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• W2053253135 abstract "Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. CCK exerts its growth modulatory effects through Gq-coupled receptors (CCKA and CCKB) and activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). In the present study, we investigated the different mechanisms participating in CCK-induced activation of ERK1/2 in pancreatic AR42J cells expressing both CCKA and CCKB. CCK activated ERK1/2 and Raf-1 to a similar extent as epidermal growth factor (EGF). Inhibition of EGF receptor (EGFR) tyrosine kinase or expression of dominant-negative Ras reduced CCK-induced ERK1/2 activation, indicating participation of the EGFR and Ras in CCK-induced ERK1/2 activation. However, compared with EGF, CCK caused only small increases in tyrosine phosphorylation of the EGFR and Shc, Shc-Grb2 complex formation, and Ras activation. Signal amplification between Ras and Raf in a CCK-induced ERK cascade appears to be mediated by activation of protein kinase Cε (PKCε), because 1) down-modulation of phorbol ester-sensitive PKCs inhibited CCK-induced activation of Ras, Raf, and ERK1/2 without influencing Shc-Grb2 complex formation; 2) PKCε, but not PKCα or PKCδ, was detectable in Raf-1 immunoprecipitates, although CCK activated all three PKC isoenzymes. In addition, the present study provides evidence that the Src family tyrosine kinase Yes is activated by CCK and mediates CCK-induced tyrosine phosphorylation of Shc. Furthermore, we show that CCK-induced activation of the EGFR and Yes is achieved through the CCKB receptor. Together, our data show that different signals emanating from the CCK receptors mediate ERK1/2 activation; activation of Yes and the EGFR mediate Shc-Grb2 recruitment, and activation of PKC, most likely PKCε, augments CCK-stimulated ERK1/2 activation at the Ras/Raf level. Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. CCK exerts its growth modulatory effects through Gq-coupled receptors (CCKA and CCKB) and activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). In the present study, we investigated the different mechanisms participating in CCK-induced activation of ERK1/2 in pancreatic AR42J cells expressing both CCKA and CCKB. CCK activated ERK1/2 and Raf-1 to a similar extent as epidermal growth factor (EGF). Inhibition of EGF receptor (EGFR) tyrosine kinase or expression of dominant-negative Ras reduced CCK-induced ERK1/2 activation, indicating participation of the EGFR and Ras in CCK-induced ERK1/2 activation. However, compared with EGF, CCK caused only small increases in tyrosine phosphorylation of the EGFR and Shc, Shc-Grb2 complex formation, and Ras activation. Signal amplification between Ras and Raf in a CCK-induced ERK cascade appears to be mediated by activation of protein kinase Cε (PKCε), because 1) down-modulation of phorbol ester-sensitive PKCs inhibited CCK-induced activation of Ras, Raf, and ERK1/2 without influencing Shc-Grb2 complex formation; 2) PKCε, but not PKCα or PKCδ, was detectable in Raf-1 immunoprecipitates, although CCK activated all three PKC isoenzymes. In addition, the present study provides evidence that the Src family tyrosine kinase Yes is activated by CCK and mediates CCK-induced tyrosine phosphorylation of Shc. Furthermore, we show that CCK-induced activation of the EGFR and Yes is achieved through the CCKB receptor. Together, our data show that different signals emanating from the CCK receptors mediate ERK1/2 activation; activation of Yes and the EGFR mediate Shc-Grb2 recruitment, and activation of PKC, most likely PKCε, augments CCK-stimulated ERK1/2 activation at the Ras/Raf level. cholecystokinin CCK-A receptor CCK-B receptor Dulbecco's modified Eagle's medium epidermal growth factor EGF receptor G protein-coupled receptor heparin-binding EGF-like growth factor hepatocyte growth factor protein kinase C extracellular-regulated kinase receptor tyrosine kinase 12-O-tetradecanoylphorbol-13-acetate 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine 4-morpholinepropanesulfonic acid mitogen-activated protein kinase/extracellular signal-regulated kinase kinase Cholecystokinin (CCK)1 and related peptides are potent growth factors in the gastrointestinal tract (1Baldwin G.S. J. Gastroenterol. Hepatol. 1995; 10: 215-232Crossref PubMed Scopus (70) Google Scholar, 2Rozengurt E. Walsh J.H. Annu. Rev. Physiol. 2001; 63: 49-76Crossref PubMed Scopus (182) Google Scholar). CCK stimulates normal growth of the pancreas (3Mainz D.L. Black O. Webster P.D. J. Clin. Invest. 1973; 52: 2300-2304Crossref PubMed Scopus (257) Google Scholar, 4Solomon T.E. Vanier N. Morisset J. Am. J. Physiol. 1983; 245: G99-G105PubMed Google Scholar, 5Logsdon C.D. Williams J.A. Am. J. Physiol. 1986; 250: G440-G447PubMed Google Scholar, 6Povoski S.P. Zhou W. Longnecker D.S. Jensen R.T. Mantey S.A. Bell Jr., R.H. Gastroenterology. 1994; 107: 1135-1146Abstract Full Text PDF PubMed Scopus (48) Google Scholar) and might be involved in growth of human pancreatic cancer (7Herrington M.K. Adrian T.E. Int. J. Pancreatol. 1995; 17: 121-138PubMed Google Scholar, 8Clerc P. Leung-Theung-Long S. Wang T.C. Dockray G.J. Bouisson M. Delisle M.B. Vaysse N. Pradayrol L. Fourmy D. Dufresne M. Gastroenterology. 2002; 122: 428-437Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Exogenous administration of CCK can lead to pancreatic hyperplasia, dysplasia, and malignancies (9Folsch U.R. Winckler K. Wormsley K.G. Scand. J. Gastroenterol. 1978; 13: 663-671Crossref PubMed Scopus (141) Google Scholar) and accelerates the growth of malignant pancreatic tissue (10Howatson A.G. Carter D.C. Br. J. Cancer. 1985; 51: 107-114Crossref PubMed Scopus (120) Google Scholar, 11Lhoste E.F. Longnecker D.S. Cancer Res. 1987; 47: 3273-3277PubMed Google Scholar, 12Douglas B.R. Woutersen R.A. Jansen J.B. de Jong A.J. Rovati L.C. Lamers C.B. Gastroenterology. 1989; 96: 462-469Abstract Full Text PDF PubMed Scopus (89) Google Scholar). In several cancer cell lines, CCK promotes growth (13Smith J.P. Kramer S.T. Solomon T.E. Regul. Pept. 1991; 32: 341-349Crossref PubMed Scopus (78) Google Scholar). Furthermore, endogenous hypercholecystokininemia promotes carcinogenesis in the hamster (14Chu M. Rehfeld J.F. Borch K. Carcinogenesis. 1997; 18: 315-320Crossref PubMed Scopus (19) Google Scholar). CCK binds to and activates receptors (CCKAand CCKB) belonging to the seven-transmembrane-spanning family of G protein-coupled receptors (GPCRs) (15Wank S.A. Pisegna J.R. de Weerth A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8691-8695Crossref PubMed Scopus (463) Google Scholar, 16Piiper A. Stryjek-Kaminska D. Klengel R. Zeuzem S. Gastroenterology. 1997; 113: 1747-1755Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). A major signaling cascade stimulated by both CCKA and CCKB receptors is pertussis toxin-insensitive activation of phospholipase C-β and subsequent activation of protein kinase C (PKC) (2Rozengurt E. Walsh J.H. Annu. Rev. Physiol. 2001; 63: 49-76Crossref PubMed Scopus (182) Google Scholar, 16Piiper A. Stryjek-Kaminska D. Klengel R. Zeuzem S. Gastroenterology. 1997; 113: 1747-1755Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) plays a key role in mediating proproliferative effects of both receptor tyrosine kinases (RTKs) such as the epidermal growth factor (EGF) receptor (EGFR) and GPCR (17Gutkind J.S. Science's STKE. 2000; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/40/re1)PubMed Google Scholar). Depending on receptor and cell type, GPCR-induced ERK1/2 activation may involve stimulation of nonreceptor tyrosine kinases of the Src family and Pyk-2, receptor tyrosine kinases (most notably the EGFR), and phosphatidylinositol 3-kinase, leading to activation of Ras (17Gutkind J.S. Science's STKE. 2000; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/40/re1)PubMed Google Scholar, 18Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar, 19Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (880) Google Scholar, 20Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 21Sadoshima J. Izumo S. EMBO J. 1996; 15: 775-787Crossref PubMed Scopus (232) Google Scholar, 22Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 23Della 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, 24Leserer M. Gschwind A. Ullrich A. IUBMB Life. 2000; 49: 405-409Crossref PubMed Scopus (61) Google Scholar). Moreover, Gq-coupled receptors can activate ERK1/2 by a PKC-dependent Ras-independent pathway involving direct activation of Raf-1 (17Gutkind J.S. Science's STKE. 2000; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/40/re1)PubMed Google Scholar, 25Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1160) Google Scholar, 26Marais R. Light Y. Mason C. Paterson H. Olson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (402) Google Scholar). CCK stimulates ERK1/2 by a mechanism depending on activation of phospholipase C and phorbol ester-sensitive PKCs (27Dabrowski A. VanderKuur J.A. Carter-Su C. Williams J.A. J. Biol. Chem. 1996; 271: 27125-27129Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Piiper A. Gebhardt R. Kronenberger B. Giannini C.D. Elez R. Zeuzem S. Mol. Pharmacol. 2000; 58: 608-613Crossref PubMed Scopus (21) Google Scholar). In agreement with a PKC-dependent, Ras-independent mechanism of CCK-induced ERK1/2 activation, expression of dominant-negative Ras did not inhibit CCK-induced ERK1/2 activation in primary pancreatic acini (29Nicke B. Tseng M.J. Fenrich M. Logsdon C.D. Am. J. Physiol. 1999; 276: G499-G506PubMed Google Scholar). However, down-modulation of PKC by long term treatment with phorbol ester only partially inhibits CCK-induced ERK1/2 activation (28Piiper A. Gebhardt R. Kronenberger B. Giannini C.D. Elez R. Zeuzem S. Mol. Pharmacol. 2000; 58: 608-613Crossref PubMed Scopus (21) Google Scholar, 30Daulhac L. Kowalski-Chauvel A. Pradayrol L. Vaysse N. Seva C. Biochem. J. 1997; 325: 383-389Crossref PubMed Scopus (47) Google Scholar), indicating that additional mechanisms are involved in CCK-induced ERK1/2 activation. CCK has been shown to induce tyrosine phosphorylation of Shc and Pyk-2 as well as complex formation of Grb2 with Shc and Pyk-2 in native rat pancreatic acini (27Dabrowski A. VanderKuur J.A. Carter-Su C. Williams J.A. J. Biol. Chem. 1996; 271: 27125-27129Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 31Tapia J.A. Ferris H.A. Jensen R.T. Garcia L.J. J. Biol. Chem. 1999; 274: 31261-31271Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), events occurring in Ras-dependent ERK1/2 activation (17Gutkind J.S. Science's STKE. 2000; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/40/re1)PubMed Google Scholar, 19Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (880) Google Scholar). In CCKB-transfected Chinese hamster ovary cells, the CCKB receptor agonist gastrin activates ERK1/2 by a Shc- and Src-dependent mechanism (32Daulhac L. Kowalski-Chauvel A. Pradayrol L. Vaysse N. Seva C. J. Biol. Chem. 1999; 274: 20657-20663Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In CCKBreceptor-transfected gastric epithelial cells, gastrin induces EGFR tyrosine phosphorylation (33Miyazaki Y. Shinomura Y. Tsutsui S. Zushi S. Higashimoto Y. Kanayama S. Higashiyama S. Taniguchi N. Matsuzawa Y. Gastroenterology. 1999; 116: 78-89Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Phosphatidylinositol 3-kinase may represent an additional signaling intermediate in gastrin-induced ERK1/2 activation (34Seva C. Kowalski-Chauvel A. Daulhac L. Barthez C. Vaysse N. Pradayrol L. Biochem. Biophys. Res. Commun. 1997; 238: 202-206Crossref PubMed Scopus (29) Google Scholar, 35Zieger M. Oehrl W. Wetzker R. Henklein P. Nowak G. Kaufmann R. Biol. Chem. 2000; 381: 763-768Crossref PubMed Scopus (8) Google Scholar). Thus, a number of possible signaling intermediates of the Ras-dependent pathway have been assigned to CCK receptor-induced ERK1/2 activation, but it is unclear whether these signaling pathways operate within one cell type and whether cross-talk exists to mediate CCK-induced ERK1/2 activation. In the present study, we sought to delineate the contribution of the EGFR, Src family tyrosine kinases, and PKC as well as their cross-talk in CCK-induced activation of the ERK1/2 pathway in the pancreatic acinar carcinoma cell line AR42J. Our data show that different signals emanating from the CCK receptor cooperate to mediate stimulation of ERK1/2; activation of Yes and the EGFR mediate Shc-Grb2 recruitment and Ras activation, and activation of PKC, most likely PKCε, potentiates ERK1/2 activation at the Ras/Raf level. CCK octapeptide, human recombinant EGF, [Glu52]diphtheria toxin (CRM197), and pertussis toxin were obtained from Sigma. pUSEamp expression vector containing dominant negative Ras (N17Ras), agarose-conjugated Ras-binding domain, and glutathione S-transferase-MEK1 were from Upstate Biotechnology, Inc. (Lake Placid, NY). Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, LipofectAMINE 2000, and penicillin/streptomycin were from Invitrogen. The EGFR-specific tyrosine kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), the PKC inhibitors bisindoylmaleimide (GF109203X), Gö6976 and rottlerin, the Src family kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1), 1,2-bis(2-aminophenoxy)etheneN,N,N′,N′-tetraacetic acid acetoxymethyl ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), all dissolved in dimethyl sulfoxide, hepatocyte growth factor (HGF), and gastrin were obtained from Calbiochem. The ERK1/2 activity assay was from Cell Signaling (Beverly, MA). pEGFP-C1 was from Clontech (Palo Alto, CA). The CCKAreceptor antagonist L364,717 and the CCKB receptor antagonist L365,260 were kind gifts of ML Laboratories PLC (Liverpool, UK). Monoclonal anti-β-actin, affinity-purified horseradish peroxidase-conjugated anti-mouse, anti-rabbit, and anti-sheep IgG were obtained from Sigma. Sheep anti-EGFR used for immunoblotting and polyclonal anti-Shc IgG used for immunoprecipitation, anti-pan-Ras IgG, and agarose-conjugated Ras-binding domain were from Upstate Biotechnology. The antibody against Tyr(P)418-Src was fromBIOSOURCE (Camarillo, CA). The monoclonal anti-Grb2, anti-Fyn, anti-Yes, anti-PKCα, anti-PKCδ, and anti-PKCε as well as monoclonal anti-Shc IgGs were from Transduction Laboratories (Lexington, KY). The anti-c-Src IgG (clone 327) was obtained from Calbiochem. The antibody raised against dually phosphorylated activated ERK1/2 was from Cell Signaling. Monoclonal anti-phosphotyrosine, polyclonal goat anti-EGFR and anti-Raf-1 used for immunoprecipitation, rabbit anti-Raf-1 IgG used for reprobing of the immunoblots, and horseradish peroxidase-conjugated anti-goat IgG were from Santa Cruz. Neutralizing and anti-HB-EGF was from R & D Systems (Minneapolis, MN). AR42J cells were cultured in DMEM containing 10% fetal calf serum and antibiotics (36Piiper A. Leser J. Lutz M.P. Beil M. Zeuzem S. Biochem. Biophys. Res. Commun. 2001; 287: 746-751Crossref PubMed Scopus (22) Google Scholar). For transient transfection, cells were cultured in 8-cm2 dishes and transfected with 3 μg of Csk (C-terminalSrc kinase) in pSG5 or pUSEamp containing dominant negative Ras (N17Ras) using LipofectAMINE 2000 according to the instructions of the manufacturer. The efficiency of transfection was monitored by transfecting the cells with a plasmid coding for the green fluorescent protein (pEGFR-C1). Pertussis toxin treatment was carried out by incubating the cells with 200 ng/ml pertussis toxin for 12 h prior to the experiments (28Piiper A. Gebhardt R. Kronenberger B. Giannini C.D. Elez R. Zeuzem S. Mol. Pharmacol. 2000; 58: 608-613Crossref PubMed Scopus (21) Google Scholar). AR42J cells were incubated with appropriate agents at 37 °C. At specified times, the incubation was stopped by the addition of lysis buffer (50 mm Hepes, pH 7.0, 100 mm NaCl, 0.2 mm MgSO4, 0.5 mmNa3VO4, 0.4 mm phenylmethylsulfonyl fluoride, 1% Triton X-100, 10 μg/ml leupeptin, 10 μg/ml aprotinin). The extracts were clarified by centrifugation and incubated sequentially (2 h for each incubation at 4 °C) with antibodies as indicated and protein A/G-Sepharose (Amersham Biosciences) with gentle agitation. Immunoprecipitates were washed three times with lysis buffer, boiled for 3 min in 4× Laemmli sample buffer, separated on SDS-polyacrylamide gels under reducing conditions, and electrotransferred to nitrocellulose membranes (16Piiper A. Stryjek-Kaminska D. Klengel R. Zeuzem S. Gastroenterology. 1997; 113: 1747-1755Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 28Piiper A. Gebhardt R. Kronenberger B. Giannini C.D. Elez R. Zeuzem S. Mol. Pharmacol. 2000; 58: 608-613Crossref PubMed Scopus (21) Google Scholar). Antigen-antibody complexes were visualized using horseradish peroxidase-conjugated IgGs and the enhanced chemiluminescence system. Where appropriate, a 50-μl aliquot of clarified whole-cell lysate was mixed with an equal volume of 4× Laemmli sample buffer and resolved by SDS-PAGE for confirmation of identity of proteins after immunoprecipitation. For reprobing, blots were incubated in stripping buffer (62.5 mm Tris-HCl, pH 6.7, 2% SDS, and 100 mm 2-mercaptoethanol) at 50 °C for 30 min, washed extensively with phosphate-buffered saline, reblocked as described (28Piiper A. Gebhardt R. Kronenberger B. Giannini C.D. Elez R. Zeuzem S. Mol. Pharmacol. 2000; 58: 608-613Crossref PubMed Scopus (21) Google Scholar), and reprobed with appropriate antibodies. To determine Src family kinase activity, cell lysates were either subjected to immunoprecipitation with anti-Src, anti-Yes, or anti-Fyn or directly analyzed by immunoblotting with an antibody recognizing Tyr(P)418-Src. Tyr418 is an autophosphorylation site and thus reflects activation of Src family kinases (37Kmiecik T.E. Shalloway D. Cell. 1987; 49: 65-73Abstract Full Text PDF PubMed Scopus (411) Google Scholar). Blots were stripped and reprobed with appropriate antibodies to verify the amount of immunoprecipitated protein. Ras activation was determined by affinity precipitation of activated Ras from cell lysates using agarose-conjugated Ras-binding domain. Cells were stimulated, washed once with phosphate-buffered saline, and lysed in a buffer (Ras lysis buffer) containing 25 mm Hepes, pH 7.5, 150 mmNaCl, 1% Igepal CA-630, 10 mm MgCl2, 1 mm EDTA, 2% glycerol, and 8 μl of agarose-conjugated Ras-binding domain. After a 30-min incubation at 4 °C, immune complexes were washed three times with Ras lysis buffer, followed by analysis of the immune complexes by anti-Ras immunoblotting. The Raf-1 assay was carried out similarly as described previously (28Piiper A. Gebhardt R. Kronenberger B. Giannini C.D. Elez R. Zeuzem S. Mol. Pharmacol. 2000; 58: 608-613Crossref PubMed Scopus (21) Google Scholar) with some modifications. Cells were stimulated and lysed, and Raf-1 was immunoprecipitated with anti-Raf-1 antibody as described above in the immunoprecipitation and immunoblotting protocol. The immunoprecipitates were washed twice in lysis buffer and once in assay buffer (20 mm MOPS, pH 7.2, 25 mm 2-glycerol phosphate, 5 mm EGTA, 1 mm Na3VO4, 1 mmdithiothreitol). The immune complexes were incubated with inactive fusion proteins glutathione S-transferase-MEK1 (0.4 μg) in a buffer containing 15 mm MOPS, pH 7.2, 20 mm2-glycerol phosphate, 5 mm EGTA, 1 mmNa3VO4, 1 mm dithiothreitol, 150 μm ATP, and 25 μm MgCl2 in a final volume of 45 μl for 30 min at 30 °C with gentle agitation. Reactions were stopped by the addition of 15 μl of 4× sample buffer, followed by boiling of the samples and SDS-PAGE. Phosphorylation of MEK1 by immunoprecipitated Raf-1 was determined by immunoblotting with an antibody specific for the phosphorylated form of MEK. The PKC translocation assay was carried out as described recently (38Huwiler A. Fabbro D. Pfeilschifter J. Biochemistry. 1998; 37: 14556-14562Crossref PubMed Scopus (107) Google Scholar). Cells were incubated for 2 days in serum-free DMEM and stimulated as indicated. Thereafter, cells were washed and scraped into 1 ml of homogenization buffer (20 mm Tris/HCl, pH 7.4, 1 mm EDTA, 1 mm EGTA, 2 mm dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin), lysed by sonication, and centrifuged for 1 h at 100,000 × g at 4 °C. Supernatants were used as a source of cytosolic protein. Pellets were resonicated in 1 ml of homogenization buffer supplemented with 1% Triton X-100 and centrifuged for 1 h at 100,000 × g, yielding the solubilized particulate fractions. Protein concentration was determined, and the fractions were analyzed by immunoblotting. Results are representative of at least three experiments on different occasions giving similar results. The pancreatic cell line AR42J is a well established model for CCK-induced signaling and expresses both CCKA and CCKB receptors, both of which are activated by CCK and stimulate ERK1/2 by a PKC-dependent mechanism (27Dabrowski A. VanderKuur J.A. Carter-Su C. Williams J.A. J. Biol. Chem. 1996; 271: 27125-27129Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Piiper A. Gebhardt R. Kronenberger B. Giannini C.D. Elez R. Zeuzem S. Mol. Pharmacol. 2000; 58: 608-613Crossref PubMed Scopus (21) Google Scholar, 30Daulhac L. Kowalski-Chauvel A. Pradayrol L. Vaysse N. Seva C. Biochem. J. 1997; 325: 383-389Crossref PubMed Scopus (47) Google Scholar, 39Duan R.D. Zheng C.F. Guan K.L. Williams J.A. Am. J. Physiol. 1995; 268: G1060-G1065PubMed Google Scholar). CCK activated ERK1/2 with a maximum after 3 min as determined by immunoblotting of cell lysates with an antibody recognizing specifically the dually phosphorylated activated form of ERK1/2 (Fig.1 A). To determine whether the EGFR is involved in CCK-induced activation of ERK1/2, we tested the effect of AG1478, a well established inhibitor of the EGFR tyrosine kinase, on CCK-induced ERK1/2 activation. As shown in Fig. 1,B and C, AG1478 strongly inhibited CCK-induced ERK1/2 phosphorylation and activation. AG1478 had no effect on ERK1/2 activation in response to HGF, which activates the c-Met/HGF receptor tyrosine kinase, showing the specificity of this compound for the EGFR tyrosine kinase (Fig. 1 B). AG1478 had only a small if any effect on ERK1/2 phosphorylation in response to TPA (Fig.1 D), an activator of PKC. These data indicate that maximal ERK1/2 activation in response to CCK requires EGFR tyrosine kinase, whereas TPA-induced ERK1/2 activation is mainly EGFR-independent in AR42J cells. To differentiate which CCK receptor subtype mediates EGFR tyrosine kinase-dependent ERK1/2 activation, we investigated the effect of AG1478 on ERK1/2 phosphorylation in response to gastrin, which activates only the CCKB receptor. As shown in Fig.1 E, AG1478 strongly inhibited gastrin-induced ERK1/2 phosphorylation, indicating that ERK1/2 activation in response to CCKB receptor stimulation is EGFR-dependent. Certain GPCRs have been reported to induce tyrosine phosphorylation and activation of the EGFR (24Leserer M. Gschwind A. Ullrich A. IUBMB Life. 2000; 49: 405-409Crossref PubMed Scopus (61) Google Scholar, 40Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1327) Google Scholar). To investigate if CCK induces tyrosine phosphorylation of the EGFR, cells were stimulated with CCK or EGF followed by immunoprecipitation of the EGFR and analysis of the immunoprecipitates by anti-phosphotyrosine immunoblotting. As shown in Fig. 2, incubation of the cells with CCK caused rapid tyrosine phosphorylation of the EGFR. CCK-induced tyrosine phosphorylation of the EGFR was, however, considerably smaller than the effect of EGF. The EGFR-specific tyrosine kinase inhibitor AG1478 abolished CCK-induced EGFR tyrosine phosphorylation, indicating that the EGFR tyrosine kinase domain mediates CCK-induced EGFR tyrosine phosphorylation. EGFR activation involves complex formation of the EGFR with the adaptor proteins Grb2 and Shc and tyrosine phosphorylation of Src homology 2 domain-containing substrates such as Shc (17Gutkind J.S. Science's STKE. 2000; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/40/re1)PubMed Google Scholar). Determination of Grb2 immunoreactivity in EGFR immunoprecipitates showed that CCK increased complex formation between the EGFR and Grb2 (Fig. 2 B). Analysis of Shc immunoprecipitates by anti-phosphotyrosine, anti-Grb2, and anti-EGFR immunoblotting revealed that CCK induced tyrosine phosphorylation of p47Shc and p52Shc as well as complex formation of Shc with Grb2 and the EGFR (Fig. 2 C). Taken together, these data demonstrate that CCK induces tyrosine phosphorylation of the EGFR and Shc as well as formation of a complex between Shc, Grb2, and the EGFR, events closely related to EGFR activation. AG1478 abolished CCK-induced complex formation with Grb2 and the tyrosine-phosphorylated EGFR (Fig. 2 B), indicating the involvement of the EGFR tyrosine kinase activation in this process. In contrast, AG1478 had no effect on tyrosine phosphorylation of Shc and its complex formation with Grb2 in response to HGF (data not shown), which activates the c-Met/HGF receptor tyrosine kinase, thus confirming that AG1478 specifically antagonized the effect of the EGFR tyrosine kinase in our system. The addition of GF109203X, a chemical inhibitor of PKC, or loading of the cells with the intracellular calcium chelator 1,2-bis(2-aminophenoxy)etheneN,N,N′,N′-tetraacetic acid acetoxymethyl ester did not prevent CCK-induced tyrosine phosphorylation of the EGFR (data not shown), indicating that an increase in intracellular calcium or activation of PKC is not essential for CCK-induced EGFR transactivation. Moreover, since cleavage of pro-HB-EGF by metalloproteinases has been shown to mediate EGFR transactivation by Gi- and Gq-coupled receptors in several different cell lines (41Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1500) Google Scholar), we investigated the effect of neutralizing anti-HB-EGF antibody and of the HB-EGF inhibitor [Glu52]diphtheria toxin (CRM197) on CCK-induced tyrosine phosphorylation of the EGFR and ERK1/2 activation. Anti-HB-EGF or CRM197 had no effect on CCK responses (data not shown), indicating that HB-EGF is not involved in CCK-induced EGFR and ERK1/2 activation in AR42J cells. Whereas there is agreement concerning the requirement of Ras in EGF- and Gi-coupled receptor-induced ERK1/2 activation, Gq-coupled receptor-induced ERK1/2 activation has been reported to be mediated by both Ras-dependent or -independent pathways (17Gutkind J.S. Science's STKE. 2000; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/40/re1)PubMed Google Scholar, 23Della 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, 42Grosse R. Roelle S. Herrlich A. Höhn J. Gudermann T. J. Biol. Chem. 2000; 275: 12251-12260Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 43Benard O. Naor Z. Seger R. J. Biol. Chem. 2001; 276: 4554-4563Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 44Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). Adenoviral expression of dominant negative Ras was found to have no effect on CCK-induced activation of ERK1/2 in cultured primary pancreatic acinar cells (29Nicke B. Tseng M.J. Fenrich M. Logsdon C.D. Am. J. Physiol. 1999; 276: G499-G506PubMed Google Scholar), and CCK appears to have no significant effect on the amount of activated GTP-bound Ras in freshly prepared pancreatic acinar cells (39Duan R.D. Zheng C.F. Guan K.L. Williams J.A. Am. J. Physiol. 1995; 268: G1060-G1065PubMed Google Scholar, 45Dabrowski A. Groblewski G.E. Schäfer C. Guan K.L. Williams J.A. Am. J. Physiol. 1997; 273: C1472-C1478Crossref PubMed Google Scholar), suggesting that Ras may not be involved in CCK-induced ERK1/2 activation in native pancreatic acinar cells. If CCK-induced activation of ERK1/2 is mediated by EGFR transactivation in pancreatic AR42J cells, CCK-induced ERK1/2 activation should depend on Ras activation. Involvement of Ras in CCK-induced ERK1/2 activation was determined in AR42J cells transiently transfected with dominant-negative Ras (N17Ras) or empty vector. As shown in Fig. 3 A, N17Ras inhibited CCK-induced ERK1/2 phosphorylation. The findings that dominant-negative Ras and AG1478 inhibited CCK-induced ERK1/2 activation suggest involvement of both the EGFR and Ras in CCK-induced ERK1/2 activation. Therefore, we studied the effect of CCK, EGF, and AG1478 on Ras activity. Both CCK and EGF increased the amount of GTP-bound Ras in an AG1478-sensitive fashion (Fig.3 B)" @default.
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