrosine phosphorylation of several prominent bands with apparent molecular masses of 100, 105, and 120–130 kDa (Fig. 6 A). A similar pattern of protein tyrosine phosphorylation was observed when Gq/11-coupled m1 muscarinic receptor-expressed cells were stimulated by carbachol (data not shown). Some of the protein tyrosine phosphorylation enhanced by Gα11 Q209L were reduced by treatment with PP2 in a dose-dependent manner (Fig. 6 B). The tyrosine phosphorylation of a 100-kDa protein was completely abolished by 10 μm PP2, and that of 120–130-kDa proteins was strongly inhibited by 50 μmPP2. In contrast, PP2 had a very weak effect on the tyrosine phosphorylation of a 105-kDa protein. We previously reported that both Gα11 Q209L and Gβ1γ2 stimulate p38 MAPK activity and that the p38 MAPK activation by m1 muscarinic acetylcholine receptor is mediated by both Gαq/11 and Gβγ in HEK-293 cells (18Yamauchi J. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 27771-27777Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In addition, we showed that Gβγ effectively stimulates JNK1 activity in the cells (31Yamauchi J. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 7602-7607Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In the present study, we further found that Gα11 Q209L as well as Gβγ can activate not only p38 MAPK, but also JNK1 in HEK-293 cells (Fig. 1). Although two reports (13Heasley L.E. Storey B. Fanger G.R. Butterfield L. Zamarripa J. Blumberg D. Maue R.A. Mol. Cell. Biol. 1996; 16: 648-656Crossref PubMed Google Scholar, 23Prasad M.V.V.S.V. Dermott J.M. Heasley L.E. Johnson G.L. Dhanasekaran N. J. Biol. Chem. 1995; 270: 18655-18659Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) are consistent with our observation that Gα11Q209L stimulates the JNK1 activity, other groups (32Coso O.A. Teramoto H. Simonds W.F. Gutkind J.S. J. Biol. Chem. 1996; 271: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 33Collins L.R. Minden A. Karin M. Brown J.H. J. Biol. Chem. 1996; 271: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) have reported that a constitutively activated mutant of Gαq fails to stimulate the JNK1 activity in COS-7 and HEK-293 cells. The discrepancy may be due to the difference of the expression level of constitutively activated Gαq or the experimental conditions. It has been recently reported that Btk (Bruton'styrosine kinase) is necessary for p38 MAPK activation by m1 muscarinic acetylcholine receptor in DT40 cells (19Bence K. Ma W. Kozasa T. Huang X.-Y. Nature. 1997; 389: 296-299Crossref PubMed Scopus (169) Google Scholar). However, the signal transduction pathways linking Gαq/11with JNK and p38 MAPK activation remain largely unknown. As shown in Fig. 2, Gα11 Q209L-stimulated JNK1 and p38 MAPK activation was partially inhibited by prolonged PMA treatment and calphostin C. These results suggest that a phorbol ester-sensitive PKC may be involved in part in the Gαq/11-induced JNK1 and p38 MAPK activation. It has been shown that PKC can activate MEKK1in vivo (34Siow Y.L. Kalmar G.B. Sanghera J.S. Tai G. Oh S.S. Pelech S.L. J. Biol. Chem. 1997; 272: 7586-7594Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). MEKK1 was originally identified as a MAPK kinase kinase that has the ability to activate ERK1/2 (35Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. 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Therefore, we expect that Gαq/11 may regulate the focal adhesion proteins and the cytoskeletal proteins as well as JNK and p38 MAPK through Src family kinases. In summary, we have demonstrated that Gαq/11 can stimulate both JNK and p38 MAPK activities and that PKC and Src family kinase contribute to the activation by Gαq/11 in HEK-293 cells. We previously reported that Gβγ, as well as Gαq/11, stimulates JNK and p38 MAPK activities (18Yamauchi J. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 27771-27777Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 31Yamauchi J. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 7602-7607Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The signaling pathways from Gβγ to JNK and p38 MAPK and the regulatory mechanism of Src family kinase activation by Gαq/11 remain to be clarified. We thank Dr. A. Levitzki for the generous gifts of PP1/AG1872 and PP2/AG1879 and advice on Src family kinase assay and Drs. M. Karin, M. Okada, G. M. Cooper, E. M. Ross, Y. Fukami, and K. Matsumoto for kindly providing us with expression plasmids. We also thank Drs. H. Koide and S. Mizutani for helpful discussions.

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