Thrombin Protease-activated Receptor-1 Signals through Gq- and G13-initiated MAPK Cascades Regulating c-Jun Expression to Induce Cell Transformation
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m305709200
ISSN1083-351X
AutoresMaría Julia Marinissen, Joan‐Marc Servitja, Stefan Offermanns, Melvin I. Simon, J. Silvio Gutkind,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoAlthough the ability of G protein-coupled receptors to stimulate normal and aberrant cell growth has been intensely investigated, the precise nature of the molecular mechanisms underlying their transforming potential are still not fully understood. In this study, we have taken advantage of the potent mitogenic effect of thrombin and the focus-forming activity of one of its receptors, protease-activated receptor-1, to dissect how this receptor coupled to Gαi, Gαq/11, and Gα12/13 transduces signals from the membrane to the nucleus to initiate transcriptional events involved in cell transformation. Using endogenous and transfected thrombin receptors in NIH 3T3 cells, ectopic expression of muscarinic receptors coupled to Gαq and Gαi, and chimeric G protein α subunits and murine fibroblasts deficient in Gαq/11, and Gα12/13, we show here that, although coupling to Gαi is sufficient to induce ERK activation, the ability to couple to Gαq and/or Gα13 is necessary to induce c-jun expression and cell transformation. Furthermore, we show that Gαq and Gα13 can initiate the activation of MAPK cascades, including JNK, p38, and ERK5, which in turn regulate the activity of transcription factors controlling expression from the c-jun promoter. We also present evidence that c-Jun and the kinases regulating its expression are integral components of the transforming pathway initiated by protease-activated receptor-1. Although the ability of G protein-coupled receptors to stimulate normal and aberrant cell growth has been intensely investigated, the precise nature of the molecular mechanisms underlying their transforming potential are still not fully understood. In this study, we have taken advantage of the potent mitogenic effect of thrombin and the focus-forming activity of one of its receptors, protease-activated receptor-1, to dissect how this receptor coupled to Gαi, Gαq/11, and Gα12/13 transduces signals from the membrane to the nucleus to initiate transcriptional events involved in cell transformation. Using endogenous and transfected thrombin receptors in NIH 3T3 cells, ectopic expression of muscarinic receptors coupled to Gαq and Gαi, and chimeric G protein α subunits and murine fibroblasts deficient in Gαq/11, and Gα12/13, we show here that, although coupling to Gαi is sufficient to induce ERK activation, the ability to couple to Gαq and/or Gα13 is necessary to induce c-jun expression and cell transformation. Furthermore, we show that Gαq and Gα13 can initiate the activation of MAPK cascades, including JNK, p38, and ERK5, which in turn regulate the activity of transcription factors controlling expression from the c-jun promoter. We also present evidence that c-Jun and the kinases regulating its expression are integral components of the transforming pathway initiated by protease-activated receptor-1. Growth factors acting on cell-surface receptors possessing an intrinsic tyrosine kinase activity can initiate the activation of multiple intracellular signaling pathways, which in turn control key biological processes, including cell proliferation, differentiation, adhesion, and migration and cell fate decisions (reviewed in Ref. 1van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1245) Google Scholar). Subtle alterations in the normal activity of these tyrosine kinase receptors or their intracellular down-stream targets can have dramatic biological consequences, as they may promote the aberrant growth and survival of tumor cells (for review, see Ref. 2Aaronson S.A. Science. 1991; 254: 1146-1153Crossref PubMed Scopus (1158) Google Scholar). The discovery of the mas oncogene, the predicted structure of which resembles that of the G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCRsG protein-coupled receptorsPARprotease-activated receptorERKextracellular signal-regulated kinaseMAPKmitogen-activated protein kinaseCATchloramphenicol acetyltransferaseMEF2myocyte enhancer factor-2HAhemagglutininJNKc-Jun N-terminal kinaseMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseMKKmitogen-activated protein kinase kinaseMEKKmitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinaseGSTglutathione S-transferaseATF2activating transcription factor-2PBSphosphate-buffered salineMOPS4-morpholinepropanesulfonic acidJIP-1JNK-interacting protein-1jAP1c-jun AP1-like response element.1The abbreviations used are: GPCRsG protein-coupled receptorsPARprotease-activated receptorERKextracellular signal-regulated kinaseMAPKmitogen-activated protein kinaseCATchloramphenicol acetyltransferaseMEF2myocyte enhancer factor-2HAhemagglutininJNKc-Jun N-terminal kinaseMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseMKKmitogen-activated protein kinase kinaseMEKKmitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinaseGSTglutathione S-transferaseATF2activating transcription factor-2PBSphosphate-buffered salineMOPS4-morpholinepropanesulfonic acidJIP-1JNK-interacting protein-1jAP1c-jun AP1-like response element. rather than a tyrosine kinase receptor (3Young D. Waitches G. Birchmeier C. Fasano O. Wigler M. Cell. 1986; 45: 711-719Abstract Full Text PDF PubMed Scopus (326) Google Scholar), provided the first evidence that heptahelical receptors can also harbor transforming potential. GPCRs represent the largest family of cell-surface receptors, and they regulate intracellular signaling pathways primarily by interacting with heterotrimeric G proteins composed of α, β, and γ subunits. Upon receptor activation, there is a conformational change that promotes the exchange of GDP bound to the α subunit for GTP and the release of βγ dimers, thereby initiating a series of signaling events that culminate in a wide variety of cellular responses (4Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (938) Google Scholar, 5Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1585) Google Scholar). Constitutively activated mutant receptors (6Allen L.F. Lefkowitz R.J. Caron M.G. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11354-11358Crossref PubMed Scopus (292) Google Scholar) and receptors persistently activated by agonists (7Julius D. Livelli T.J. Jessell T.M. Axel R. Science. 1989; 244: 1057-1062Crossref PubMed Scopus (287) Google Scholar, 8Gutkind J.S. Novotny E.A. Brann M.R. Robbins K.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4703-4707Crossref PubMed Scopus (261) Google Scholar) were found to cause cell transformation. Furthermore, paracrine and autocrine stimulation of GPCRs by tumor-released agonists has been implicated in different types of neoplasias such as small cell lung carcinoma and prostate and gastric cancer (for review, see Refs. 9Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar and 10Heasley L.E. Oncogene. 2001; 20: 1563-1569Crossref PubMed Scopus (180) Google Scholar), thus highlighting a role for the large GPCR family in carcinogenesis. However, the molecular mechanisms underlying the transforming potential of GPCRs are still not fully understood. G protein-coupled receptors protease-activated receptor extracellular signal-regulated kinase mitogen-activated protein kinase chloramphenicol acetyltransferase myocyte enhancer factor-2 hemagglutinin c-Jun N-terminal kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase mitogen-activated protein kinase kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase glutathione S-transferase activating transcription factor-2 phosphate-buffered saline 4-morpholinepropanesulfonic acid JNK-interacting protein-1 c-jun AP1-like response element. G protein-coupled receptors protease-activated receptor extracellular signal-regulated kinase mitogen-activated protein kinase chloramphenicol acetyltransferase myocyte enhancer factor-2 hemagglutinin c-Jun N-terminal kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase mitogen-activated protein kinase kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase glutathione S-transferase activating transcription factor-2 phosphate-buffered saline 4-morpholinepropanesulfonic acid JNK-interacting protein-1 c-jun AP1-like response element. Using a retroviral expression library approach to identify novel oncogenes from a mouse myeloid progenitor cell line, Whitehead et al. (11Whitehead I. Kirk H. Kay R. Mol. Cell. Biol. 1995; 15: 704-710Crossref PubMed Google Scholar) identified several independent cDNAs encoding murine PAR-1, a thrombin-stimulated GPCR. Thrombin is a serine protease that exerts multiple physiological effects (12Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2129) Google Scholar). Among them, the best known function of thrombin is its key role in blood coagulation. In addition, thrombin can act on many cell types, eliciting a large variety of cellular responses, including the regulation of cell proliferation and invasion and tumor growth (13O'Brien P.J. Molino M. Kahn M. Brass L.F. Oncogene. 2001; 20: 1570-1581Crossref PubMed Scopus (200) Google Scholar, 14Van Obberghen-Schilling E. Vouret-Craviari V. Chen Y.H. Grall D. Chambard J.C. Pouyssegur J. Ann. N. Y. Acad. Sci. 1995; 766: 431-441Crossref PubMed Scopus (16) Google Scholar). At least three receptors for thrombin, PAR-1, PAR-3, and PAR-4, have been cloned thus far and found to belong to the GPCR superfamily (12Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2129) Google Scholar). Rather than being a direct agonist for these receptors, thrombin acts by cleaving an Arg–Ser bond in their N-terminal extracellular domain, thereby generating a new N terminus that functions as a tethered agonist. The potent transforming potential of PAR-1 suggests that its deregulated expression can promote the aberrant activation of growth-promoting pathways. This receptor is known to couple effectively to the Gαi, Gαq, and Gα13 families of G protein α subunits (reviewed in Ref. 12Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2129) Google Scholar). Indeed, activated forms of the Gαq and Gα13 families can themselves transform NIH 3T3 cells (15Kalinec G. Nazarali A.J. Hermouet S. Xu N. Gutkind J.S. Mol. Cell. Biol. 1992; 12: 4687-4693Crossref PubMed Google Scholar, 16Xu N. Bradley L. Ambdukar I. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6741-6745Crossref PubMed Scopus (174) Google Scholar), supporting that at least these G proteins and their coupled receptors can promote cell-transforming pathways. Of interest, thrombin can potently induce the nuclear expression of members of the AP1 transcription factor family (17Trejo J. Chambard J.C. Karin M. Brown J.H. Mol. Cell. Biol. 1992; 12: 4742-4750Crossref PubMed Scopus (60) Google Scholar), which is composed of members of the Jun and Fos families of nuclear proteins that bind as Jun dimers or Jun-Fos heterodimers to DNA sequences known as 12-O-tetradecanoylphorbol-13-acetate response elements on the regulatory region of target genes, thereby enhancing or inhibiting their expression (18Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3262) Google Scholar, 19Shaulian E. Karin M. Nat. Cell Biol. 2002; 4: E131-E136Crossref PubMed Scopus (2205) Google Scholar). In particular, thrombin can induce the rapid expression of the c-jun proto-oncogene (17Trejo J. Chambard J.C. Karin M. Brown J.H. Mol. Cell. Biol. 1992; 12: 4742-4750Crossref PubMed Scopus (60) Google Scholar), which is a critical molecule in the regulation of cell proliferation and neoplastic transformation (18Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3262) Google Scholar, 19Shaulian E. Karin M. Nat. Cell Biol. 2002; 4: E131-E136Crossref PubMed Scopus (2205) Google Scholar, 20Kovary K. Bravo R. Mol. Cell. Biol. 1991; 11: 4466-4472Crossref PubMed Scopus (393) Google Scholar). However, the nature of the intracellular signaling route by which thrombin stimulates c-jun expression and whether its protein product, c-Jun, contributes to the transforming ability of PAR-1 are still unknown. In this study, we have explored the molecular mechanisms by which endogenously expressed or overexpressed thrombin receptors can transduce signals from the membrane into nuclear events participating in cell transformation. For this work, we have used endogenous and transfected thrombin receptors, ectopic expression of muscarinic receptors coupled to Gαq and Gαi (m1 and m2, respectively), and chimeric G protein α subunits and murine fibroblasts deficient in Gαq/11 and Gα12/13. We show here that, although coupling to Gαi is sufficient to induce ERK activation, the ability to couple to Gαq and/or Gα13 is necessary to induce c-jun expression and cell transformation. Furthermore, we show that Gαq and Gα13 can initiate the activation of MAPK cascades regulating the activity of transcription factors controlling the activity of the c-jun promoter. We also present evidence that c-Jun and kinases regulating its expression are integral components of the transforming pathways initiated by PAR-1. NIH 3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% calf serum. Human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Mouse embryonic fibroblasts from wild-type and Gαq/11 and Gα12/13 knockout animals were kept in the same media supplemented with 1 mm sodium pyruvate and nonessential amino acids. A plasmid encoding a luciferase gene driven by a murine wild-type c-jun promoter, pJLuc, was kindly provided by R. Prywes (21Han T.H. Prywes R. Mol. Cell. Biol. 1995; 15: 2907-2915Crossref PubMed Scopus (167) Google Scholar). Plasmids pJC6, pJC9, pJTX, pJSX, and pJSTX are pBLCAT3-based reporter constructs carrying a chloramphenicol acetyltransferase (CAT) gene controlled by the murine full-length c-jun promoter and its mutants, as previously described (22Han T.H. Lamph W.W. Prywes R. Mol. Cell. Biol. 1992; 12: 4472-4477Crossref PubMed Scopus (90) Google Scholar). A pGL3 reporter plasmid (Promega) containing the jAP1 (TGACATCA) and MEF2 (CTATTTTTAG) sites from the murine c-jun promoter, designated pjAP1-MEF2, was engineered by inserting the oligonucleotide sequence 5′-GTACCGTCGACTCGGGGTGACATCATGGGCTATTTTTAGGGAGATC-3′ as an Asp718/BglII fragment upstream of an SV40 minimal promoter and a luciferase gene. Reporter plasmids with mutations in the jAP1 (pjAP1m-MEF2) or MEF2 (pjAP1-MEF2m) site and a double mutant (pjAP1m-MEF2m) and a plasmid carrying two jAP1 sites were prepared following the same strategy. A similar reporter plasmid carrying an MEF2 site has been previously reported (23Coso O.A. Montaner S. Fromm C. Lacal J.C. Prywes R. Teramoto H. Gutkind J.S. J. Biol. Chem. 1997; 272: 20691-20697Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Expression vectors for HA-ERK2, HA-JNK, HA-ERK5, HA-p38α, HA-p38γ (ERK6), pCEFL-MEK5DD, pCEFL-MEK5AA, pCEV29-MEKEE, pCEFL-MEKAA, pCEFL-GST-MKK6, pCEFL-GST-MKK6KR, pCEFL-MEKK, and constitutively activated small G proteins Ras, RhoA, Rac1, and Cdc42 have also been described (23Coso O.A. Montaner S. Fromm C. Lacal J.C. Prywes R. Teramoto H. Gutkind J.S. J. Biol. Chem. 1997; 272: 20691-20697Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 24Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1566) Google Scholar, 25Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (190) Google Scholar, 26Teramoto H. Salem P. Robbins K.C. Bustelo X.R. Gutkind J.S. J. Biol. Chem. 1997; 272: 10751-10755Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). H-RasV12 and a dominant-negative mutant of RhoA, RhoAN19 have been described (24Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1566) Google Scholar). Gal4 fusion proteins, including the transactivating domains of ATF2 (amino acids 1–96) and MEF2A (amino acids 266–360) and a TATA-Gal4-driven luciferase reporter plasmid (pGal4-Luc), and bacterially expressed GST-ATF2 and GST-MEF2C fusion proteins were described previously (25Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (190) Google Scholar). pcDNAIII-MKK3b-WT and its constitutively activated (EE) and dominant-negative (AA) mutants were kindly provided by J. Han (27Han J. Wang X. Jiang Y. Ulevitch R.J. Lin S. FEBS Lett. 1997; 403: 19-22Crossref PubMed Scopus (55) Google Scholar). pCEFL-AU5-JunTAM67 has been described (28Chiariello M. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2000; 20: 1747-1758Crossref PubMed Scopus (168) Google Scholar). PAR-1, kindly provided by Dr. L. F. Brass, was subcloned into the pCEFL vector as an EcoRI fragment. DNA encoding a Gα13i5 chimera, in which five amino acids at the C terminus of Gαq were replaced with the corresponding sequence of Gαi2, was prepared by PCR amplification using pcDNA3-HA-Gα13 (29Fukuhara S. Marinissen M.J. Chiariello M. Gutkind J.S. J. Biol. Chem. 2000; 275: 21730-21736Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) as a template, and the resulting DNA was subcloned into the pCEFL-HA vector (25Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (190) Google Scholar) as a BglII/EcoRI fragment. A DNA plasmid encoding a Gαqi5 chimeric protein, in which five amino acids at the C terminus of Gαq were replaced with the corresponding sequence of Gαi2, was a gift from Dr. B. R. Conklin (30Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (606) Google Scholar). Expression plasmids for constitutively activated forms of Gαq, Gαi2, Gαs, Gα12, and Gα13; G protein β and γ subunits; and m1 and m2 muscarinic receptors were described previously (24Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1566) Google Scholar, 25Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (190) Google Scholar, 29Fukuhara S. Marinissen M.J. Chiariello M. Gutkind J.S. J. Biol. Chem. 2000; 275: 21730-21736Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 31Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Transient transfections of NIH 3T3 and human embryonic kidney 293T cells cultured in 6-well plates were performed using the LipofectAMINE Plus reagent (Invitrogen) following the manufacturer's instructions. Stable transfections of NIH 3T3 cells expressing the m1 or m2 receptor (NIH-m1 and NIH-m2 cells, respectively; each expressing ∼100,000 receptors/cell) (8Gutkind J.S. Novotny E.A. Brann M.R. Robbins K.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4703-4707Crossref PubMed Scopus (261) Google Scholar, 32Stephens E.V. Kalinec G. Brann M.R. Gutkind J.S. Oncogene. 1993; 8: 19-26PubMed Google Scholar) and the m2 receptor plus the Gα13i5 or Gαqi5 chimera (NIH-m2Gα13i5 and NIH-m2Gαqi5 cells, respectively) were performed using the same protocol as described above, and cells were selected in culture medium containing Geneticin (750 μg/ml). Cells were grown to 70% confluence in 10-cm plates and serum-starved for 20 h. They were left untreated (controls) or were treated with 1 mm carbachol or 5 units/ml thrombin for different times. After treatment, they were washed with cold PBS, and total RNA was extracted by homogenization in TRIzol (Invitrogen) according to the manufacturer's specifications. For Northern blotting, 10–20 μg of total RNA was fractionated on 2% formaldehyde-agarose gels, transferred to nylon membranes, and hybridized with murine full-length 32P-labeled c-jun cDNA probe prepared using a Prime-a-Gene labeling system (Promega). Accuracy in gel loading and transfer was confirmed by fluorescence under UV light upon ethidium bromide staining. Luciferase Assays—Cells were transfected with different expression plasmids together with 0.1 μg of each reporter plasmid and 0.01 μg of pRL-null (a plasmid expressing luciferase from Renilla reniformis) as an internal control. In all cases, the total amount of plasmid DNA was adjusted with pcDNAIII-β-gal (a plasmid expressing β-galactosidase). Firefly and Renilla luciferase activities present in cell lysates were assayed using a dual-luciferase reporter system (Promega), and light emission was quantitated using a Monolight 2010 luminometer (Analytical Luminescence Laboratory) as specified by the manufacturer. CAT Assays—NIH 3T3 cells were transfected with different expression plasmids together with 0.1 μg of each reporter plasmid and 0.5 μg of pcDNAIII-β-gal. After a 24-h incubation, cells were washed and lysed using reporter lysis buffer (Promega). CAT activity was assayed in cell extracts by incubation for 1 h in the presence of 0.25 μCi of [14C]chloramphenicol (100 mCi/mmol) and 200 μg/ml butyryl-CoA in 0.25 m Tris-HCl (pH 7.4). Labeled butyrylated products were extracted using a 1:2 mixture of xylenes and 2,6,10,14-tetramethylpentadecane (Sigma), and incorporated radioactivity was counted by liquid scintillation. Cells were seeded at 70–80% confluence and transfected with expression vectors for HA-tagged kinases alone or in combination with different upstream molecules. After transfection, cells were cultured for 24 h and incubated in serum-free medium overnight for ERK2 and ERK5 and for 2 h for JNK, p38α, and p38γ. Cells were washed with cold PBS and lysed at 4 °C in buffer containing 25 mm HEPES (pH 7.5), 0.3 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, 20 mm β-glycerophosphate, 1 mm vanadate, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 20 μg/ml leupeptin. Cleared lysates containing HA-tagged kinases were immunoprecipitated at 4 °C for 2 h with anti-HA monoclonal antibody HA.11 (Berkeley Antibody Co.). Immunocomplexes were recovered with protein G-Sepharose (Amersham Biosciences). Beads were washed three times with PBS containing 1% Nonidet P-40 and 2 mm vanadate, once with 100 mm Tris (pH 7.5) and 0.5 m LiCl, and once with kinase reaction buffer (12.5 mm MOPS (pH 7.5), 12.5 mm β-glycerophosphate, 7.5 mm MgCl2, 0.5 mm EGTA, 0.5 mm sodium fluoride, and 0.5 mm vanadate). Samples were resuspended in 30 μl of kinase reaction buffer containing 1 μCi of [γ-32P]ATP/reaction and 20 μm unlabeled ATP. After 20 min at 30 °C, the reactions were terminated by addition of 10 μlof5× Laemmli buffer. In vitro kinase assays were performed using 1.5 μg/μl myelin basic protein (Sigma) for ERK2 and 1 μg of purified, bacterially expressed GST-ATF2 for JNK, p38α, and p38γ and GST-MEF2C for ERK5 as substrates, as indicated. Samples were analyzed by SDS-gel electrophoresis on 12% (or 15% for myelin basic protein) acrylamide gels, and autoradiography was performed with the aid of an intensifying screen. HA-tagged immunoprecipitates from transiently transfected NIH 3T3 cells carrying HA-MAPK, HA-JNK, HA-ERK5, HA-p38α, and HA-p38γ cDNAs were analyzed by Western blotting after SDS-PAGE using anti-HA monoclonal antibody HA.11. Gαq and Gα11 were detected by rabbit anti-Gαq/11 antibody (Santa Cruz Biotechnology, Inc.). Gα12 and Gα13 were detected by a mixture of anti-Gα12 antibody (Santa Cruz Biotechnology, Inc.) and anti-Gα13 antibody (33Xu N. Voyno-Yasenetskaya T. Gutkind J.S. Biochem. Biophys. Res. Commun. 1994; 201: 603-609Crossref PubMed Scopus (88) Google Scholar). Proteins were visualized by enhanced chemiluminescence detection (Amersham Biosciences) using horseradish peroxidase-coupled goat anti-mouse and anti-rabbit IgGs as the secondary antibodies (Cappel). NIH 3T3 cells and these cells stably transfected with the m1 or m2 receptor were seeded on glass coverslips and transfected using LipofectAMINE Plus reagent as described above. 24-h serum-starved cells were treated with 1 mm carbachol and 5 units/ml thrombin, washed twice with 1× PBS, and then fixed and permeabilized with 4% formaldehyde and 0.5% Triton X-100 in 1× PBS for 10 min. After washing with PBS, cells were blocked with 1% bovine serum albumin and incubated with the indicated primary antibodies for 1 h. c-Jun was detected using rabbit anti-c-Jun antibody (Santa Cruz Biotechnology, Inc.). Following incubation, cells were washed three times with 1× PBS and incubated with the corresponding fluorescein isothiocyanate-conjugated secondary antibodies (1:200 dilution; Jackson ImmunoResearch Laboratories, Inc.). Coverslips were washed three times and mounted in Vectashield mounting medium with 4,6-diamidino-2-phenylindole (Vector Laboratories, Inc.) and viewed using a Zeiss Axiophot photomicroscope equipped with epifluorescence. Immunofluorescence was photographed using Eastman Kodak TMAX 3200 film. NIH 3T3 cells were transfected by the calcium phosphate precipitation technique with different expression plasmids together with 1 μg of pcDNAIII-β-gal, adjusting the total amount of plasmid DNA with empty vector. The day after transfection, cells were washed with medium supplemented with 5% calf serum and then maintained in the same medium until foci were scored, 2–3 weeks later. Duplicate plates were fixed with 1× PBS containing 2% (v/v) formaldehyde and 0.2% (v/v) glutaraldehyde and stained at 37 °C for β-galactosidase activity with 1× PBS containing 2 mm MgCl2, 5 mm K3Fe(CN)6, 5 mm K4Fe(CN)6 and 0.1% 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) to evaluate the transfection efficiency. Human PAR-1 and m1, but Not m2, Harbor Oncogenic Potential—PAR-1, a GPCR activated by thrombin and other proteases (34Martin C.B. Mahon G.M. Klinger M.B. Kay R.J. Symons M. Der C.J. Whitehead I.P. Oncogene. 2001; 20: 1953-1963Crossref PubMed Scopus (109) Google Scholar, 35Camerer E. Huang W. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5255-5260Crossref PubMed Scopus (605) Google Scholar) that is linked to Gαi, Gαq, and Gα12/13 subunits (12Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2129) Google Scholar), was cloned as an oncogene using an expression library approach (11Whitehead I. Kirk H. Kay R. Mol. Cell. Biol. 1995; 15: 704-710Crossref PubMed Google Scholar). Indeed, as previously reported for the murine PAR-1 gene, human PAR-1 readily induced the appearance of foci of transformation after 2–3 weeks of culture, as shown in Fig. 1. Interestingly, PAR-1 was even more potent than a Gq-coupled receptor, the m1 muscarinic receptor, which transforms NIH 3T3 effectively when cells are cultured in the presence of the cholinergic agonist carbachol (8Gutkind J.S. Novotny E.A. Brann M.R. Robbins K.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4703-4707Crossref PubMed Scopus (261) Google Scholar). In contrast, m2 receptors that are coupled to Gi proteins do not transform cells in culture, thus suggesting that Gαq and Gα13, but not Gαi, can stimulate transforming pathways in these murine fibroblasts. c-jun Expression Is Stimulated by Transforming GPCRs, and a Dominant Inhibitory Mutant of c-Jun Prevents Their Focus-forming Activity—To begin addressing the molecular mechanism underlying the transforming ability of these GPCRs, we first examined whether they could stimulate the ERK signaling route, a key component of cell growth-promoting pathways (36Pearson G. Robinson F. Beers Gibson T. Xu B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3528) Google Scholar), using wild-type NIH 3T3 cells, which express PAR-1 endogenously, and the same cells stably transfected with the m1 or m2 receptor (NIH-m1 and NIH-m2 cells, respectively). As shown in Fig. 2, agonist addition to NIH 3T3 cells resulted in the potent activation of ERK. However, in repeated experiments, there were no remarkable differences in the strength and duration of the ERK signal elicited by transforming and non-transforming GPCRs, suggesting that the ability to stimulate ERK does not correlate with their transforming activity. In the search for the molecular mechanisms underlying the distinct biological activities of these GPCRs, we focused our attention on nuclear responses, in particular on the expression of the transcription factor c-Jun, the function of which has often been associated with malignant conversion (reviewed in Ref. 19Shaulian E. Karin M. Nat. Cell Biol. 2002; 4: E131-E136Crossref PubMed Scopus (2205) Google Scholar). As shown in Fig. 2 (lower panels), activation of PAR-1 and m1 receptors induced the rapid accumulation of c-jun mRNA. Consistently, the expression of the c-Jun protein was also increased as revealed by the nuclear c-Jun immunostaining of thrombin- and carbachol-stimulated cells (Fig. 2B). Of interest, carbachol did not induce c-jun message or c-Jun protein expression in NIH-m2 cells, suggesting that only transforming GPCRs can stimulate this particular nuclear response. We next investigated whether the ability to trigger c-Jun expression and transformation by PAR-1 and the m1 receptor is two functionally related events. As shown in Fig. 3, transformation induced by these receptors was potently inhibited by the coexpression of a dominant-negative mutant form of c-Jun, c-Jun TAM67 (37Brown P.H. Alani R. Preis L.H. Szabo E. Birrer M.J. Oncogene. 1993; 8: 877-886PubMed Google Scholar
Referência(s)