Prostaglandin F2αStimulates Formation of p21 -GTP Complex and Mitogen-activated Protein Kinase in NIH-3T3 Cells via Gq-protein-coupled Pathway
1995; Elsevier BV; Volume: 270; Issue: 15 Linguagem: Inglês
10.1074/jbc.270.15.8984
ISSN1083-351X
AutoresTsuyoshi Watanabe, Iwao Waga, Zen‐ichiro Honda, Kiyoshi Kurokawa, Takao Shimizu,
Tópico(s)Melanoma and MAPK Pathways
ResumoProstaglandin (PG) F2αactivated mitogen-activated protein (MAP) kinase and MAP kinase kinase in NIH-3T3 cells by a mechanism that was completely inhibited by protein kinase inhibitors, staurosporine (20 n M) or H-7 (20 μM), but was insensitive to pretreatment with islet-activating protein (100 ng/ml; 24 h) or 12- O-tetradecanoylphorbol 13-acetate (2.5 μM; 24 h). PGF2αstimulation also led to a significant increase in Ras·GTP complex. Transfection of a cDNA encoding a constitutively active mutant of Gqα-subunit (Q209L) mimicked PGF2α-induced MAP kinase activation, increase in Ras·GTP complex, and DNA synthesis in these cells, suggesting that activation of Gq mediates the PGF2α-activation of Ras-MAP kinase pathway and mitogenesis in NIH-3T3 cells.These data provide a new insight into regulatory mechanisms of Ras-MAP kinase pathway through heterotrimeric G-protein-mediated pathways. Prostaglandin (PG) F2αactivated mitogen-activated protein (MAP) kinase and MAP kinase kinase in NIH-3T3 cells by a mechanism that was completely inhibited by protein kinase inhibitors, staurosporine (20 n M) or H-7 (20 μM), but was insensitive to pretreatment with islet-activating protein (100 ng/ml; 24 h) or 12- O-tetradecanoylphorbol 13-acetate (2.5 μM; 24 h). PGF2αstimulation also led to a significant increase in Ras·GTP complex. Transfection of a cDNA encoding a constitutively active mutant of Gqα-subunit (Q209L) mimicked PGF2α-induced MAP kinase activation, increase in Ras·GTP complex, and DNA synthesis in these cells, suggesting that activation of Gq mediates the PGF2α-activation of Ras-MAP kinase pathway and mitogenesis in NIH-3T3 cells. These data provide a new insight into regulatory mechanisms of Ras-MAP kinase pathway through heterotrimeric G-protein-mediated pathways. Prostaglandin (PG) 1The abbreviations used are:PGprostaglandinPKprotein kinaseMAPmitogen-activated proteinMAPKMAP kinaseMAPKKMAP kinase kinaseMEKKa protein with MAPKK kinase activity independent of the p21-p71 pathwayMBPmyelin basic proteinTPA12- O-tetradecanoylphorbol 13-acetateBAPTA-AM1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetraacetoxymethyl esterIAPislet-activating proteinFCSfetal calf serumEGFepidermal growth factorrMAPa kinase-negative mutant of a recombinant Xenopus MAPKGαα-subunit of G-proteinIPsinositol phosphatesDMEMDulbecco's modified Eagle's medium. 1The abbreviations used are:PGprostaglandinPKprotein kinaseMAPmitogen-activated proteinMAPKMAP kinaseMAPKKMAP kinase kinaseMEKKa protein with MAPKK kinase activity independent of the p21-p71 pathwayMBPmyelin basic proteinTPA12- O-tetradecanoylphorbol 13-acetateBAPTA-AM1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetraacetoxymethyl esterIAPislet-activating proteinFCSfetal calf serumEGFepidermal growth factorrMAPa kinase-negative mutant of a recombinant Xenopus MAPKGαα-subunit of G-proteinIPsinositol phosphatesDMEMDulbecco's modified Eagle's medium. F2αstimulates cell proliferation in NIH-3T3 cells (1De Asua L.J. Clingan D. Rudland P.S. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2724-2728Crossref PubMed Scopus (182) Google Scholar, 2Nakao A. Watanabe T. Taniguchi S. Nakamura M. Honda Z. Shimizu T. Kurokawa K. J. Cell. Physiol. 1993; 155: 257-264Crossref PubMed Scopus (31) Google Scholar). Activation of phospholipase C is to date the only known biochemical signal via the Gq-coupled PGF2αreceptor (2Nakao A. Watanabe T. Taniguchi S. Nakamura M. Honda Z. Shimizu T. Kurokawa K. J. Cell. Physiol. 1993; 155: 257-264Crossref PubMed Scopus (31) Google Scholar). Such being the case, this Gq-coupled pathway is likely to be linked to the mitogenic response, and NIH-3T3 cells may be a useful model system to examine G-protein-mediated intracellular mechanisms linked to mitogenic responses. Possible intracellular mechanisms that may explain PGF2αeffects are the activation of specific serine/threonine and/or tyrosine protein kinases (PKs). Activation of phospholipase C leads to elevation of intracellular Ca2+([Ca2+]i) and/or formation of diacylglycerol, which in turn leads to activation of [Ca2+]i/calmodulin-dependent PKs and/or PKC, a family of multipotent serine/threonine kinases that elicits cellular responses, including mitogenesis (3Nishizuka Y. Science. 1986; 233: 305-312Crossref PubMed Scopus (4033) Google Scholar), respectively. We recently found that PGF2α receptor-mediated, [Ca2+]i-dependent tyrosine phosphorylation of cellular components including p125FAKcorrelated well with PGF2α-induced mitogenesis (4Watanabe T. Nakao A. Emerling D. Hashimoto Y. Tsukamoto K. Horie Y. Kinoshita M. Kurokawa K. J. Biol. Chem. 1994; 269: 17619-17625Abstract Full Text PDF PubMed Google Scholar). However, the entire spectra of intracellular PK cascades and their target cellular proteins remained to be determined. prostaglandin protein kinase mitogen-activated protein MAP kinase MAP kinase kinase a protein with MAPKK kinase activity independent of the p21-p71 pathway myelin basic protein 12- O-tetradecanoylphorbol 13-acetate 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetraacetoxymethyl ester islet-activating protein fetal calf serum epidermal growth factor a kinase-negative mutant of a recombinant Xenopus MAPK α-subunit of G-protein inositol phosphates Dulbecco's modified Eagle's medium. prostaglandin protein kinase mitogen-activated protein MAP kinase MAP kinase kinase a protein with MAPKK kinase activity independent of the p21-p71 pathway myelin basic protein 12- O-tetradecanoylphorbol 13-acetate 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetraacetoxymethyl ester islet-activating protein fetal calf serum epidermal growth factor a kinase-negative mutant of a recombinant Xenopus MAPK α-subunit of G-protein inositol phosphates Dulbecco's modified Eagle's medium. Mitogen-activated protein (MAP) kinases (MAPKs) are activated during differentiation and cell cycle transition triggered by a variety of stimuli (5Ray L.B. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1502-1506Crossref PubMed Scopus (414) Google Scholar), thereby playing a key role in the kinase cascade originating from receptor activation (6Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (959) Google Scholar). MAPK seems to transmit mitogenic signals by phosphorylating downstream components such as transcription factors (c- myc (7Seth A. Alvatez E. Gupta S. Davis R.J. J. Biol. Chem. 1991; 266: 23521-23524Abstract Full Text PDF PubMed Google Scholar), c-jun (8Baker S.J. Kerppola T.K. Luk D. Vanderberg M.T. Marshak D.R. Curran T. Abate C. Mol. Cell. Biol. 1992; 12: 4694-4705Crossref PubMed Scopus (98) Google Scholar), and p62TCF(9Gille H. Sharrocks A.D. Shaw P.E. Nature. 1992; 358: 414-417Crossref PubMed Scopus (814) Google Scholar)). A pathway leading from the tyrosine kinase receptor to MAP kinase activation has been elucidated; ligand-receptor interaction causes formation of the Ras·GTP complex, which in turn activates a kinase cascade comprising p74 (10Wood K.W. Sarnecki C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (661) Google Scholar), MAP kinase kinase (MAPKK), and MAPK. However, another protein with MAP kinase kinase kinase activity (MEKK) has been cloned (11Lange-Carter C.A. Pleoman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (873) Google Scholar). As overexpressed MEKK can activate MAPKK without activating p74, p74 and MEKK may converge on MAPKK. Recent studies revealed that MAPK is also activated through heterotrimeric G-protein-mediated mechanisms (12Gupta S.K. Gallego C. Johnson G.L. Heasley L.E. J. Biol. Chem. 1992; 267: 7987-7990Abstract Full Text PDF PubMed Google Scholar, 13Kahan C. Seuwen K. Meloche S. Pouyssegur J. J. Biol. Chem. 1992; 267: 13369-13375Abstract Full Text PDF PubMed Google Scholar). It was suggested that receptor tyrosine kinase may activate MAPKK via p21 and p72, while G-protein coupled receptors may be linked to MEKK (11Lange-Carter C.A. Pleoman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (873) Google Scholar). This hypothesis is supported by our recent observation that transfected platelet-activating factor receptor cDNA into Chinese hamster ovary cells mediates platelet-activating factor-induced activation of MAPK and MAPKK without detectable increase in GTP form of Ras (14Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar). However, lysophosphatidic acid (15Howe L.R. Marshall C.J. J. Biol. Chem. 1993; 268: 20717-20720Abstract Full Text PDF PubMed Google Scholar, 16van Corven E.J. Hordijk P.L. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (337) Google Scholar, 17Hordijk P.L. Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar), thrombin (16van Corven E.J. Hordijk P.L. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (337) Google Scholar, 18LaMorte V.J. Kennedy E.D. Collins L.R. Goldstein D. Harootunian A.T. Brown J.H. Feramisco J.R. J. Biol. Chem. 1993; 268: 19411-19415Abstract Full Text PDF PubMed Google Scholar), α2 adrenergic (19Alblas J. van Corven E.J. Hordijk P.L. Milligan G. Moolenaar W.H. J. Biol. Chem. 1993; 268: 22235-22238Abstract Full Text PDF PubMed Google Scholar), and M2 muscarinic (20Winitz S. Russell M. Qian N.-X. Gardner A. Dwyer L. Johnson G.L. J. Biol. Chem. 1993; 268: 19196-19199Abstract Full Text PDF PubMed Google Scholar) agonists stimulate formation of the GTP form of Ras and MAPK activity via an islet-activating protein (IAP)-sensitive pathway. Moreover, MAPK activation by lysophosphatidic acid can be blocked by dominant negative p21 or p74 (15Howe L.R. Marshall C.J. J. Biol. Chem. 1993; 268: 20717-20720Abstract Full Text PDF PubMed Google Scholar). Therefore, MAPK can be activated by an IAP-sensitive G-protein-coupled pathway that requires both p21 and p74. Even though MAPK activation induced by endothelin via an IAP-insensitive G-protein has been reported (21Wang Y. Simonson M.S. Pouyssegur J. Dunn M.J. Biochem. J. 1992; 287: 589-594Crossref PubMed Scopus (161) Google Scholar, 22Caraubon S. Parker P.J. Strosberg A.D. Couraud P.O. Biochem. J. 1993; 293: 381-386Crossref PubMed Scopus (68) Google Scholar), much less is known of the involvement of p21 in MAPK activation dependent upon an IAP-insensitive G-protein. Several subtypes of α-subunit of IAP-insensitive G-proteins have shown to link to phospholipase C-β; these include α-subunit of Gq(Gqα) family (Gqα, G11α, G14α, and G16α) (23Strathmann M. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9113-9117Crossref PubMed Scopus (386) Google Scholar, 24Taylor S.J. Chae H.Z. Rhee S.G. Exton J.H. Nature. 1991; 350: 516-518Crossref PubMed Scopus (610) Google Scholar, 25Smrcka A.V. Hepler J.R. Brown K.O. Sternweis P.C. Science. 1991; 251: 804-807Crossref PubMed Scopus (695) Google Scholar, 26Birnbaumer L. Cell. 1992; 71: 1069-1072Abstract Full Text PDF PubMed Scopus (377) Google Scholar). Subtypes of phospholipase C; phospholipase C-β1 and phospholipase C-β2, are known to be stimulated by specific types of Gqα family. Phospholipase C-β2 is also activated by βγ dimers of G-protein (26Birnbaumer L. Cell. 1992; 71: 1069-1072Abstract Full Text PDF PubMed Scopus (377) Google Scholar). The activity of Gα is regulated by the exchange of GDP and GTP and by intrinsic GTPase activity. Agonist binding to cell surface receptors stimulate Gα by enhancing GDP-GTP exchange, and the intrinsic GTPase activity reverses this state to inactivate Gα. It was reported that substitution of arginine 183 or glutamine 209 of Gqα with cysteine or leucine, respectively, constitutively activates Gqα by inhibiting intrinsic GTP hydrolysis (27Conklin B.R. Chabre O. Wong Y.H. Federman A.D. Bournet H.R. J. Biol. Chem. 1992; 267: 31-34Abstract Full Text PDF PubMed Google Scholar, 28Wu D. Lee C.H. Rhee S.G. Simon M.I. J. Biol. Chem. 1992; 267: 1811-1817Abstract Full Text PDF PubMed Google Scholar). It was also reported that NIH-3T3 cells stably expressing Gqα with a mutation of conserved glutamine residue or overexpressing the wild-type of Gqα exhibited transformation of the cells (29Kalinec G. Nazarali A.J. Hermouet S. Xu N. Gutkind J.S. Mol. Cell. Biol. 1992; 12: 4687-4693Crossref PubMed Google Scholar, 30de Vivo M. Chen J. Codina J. Iyengar R. J. Biol. Chem. 1992; 267: 18263-18266Abstract Full Text PDF PubMed Google Scholar). These recent advances have led us to direct examination of the interrelationship between activation of Gqα and PGF2α-induced cellular responses in NIH-3T3 cells. We report here that in NIH-3T3 cells, PGF2α stimulates MAPKK and MAPK by a mechanism that is inhibited by staurosporine and H-7 but is independent of classical TPA-sensitive PKC or Ca2+/calmodulin-dependent PKs and that Gqα and Ras may mediate this MAPK activation. Materials were obtained from the following sources: [γ-32P]ATP (specific activity, >5,000 Ci/mmol) from Amersham Corp.; [3H]thymidine (specific activity, 20.1 Ci/mmol) and myo-[3H]inositol (specific activity, 45.1 Ci/mmol) from DuPont NEN; 32P-labeled carrier-free Pifrom ICN; myelin basic protein (MBP), 12- O-tetradecanoylphorbol 13-acetate (TPA), epidermal growth factor (EGF), insulin, forskolin, staurosporine, H-7, W-7, and KN-62 from Sigma; IAP from Funakoshi Biochemicals (Tokyo); 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) from Dojin (Kumamoto, Japan); protein G-Sepharose and Q-Sepharose Fast Flow from Pharmacia LKB Biotechnology Inc; anti-v-H-ras monoclonal antibody Y13-259 from Oncogene Science; anti-MAPK (erk2) monoclonal antibody against murine recombinant p42, anti-rat MAPK R2 (erk1-CT), and anti-phosphotyrosine-conjugated Sepharose from Upstate Biotechnology (Lake Placid, NY); Pansorbin from Calbiochem; Transfectam from Sepracor (Marlborough, MA); P-81 phosphocellulose papers from Whatman; polyethyleneimine-cellulose plates from Merck. PGF2α, PGE2, and PGD2were donated by Ono Pharmaceuticals (Osaka, Japan). A cDNA encoding guinea pig Gqα and that encoding a GTPase deficient mutant, in which glutamine 209 was replaced with leucine (27Conklin B.R. Chabre O. Wong Y.H. Federman A.D. Bournet H.R. J. Biol. Chem. 1992; 267: 31-34Abstract Full Text PDF PubMed Google Scholar, 28Wu D. Lee C.H. Rhee S.G. Simon M.I. J. Biol. Chem. 1992; 267: 1811-1817Abstract Full Text PDF PubMed Google Scholar) (M6 mutant), were subcloned into an expression vector (pCDNA-1: Invitrogen Corp., San Diego, CA). All other chemicals were of analytical grade. Reagents for cell culture were from Nissui (Tokyo) and Life Technologies, Inc. NIH-3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), under the conditions described elsewhere (2Nakao A. Watanabe T. Taniguchi S. Nakamura M. Honda Z. Shimizu T. Kurokawa K. J. Cell. Physiol. 1993; 155: 257-264Crossref PubMed Scopus (31) Google Scholar). The cells were washed 3 times with FCS-free DMEM and cultured for 24 h before the assays of MAPKK and MAPK, analysis of GTP-bound Ras, or Western blot analysis. Each plasmid DNA was transfected into NIH-3T3 cells by DEAE-dextran method, as described (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.42-16.44Google Scholar) or using Transfectam, as described in the manual provided by the supplier (Sepracor, Marlborough, MA). Typically, 2 μg of plasmid DNA was transfected into 5 × 105cells (counted 1 day before transfection)/60-mm2dish. One day after transfection, cells from one 60-mm2dish were seeded onto two wells on a six-well coaster for the measurement of [3H]thymidine incorporation and phosphoinositide breakdown. Three days after transfection, the cells were washed 3 times with DMEM without FCS and cultured in FCS-free DMEM for 24 h prior to the measurement of [3H]thymidine incorporation, phosphoinositide breakdown, MAPK assay, and analysis of Ras-bound GTP and GDP. Quiescent Cells were washed twice with Tyrode buffer containing 20 m M HEPES pH 7.4 and 1 m M CaCl2(HEPES-Tyrode) and then stimulated with agonists in HEPES-Tyrode for the indicated times. After a wash with ice-cold phosphate-buffered saline, the cells were lysed in ice-cold lysis buffer (20 m M Tris-HCl, pH 7.5, 25 m Mβ-glycerophosphate, 2 m M EGTA, 1 m M phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 2 m M dithiothereitol, and 1 m M sodium orthovanadate) and centrifuged at 10,000 × g for 10 min. The supernatant was used as the source of MAPK and MAPKK. The immune complex MAPK assay was carried out essentially as described (32Tobe K. Kadowaki T. Tamemoto H. Ueki K. Hara K. Koshio O. Momomura K. Gotoh Y. Nishida E. Akamuma Y. Yazaki Y. Kasuga M. J. Biol. Chem. 1991; 266: 24793-24803Abstract Full Text PDF PubMed Google Scholar). MAPK was immunoprecipitated with anti-MAPK (erk2) monoclonal antibody, with the aid of Pansorbin, washed with lysis buffer, and resuspended in the same buffer. In the transfection experiments, MAPK was partially purified by batch treatment with Q-Sepharose; the cell lysate was mixed with 0.5 volume of Q-Sepharose beads equilibrated with lysis buffer containing 0.12 M NaCl for 30 min at 4°C and then briefly centrifuged (3,000 × g for 5 min). The resultant pellet was washed twice with the same buffer and incubated with the original volume of lysis buffer containing 0.3 M NaCl for 30 min at 4°C. MAPK was recovered in the supernatant by centrifugation. The sample for MAPK assay was incubated with MBP (1 mg/ml) in 25 μl of kinase buffer (20 m M Tris-HCl, pH 7.5, 10 m M MgCl2, 1 m M MnCl2, and 40 m M ATP) containing 1 μCi of [γ-32P]ATP for 25 min at 25°C. A 12-μl aliquot was spotted onto P-81 phosphocellulose paper and extensively washed with 0.5% phosphoric acid. The paper was dried, and 32P incorporation into MBP was measured by Cerenkov counting (14Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar). In the kinase detection assay in the MBP-containing gel (gel kinase assay), the supernatant of the cell lysate was electrophoresed onto an SDS-polyacylamide gel containing 1 mg/ml MBP. Proteins were denatured in 6 M guanidine-HCl and renatured as described previously (33Gotoh Y. Nishida E. Yamashita T. Hoshi M. Kawasaki M. Sakai H. Eur. J. Biochem. 1990; 193: 661-669Crossref PubMed Scopus (321) Google Scholar). Phosphorylation of MBP was carried out in 5 ml of kinase buffer containing 25 μCi of [γ-32P]ATP, and the gel was extensively washed with 7% acetic acid. The dried gel was subjected to an image analyzing system using FUJI BAS 2000. MAPKK activity was assayed by measuring 32P incorporation into a kinase-negative mutant of a recombinant Xenopus MAPK (rMAPK) (34Kosako H. Nishida E. Gotoh Y. EMBO J. 1993; 12: 787-794Crossref PubMed Scopus (120) Google Scholar, 35Matsuda S. Gotoh Y. Nishida E. J. Biol. Chem. 1993; 268: 3277-3281Abstract Full Text PDF PubMed Google Scholar), which was kindly provided by Drs. Y. Gotoh and E. Nishida of Kyoto University. MAPKK was partially purified by batch treatment with Q-Sepharose; the cell lysate was mixed with 0.5 volume of Q-Sepharose beads equilibrated with lysis buffer containing 0.12 M NaCl and briefly centrifuged. Under these conditions, MAPKK activity was recovered in the supernatant. The supernatant was incubated with rMAPK (final concentration, 50 μg/ml) in 12.5 μl of the kinase buffer containing 1 μCi of [γ-32P]ATP for 20 min at 25°C. The sample was subjected to SDS-polyacrylamide gel electrophoresis, and 32P incorporation into the rMAPK band was measured using a Fuji image analyzer (FUJI BAS 2000). Immunoprecipitation and Western blot Analysis- Quiescent cultures of NIH-3T3 cells on a 60-mm2culture dish (3 × 106) were washed twice with HEPES-Tyrode, treated with each agonist in HEPES-Tyrode at 37°C for 3 min and then frozen in liquid nitrogen. The cells were lysed on ice in 1 ml of solution containing 10 m M Tris-HCl, pH 7.6, 5 m M EDTA, 50 m M NaCl, 30 m M sodium pyrophosphate, 50 m M NaF, 100 μM sodium orthovanadate, and 1% Triton X-100 (immunoprecipitaion buffer). Cell lysates were centrifuged at 15,000 rpm for 10 min. Resultant supernatants were precleared by incubation with agarose at 4°C for 1 h. After removal of agarose by centrifugation at 15,000 rpm for 5 min, the supernatants were incubated with 100 μl of anti-phosphotyrosine conjugated to agarose at 4°C for 4 h and then cleared by centrifugation at 15,000 rpm for 5 min. Immunoprecipitates were washed 3 times with 1 ml of immunoprecipitaion buffer and then treated with 30 μl of Laemmli's sample buffer. After proteins were separated by 8% SDS-polyacrylamide gel electrophoresis, Western blot analysis was performed using 2 μg/ml of anti-Rat MAPK (erk1-CT) as the first antibody and goat anti-rabbit IgG conjugated to horseradish peroxidase (200-fold dilution) as the second antibody as described (36Watanabe T. Shimizu T. Nakao A. Taniguchi S. Arata Y. Teramoto T. Seyama Y. Ui M. Kurokawa K. Biochim. Biophys. Acta. 1991; 1074: 398-405Crossref PubMed Scopus (11) Google Scholar). Quiescent cells were labeled with 0.1 mCi/ml 32P-labeled carrier-free Piin phosphate-free DMEM medium for 4 h, and then stimulated with agonists for 3 min, unless otherwise stated. Next, the cells were lysed in Triton X-114 buffer (50 m M HEPES-NaOH, pH 7.4, 1% Triton X-114, 100 m M NaCl, 5 m M MgCl2, 1 mg/ml bovine serum albumin, 1 m M phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 100 μM GTP, 100 μM GDP, and 1 m M ATP) supplemented with phosphatase inhibitors (1 m M sodium pyrophosphate and 1 m M sodium orthovanadate). Membrane-bound Ras was recovered by detergent phase splitting, as described (37Burgering B.M.T. Medema R.H. Maassen J.A. van de Wetering M.L. van der Eb A.J. McCormic F. Bos J.L. EMBO J. 1991; 10: 1103-1109Crossref PubMed Scopus (208) Google Scholar) and immunoprecipitated with a monoclonal antibody Y13-259 with the aid of protein G-Sepharose (38Satoh T. Endo M. Nakafuku M. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5993-5997Crossref PubMed Scopus (199) Google Scholar). The immune complex was extensively washed with washing buffer (50 m M HEPES-NaOH, pH 7.4, 0.1% Triton X-100, 0.05 M NaCl, 5 m M MgCl2, and 1 mg/ml bovine serum albumin) (37Burgering B.M.T. Medema R.H. Maassen J.A. van de Wetering M.L. van der Eb A.J. McCormic F. Bos J.L. EMBO J. 1991; 10: 1103-1109Crossref PubMed Scopus (208) Google Scholar) supplemented with phosphatase inhibitors (1 m M sodium pyrophosphate and 1 m M sodium orthovanadate). Guanine nucleotides bound to Ras were eluted and analyzed by thin-layer chromatography on a polyethyleneimine-cellulose plate. The GTP/(GTP + GDP) ratio was measured using an image analyzer (FUJI BAS 2000). Formation of inositol phosphates (IPs) for 1 min in the presence or absence of PGF2α was examined as described (2Nakao A. Watanabe T. Taniguchi S. Nakamura M. Honda Z. Shimizu T. Kurokawa K. J. Cell. Physiol. 1993; 155: 257-264Crossref PubMed Scopus (31) Google Scholar) using the methods of Bijsterbosch et al. (39Bijsterbosch M.K. Meade C.J. Turner G.A. Klaus G.G. Cell. 1985; 41: 999-1006Abstract Full Text PDF PubMed Scopus (254) Google Scholar). [3H]Thymidine incorporation into DNA was measured by the method of Nakamura et al. (40Nakamura T. Tomita Y. Ichihara A. J. Biochem. (Tokyo). 1983; 94: 1029-1035Crossref PubMed Scopus (194) Google Scholar), with slight modifications. Quiescent cells (6-well coaster) were washed twice with DMEM at 37°C and then incubated for 24 h in 1 ml of DMEM in the presence or absence of PGF2α. One μCi of [3H]thymidine was added to each dish 6 h before harvest. [3H]Thymidine incorporation into trichloroacetic acid-insoluble materials was determined. Protein concentration was measured by Bio-Rad protein assay kits using bovine serum albumin as the standard. Statistical analyses were made by the procedure of analysis of variance. The addition of 1 μM PGF2α caused a transient activation of MAPK and MAPKK with a peak at around 3 min, while 1 μM TPA induced a time-dependent increase of MAPK activity up to 30 min and EGF/insulin activated MAPK with a peak value at around 5 min and a sustained phase within 60 min (Fig. 1). Dose dependence of PGF2α on MAPK and MAPKK activation with ED50of around 10−8M was similar to that for elevation of [Ca2+]i, formation of IPs, and [3H]thymidine incorporation, as described previously (2Nakao A. Watanabe T. Taniguchi S. Nakamura M. Honda Z. Shimizu T. Kurokawa K. J. Cell. Physiol. 1993; 155: 257-264Crossref PubMed Scopus (31) Google Scholar) (Fig. 2). Pretreatment of the cells with IAP did not affect the dose-dependence of PGF2α on MAPKK activation, even under conditions that are assumed to ADP ribosylate almost all of the IAP substrate, as described (2Nakao A. Watanabe T. Taniguchi S. Nakamura M. Honda Z. Shimizu T. Kurokawa K. J. Cell. Physiol. 1993; 155: 257-264Crossref PubMed Scopus (31) Google Scholar) (Fig. 2). A gel MAPK assay of the supernatant of cell lysate (see "Experimental Procedures") (Fig. 3 A) and that of anti-42-kDa MAPK immunoprecipitates (data not shown) showed that a 42-kDa MAPK was the main MAPK activated by PGs in the NIH-3T3 cells. We also found that MAPK and MAPKK were activated by PGs (1 μM); PGF2α > PGE2, PGD2, which correlates with the order of potencies of these PGs to activate the elevation of [Ca2+]i, formation of IPs, and [3H]thymidine incorporation, as described previously (2Nakao A. Watanabe T. Taniguchi S. Nakamura M. Honda Z. Shimizu T. Kurokawa K. J. Cell. Physiol. 1993; 155: 257-264Crossref PubMed Scopus (31) Google Scholar) (Fig. 3, A and B). These two kinases (MAPK and MAPKK) were also activated by EGF (100 ng/ml)/insulin (1 μM) ≫ TPA (1 μM) > ionomycin (100 n M), but not by forskolin (10 μM) (Fig. 3, A and B). Western blot analysis with anti-rat MAPK (erk1-CT) of immunoprecipitates with antiphosphotyrosine-conjugated Sepharose of cellular lysates demonstrated that a MAPK with a molecular mass around 42-44 kDa was tyrosine phosphorylated (Fig. 4). The PGF2α -induced activation of MAPK was almost completely inhibited by staurosporine (20 n M) and by H-7 (20 μM) at concentrations that inhibit PKC (41Tamaoki T. Nomoto H. Takahashi I. Kato Y. Morimoto M. Tomita F. Biochem. Biophys. Res. Commun. 1986; 135: 397-402Crossref PubMed Scopus (2215) Google Scholar, 42Hidaka H. Inagaki M. Kawamoto S. Sasaki Y. Biochemistry. 1984; 23: 5036-5041Crossref PubMed Scopus (2327) Google Scholar), but not by Ca2+/calmodulin kinase inhibitors; W-7 or KN-62, even at concentrations that inhibit calmodulin-dependent kinases (20 μM) (43Kanamori M. Naka M. Asano M. Hidaka H. J. Pharmacol. Exp. Ther. 1981; 217: 494-499PubMed Google Scholar, 44Tokumitsu H. Chijiwa T. Hagiwara M. Mitsutani A. Terasawa M. Hidaka H. J. Biol. Chem. 1990; 265: 4315-4320Abstract Full Text PDF PubMed Google Scholar); nor by removing extracellular Ca2+. The [Ca2+]ichelator, BAPTA, only partially attenuated PGF2α -induced activation of MAPK activity, even under conditions that almost completely prevented the PGF2α -induced elevation of [Ca2+]i(50 μM, 15 min) (4Watanabe T. Nakao A. Emerling D. Hashimoto Y. Tsukamoto K. Horie Y. Kinoshita M. Kurokawa K. J. Biol. Chem. 1994; 269: 17619-17625Abstract Full Text PDF PubMed Google Scholar) (Table I). Pretreatment of the cells with TPA (2.5 μM, 24 h), which is assumed to down-regulate classical PKC, did not affect the PGF2α -induced activation of MAPK (Table II).FIG. 2Dose-response of PGF2α on MAPK and MAPKK activities. Quiescent NIH-3T3 cells (3 × 106/60-mm diameter culture dish) in HEPES-Tyrode stimulated with the indicated concentrations of PGF2α at 37°C for 3 min, and then immersed in liquid nitrogen. MAPK activity was measured by immune complex assay (○) and MAPKK assay in control cells (●) or in cells pretreated with IAP (100 ng/ml; 18 h) (⊙) were performed as described under "Experimental Procedures." Each symbol represents the mean of duplicate samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIG. 3Effects of various agents on MAPK (A) and MAPKK (B) activities. Quiescent NIH-3T3 cells (3 × 106/60-mm diameter culture dish) in HEPES-Tyrode were stimulated with various agonists at 37°C for 3 min. MAPK activity of the supernatant of cell lysate measured by gel kinase assay, and MAPKK assays were performed as described under "Experimental Procedures." One μM of PGF2α, PGD2, PGE2, U46619, iloprost or TPA, a combined use of 100 ng/ml EGF and 1 μM insulin (EGF/Ins.), 100 n M ionomycin (Iono.), or 10 μM forskolin (Forsk.) was used as an agonist. MAPKK assay was done in the presence (+rMAPK) or absence (-rMAPK) of rMAPK, as described under "Experimental Procedures." Results typical of experiments repeated at least three times are depicted.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIG. 4Western blot analysis with anti-rat MAPK monoclonal antibody (erk-1 CT) of anti-phosphotyrosine immunoprecipitates of NIH-3T3 cell lysates. Quiescent NIH-3T3 cells (3 × 106/60-mm diameter culture dish) in HEPES-Tyrode buffer were stimulated with 1 μM PGF2α or the combined use of 100 ng/ml EGF and 1 μM insulin at 37°C for 3 min. Immunoprecipitation of cell lysates wit
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