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• W2040969916 abstract "The small GTP-binding proteins Ras, Rac, and Cdc42 link protein-tyrosine kinases with mitogen-activated protein kinase (MAPK) signaling cascades. Ras controls the activation of extracellular signal-regulated kinases (ERKs), while Rac and Cdc42 regulate the c-Jun N-terminal kinases (JNKs). In this study, we investigated whether small G protein/MAPK cascades contribute to signal transduction by transforming variants of c-Fes, a nonreceptor tyrosine kinase implicated in cytokine signaling and myeloid differentiation. First, we investigated the effects of dominant-negative small G proteins on Rat-2 fibroblast transformation by a retroviral homolog of c-Fes (v-Fps) and by c-Fes activated via N-terminal addition of the v-Src myristylation signal (Myr-Fes). We observed that dominant-negative Ras, Rac, and Cdc42 inhibited v-Fps- and Myr-Fes-induced growth of Rat-2 cells in soft agar, indicating that activation of these small GTP-binding proteins is required for fibroblast transformation by Fps/Fes tyrosine kinases. To determine whether MAPK pathways are activated downstream of these small G proteins, we measured ERK and JNK activity in the v-Fps- and Myr-Fes-transformed Rat-2 cells. Both ERK and JNK activities were elevated in the transformed cells, suggesting that these pathways are involved in cellular transformation. Dominant-negative mutants of Ras (but not Rac or Cdc42) specifically inhibited ERK activation by v-Fps and Myr-Fes, demonstrating that ERK activation occurs exclusively downstream of Ras. All three dominant-negative small G proteins inhibited JNK activation by v-Fps and Myr-Fes, indicating that JNK activation by these tyrosine kinases requires both Ras and Rho family GTPases. These data demonstrate that multiple small G protein/MAPK cascades are involved in downstream signal transduction by Fps/Fes tyrosine kinases. The small GTP-binding proteins Ras, Rac, and Cdc42 link protein-tyrosine kinases with mitogen-activated protein kinase (MAPK) signaling cascades. Ras controls the activation of extracellular signal-regulated kinases (ERKs), while Rac and Cdc42 regulate the c-Jun N-terminal kinases (JNKs). In this study, we investigated whether small G protein/MAPK cascades contribute to signal transduction by transforming variants of c-Fes, a nonreceptor tyrosine kinase implicated in cytokine signaling and myeloid differentiation. First, we investigated the effects of dominant-negative small G proteins on Rat-2 fibroblast transformation by a retroviral homolog of c-Fes (v-Fps) and by c-Fes activated via N-terminal addition of the v-Src myristylation signal (Myr-Fes). We observed that dominant-negative Ras, Rac, and Cdc42 inhibited v-Fps- and Myr-Fes-induced growth of Rat-2 cells in soft agar, indicating that activation of these small GTP-binding proteins is required for fibroblast transformation by Fps/Fes tyrosine kinases. To determine whether MAPK pathways are activated downstream of these small G proteins, we measured ERK and JNK activity in the v-Fps- and Myr-Fes-transformed Rat-2 cells. Both ERK and JNK activities were elevated in the transformed cells, suggesting that these pathways are involved in cellular transformation. Dominant-negative mutants of Ras (but not Rac or Cdc42) specifically inhibited ERK activation by v-Fps and Myr-Fes, demonstrating that ERK activation occurs exclusively downstream of Ras. All three dominant-negative small G proteins inhibited JNK activation by v-Fps and Myr-Fes, indicating that JNK activation by these tyrosine kinases requires both Ras and Rho family GTPases. These data demonstrate that multiple small G protein/MAPK cascades are involved in downstream signal transduction by Fps/Fes tyrosine kinases. Members of the Ras superfamily of small GTP-binding proteins regulate diverse cellular functions, including cell proliferation, differentiation, and organization of the actin cytoskeleton (1Katz M.E. McCormick F. Curr. Opin. Genet. Dev. 1997; 7: 75-79Crossref PubMed Scopus (273) Google Scholar, 2McCormick F. Wittinghofer A. Curr. Opin. Biotechnol. 1996; 7: 449-456Crossref PubMed Scopus (76) Google Scholar, 3Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (686) Google Scholar, 4Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5164) Google Scholar). One key function of these small GTP-binding proteins is to link upstream protein-tyrosine kinases with mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MBP, myelin basic protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MEK, MAPK/ERK kinase; MEKK, MEK kinase.1The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MBP, myelin basic protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MEK, MAPK/ERK kinase; MEKK, MEK kinase. pathways. Perhaps the best characterized MAPK pathway involves Raf/MEK/ERK activation downstream of Ras (5Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1156) Google Scholar, 6Morrison D.K. Cutler Jr., R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (531) Google Scholar, 7Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2266) Google Scholar). Raf binds to the GTP-bound, active form of Ras at the plasma membrane and becomes activated. Active Raf phosphorylates and activates the dual specificity MAPK kinases MEK1 and MEK2 which in turn activate the MAPKs ERK1 and ERK2. Activated ERKs can then translocate to the nucleus and phosphorylate a number of transcription factors. In this way, the Ras/ERK pathway provides a critical link between tyrosine kinase signal transduction and transcriptional events in the nucleus. In addition to the Ras/ERK cascade, recent work has revealed parallel small G protein/MAPK pathways to the nucleus. Small GTPases of the Rho family including Rac and Cdc42 (but not Rho itself) are linked to a kinase cascade leading to the activation of the c-Jun N-terminal kinase (JNK), an ERK-related serine kinase that specifically phosphorylates and activates the c-Jun transcription factor (8Vojtek A.B. Cooper J.A. Cell. 1995; 82: 527-529Abstract Full Text PDF PubMed Scopus (252) Google Scholar, 9Coso 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 (1553) Google Scholar, 10Minden A. Lin A. Claret F.-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1441) Google Scholar). This cascade is also linked to a related kinase known as p38. Both p38 and JNK are activated in response to cytokines as well as cellular stress. Rac and Cdc42 are connected to JNK via intermediate MEKK (analogous to Raf) and SEK (analogous to MEK) activities. The Rac/Cdc42/JNK cascade also includes additional kinases upstream of MEKK, such as p21-activated kinase 65, which may represent the direct targets of active Rac and Cdc42 (7Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2266) Google Scholar, 8Vojtek A.B. Cooper J.A. Cell. 1995; 82: 527-529Abstract Full Text PDF PubMed Scopus (252) Google Scholar). Thus in addition to their roles in the regulation of cytoskeletal architecture (3Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (686) Google Scholar, 4Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5164) Google Scholar, 11Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3678) Google Scholar), Rac and Cdc42 can control transcriptional events in the nucleus as well. Activation of small GTPase/MAPK pathways is a common feature of both normal and oncogenic protein-tyrosine kinase signal transduction (12Hunter T. Cell. 1997; 88: 333-346Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). The biochemical connection between tyrosine kinases and the activation of small GTPases downstream often involves recruitment and activation of guanine nucleotide exchange factors. In the case of Ras, autophosphorylation of activated tyrosine kinases can create binding sites for the SH2 domains of adaptor proteins such as Shc and Grb-2, which serve as intermediates in the recruitment of the SOS guanine nucleotide exchange factor to the plasma membrane, focal adhesions, or other sites of tyrosine kinase signaling (13Schlessinger J. Trends Biochem. Sci. 1993; 18: 273-275Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 14Hanks S.K. Polte T.R. BioEssays. 1996; 19: 130-140Google Scholar). The translocation of SOS to these sites allows for Ras activation and subsequent stimulation of the MAPK pathway downstream. In the case of Rho family GTPases, tyrosine phosphorylation of the guanine nucleotide exchange factor may be essential for activation. For example, recent studies have shown that tyrosine phosphorylation stimulates the exchange activity of Vav, leading to activation of the JNK pathway (15Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (676) Google Scholar, 16Han J.W. Das B. Wei W. Van Aelst L. Mosteller R.D. Khosravi-Far R. Westwick J.K. Der C.J. Broek D. Mol. Cell. Biol. 1997; 17: 1346-1353Crossref PubMed Scopus (276) Google Scholar). In addition, products of the phosphatidylinositol 3′-kinase pathway have recently been shown to regulate the exchange activity of Vav and SOS toward Rho family GTPases (17Han J.W. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krystal G. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (708) Google Scholar, 18Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (387) Google Scholar). The Fps/Fes family of cytoplasmic protein-tyrosine kinases are expressed primarily in myeloid hematopoietic cells and the vascular endothelium (19Smithgall T.E. Yu G. Glazer R.I. J. Biol. Chem. 1988; 263: 15050-15055Abstract Full Text PDF PubMed Google Scholar, 20Feldman R.A. Gabrilove J.L. Tam J.P. Moore M.A.S. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2379-2383Crossref PubMed Scopus (109) Google Scholar, 21MacDonald I. Levy J. Pawson T. Mol. Cell. Biol. 1985; 5: 2543-2551Crossref PubMed Scopus (96) Google Scholar, 22Greer P. Haigh J. Mbamalu G. Khoo W. Bernstein A. Pawson T. Mol. Cell. Biol. 1994; 14: 6755-6763Crossref PubMed Scopus (93) Google Scholar, 23Haigh J. McVeigh J. Greer P. Cell Growth Differ. 1996; 7: 931-944PubMed Google Scholar). Human c-Fes has been linked to multiple cytokine receptors (24Hanazono Y. Chiba S. Sasaki K. Mano H. Yazaki Y. Hirai H. Blood. 1993; 81: 3193-3196Crossref PubMed Google Scholar, 25Hanazono Y. Chiba S. Sasaki K. Mano H. Miyajima A. Arai K. Yazaki Y. Hirai H. EMBO J. 1993; 12: 1641-1646Crossref PubMed Scopus (144) Google Scholar, 26Izuhara K. Feldman R.A. Greer P. Harada N. J. Biol. Chem. 1994; 269: 18623-18629Abstract Full Text PDF PubMed Google Scholar, 27Matsuda T. Fukada T. Takahashi-Tezuka M. Okuyama Y. Fujitani Y. Hanazono Y. Hirai H. Hirano T. J. Biol. Chem. 1995; 270: 11037-11091Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 28Brizzi M.F. Aronica M.G. Rosso A. Bagnara G.P. Yarden Y. Pegoraro L. J. Biol. Chem. 1996; 271: 3562-3567Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and may play a direct role in myeloid differentiation and angiogenesis (22Greer P. Haigh J. Mbamalu G. Khoo W. Bernstein A. Pawson T. Mol. Cell. Biol. 1994; 14: 6755-6763Crossref PubMed Scopus (93) Google Scholar, 29Yu G. Smithgall T.E. Glazer R.I. J. Biol. Chem. 1989; 264: 10276-10281Abstract Full Text PDF PubMed Google Scholar, 30Manfredini R. Balestri R. Tagliafico E. Trevisan F. Pizzanelli M. Grande A. Barbieri D. Zucchini P. Citro G. Franceschi C. Ferrari S. Blood. 1997; 89: 135-145Crossref PubMed Google Scholar). However, the critical downstream signaling pathways regulating the biological activities of Fes have not been fully characterized. Previous studies have identified Bcr, Ras GAP, and Shc as substrates for c-Fes and its transforming viral homolog, v-Fps (31Ellis C. Moran M. McCormick F. Pawson T. Nature. 1990; 343: 377-381Crossref PubMed Scopus (523) Google Scholar, 32McGlade J. Cheng A. Pelicci G. Pelicci P.G. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8869-8873Crossref PubMed Scopus (237) Google Scholar, 33Hjermstad S.J. Briggs S.D. Smithgall T.E. Biochemistry. 1993; 32: 10519-10525Crossref PubMed Scopus (25) Google Scholar, 34Maru Y. Peters K.L. Afar D.E.H. Shibuya M. Witte O.N. Smithgall T.E. Mol. Cell. Biol. 1995; 15: 835-842Crossref PubMed Google Scholar, 35Li J. Smithgall T.E. J. Biol. Chem. 1996; 271: 32930-32936Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). All of these proteins have been linked to the regulation of the Ras and Rho families of small GTPases. For example, Bcr has a C-terminal GAP domain for Rac and Cdc42 (36Diekmann D. Brill S. Garrett M.D. Totty N. Hsuan J. Monfries C. Hall C. Lim L. Hall A. Nature. 1991; 351: 400-402Crossref PubMed Scopus (352) Google Scholar) as well as a central guanine nucleotide exchange region with homology to Dbl (37Chuang T.H. Xu X. Kaartinen V. Heisterkamp N. Groffen J. Bokoch G.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10282-10286Crossref PubMed Scopus (149) Google Scholar, 38Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (463) Google Scholar). Previous work from our laboratory has shown that tyrosine phosphorylation of Bcr in v-Fps-transformed fibroblasts induces its association with Grb-2/SOS, suggesting that Bcr may couple Fps/Fes tyrosine kinases to both the Ras and Rho signaling pathways downstream (34Maru Y. Peters K.L. Afar D.E.H. Shibuya M. Witte O.N. Smithgall T.E. Mol. Cell. Biol. 1995; 15: 835-842Crossref PubMed Google Scholar). In this study, we provide evidence that small G proteins of both the Ras and Rho families are required for Fps/Fes-induced fibroblast transformation and that small G protein activation is coupled to the ERK and JNK pathways downstream. These data provide strong evidence that activation of multiple small G protein/MAPK cascades is critical to Fps/Fes signal transduction. The full-length cDNAs for N17Ras, V12N17Rac, and N17Cdc42 containing a C-terminal Myc epitope tag were the generous gift of Dr. Alan Hall, MRC, London (39Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1053) Google Scholar). These cDNAs were subcloned into the retroviral vector pSRαMSVtkneo (40Muller A.J. Young J.C. Pendergast A.M. Pondel M. Landau R.N. Littman D.R. Witte O.N. Mol. Cell. Biol. 1991; 11: 1785-1792Crossref PubMed Scopus (353) Google Scholar) and into the mammalian expression vector pCDNA3 (Invitrogen). Addition of the N-terminal myristylation signal sequence of v-Src to the N-terminal region of c-Fes to create the transforming oncoprotein Myr-Fes was accomplished using a polymerase chain reaction-based approach and will be described in detail elsewhere. 2J. A. Rogers, N. A. Dunham, and T. E. Smithgall, manuscript in preparation. Construction of a full-length human c-fes cDNA with a C-terminal FLAG epitope tag is described elsewhere (42Rogers J.A. Read R.D. Li J. Peters K.L. Smithgall T.E. J. Biol. Chem. 1996; 271: 17519-17525Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). A similar polymerase chain reaction-based procedure was used to add the FLAG coding sequence to the C-terminal region of v-Fps and Myr-Fes. The coding sequence of the v-src oncogene was amplified by polymerase chain reaction from a Rous sarcoma virus template obtained from the ATCC and subcloned into the pSRαMSVtkneo and pCDNA3 expression vectors described above. Retroviral stocks were prepared by transient transfection of 293T cells (43Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2274) Google Scholar) with the pSRαMSVtkneo constructs together with an ecotropic packaging vector (40Muller A.J. Young J.C. Pendergast A.M. Pondel M. Landau R.N. Littman D.R. Witte O.N. Mol. Cell. Biol. 1991; 11: 1785-1792Crossref PubMed Scopus (353) Google Scholar). Briefly, 293T cells were grown to 50–80% confluence in 10-cm2 dishes and transfected using a calcium phosphate method as described previously (42Rogers J.A. Read R.D. Li J. Peters K.L. Smithgall T.E. J. Biol. Chem. 1996; 271: 17519-17525Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Culture medium containing the recombinant retroviruses was harvested from 2 to 5 days post-transfection. Viral supernatants were pooled and stored in frozen aliquots at −80 °C. Negative control retroviruses were prepared using the parent vector alone. Rat-2 cells were infected with recombinant retroviruses in the presence of polybrene as described (44Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). G418 was added to a final concentration of 0.8 mg/ml 48 h later, and the cells were grown under G418 selection for 2–4 weeks. The expression of N17Ras, V12N17Rac, and N17Cdc42 was confirmed by immunoprecipitation followed by immunoblotting with the anti-Myc monoclonal antibody, 9E10 (39Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1053) Google Scholar). Stable expression of c-Fes was confirmed by immunoprecipitation and immunoblotting with the anti-FLAG monoclonal antibody, M2 (Kodak Scientific Imaging Systems). Rat-2 cells (5 × 104) stably expressing N17Ras, V12N17Rac, N17Cdc42, or theneo marker alone were plated in 6-well dishes. Cells were infected with v-Src, v-Fps, or Myr-Fes retroviruses 24 h later as described above and incubated for 2 additional days. The cells were then trypsinized, and 1 × 104 cells were replated in triplicate in Dulbecco's modified Eagle's medium containing 0.3% agar and 20% fetal calf serum. Transformed colonies were counted after 2 weeks. To establish Rat-2 cell lines stably expressing each of the tyrosine kinases, single transformed colonies were isolated from the soft agar plates and grown in Dulbecco's modified Eagle's medium with 5% fetal calf serum. The expression of v-Src was confirmed by immunoprecipitation and immunoblotting with an anti-Src antibody (N-16, Santa Cruz Biotechnology); Myr-Fes and v-Fps expression were confirmed with the M2 anti-FLAG antibody. Rat-2 or 293T cells were incubated overnight in serum-free medium prior to harvesting. Clarified cell lysates were prepared as described previously (35Li J. Smithgall T.E. J. Biol. Chem. 1996; 271: 32930-32936Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). For some experiments, a solid phase JNK assay was employed (45Westwick J.K. Brenner D.A. Methods Enzymol. 1995; 255: 342-359Crossref PubMed Scopus (46) Google Scholar). Cell lysates (75 μg of protein) were incubated with 2 μg of an immobilized GST-Jun fusion protein (Jun amino acids 1–72) in a final volume of 300 μl of HBB buffer (20 mm HEPES, pH 7.7, 50 mmNaCl, 0.1 mm EDTA, 2.5 mm MgCl2, and 0.05% Triton X-100) for 3 h at 4 °C. The GST-Jun fusion protein was then washed with four 1.0-ml aliquots of HBB buffer and incubated with 35 μl of kinase buffer (20 mm HEPES, pH 7.5, 20 mm β-glycerophosphate, 10 mmMgCl2, 40 μm ATP, 1 mmdithiothreitol, and 50 μm Na3VO4) containing 5 μCi of [γ-32P]ATP (3000 Ci/mmol, NEN Life Science Products) for 20 min at 30 °C. Phosphorylated proteins were separated by SDS-PAGE and visualized by storage phosphorimaging (Molecular Dynamics PhosphorImager). Alternatively, JNK activity was assessed by immune complex kinase assay. Clarified cell lysates (75 μg) were incubated with 0.8 μg of JNK1 antibody (C-17, Santa Cruz Biotechnology) in HBB buffer at 4 °C for 3 h. Immune complexes were precipitated with protein G-Sepharose, washed with HBB buffer, and divided into two aliquots. One aliquot was tested for JNK activity using the in vitro kinase assay conditions described above for the solid phase assay. The second aliquot was immunoblotted with the JNK antibody to verify equivalent levels of the JNK protein in each reaction. Clarified cell lysates (75 μg) were incubated with MAPK lysis buffer (40 mm Tris-HCl, pH 7.4, 120 mm NaCl, 5 mm EDTA, 2 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 0.5% Triton X-100, 10 mm NaF, 2 mm Na3VO4, 10 mm sodium pyrophosphate, 10 μg/ml leupeptin, and 5 μg/ml aprotinin) and immunoprecipitated with ERK1 and ERK2 antibodies (0.4 μg each; Santa Cruz Biotechnology). Immune complexes were precipitated with protein G-Sepharose, washed three times with 1.0 ml of MAPK lysis buffer and twice with MAP kinase buffer (40 mm Tris-HCl, pH 7.4, 20 mm MgCl2, 2 mm MnCl2, 25 μm ATP, 0.5 mm dithiothreitol), and divided into two aliquots. One aliquot was assayed for ERK activity in 40 μl of MAP kinase buffer containing 2 μg of myelin basic protein (MBP) and 5 μCi of [γ-32P]ATP. Phosphorylation reactions were incubated for 20 min at 30 °C, and labeled MBP was separated by SDS-PAGE and visualized by storage phosphorimaging. The second aliquot was immunoblotted with the ERK1 antibody to verify equivalent amounts of ERK protein in each immunoprecipitate. In this study, we used fibroblast transformation as a model system to study small G protein/MAPK signaling by v-Fps and Myr-Fes, two activated forms of the Fps/Fes tyrosine kinase family. v-Fps is a potent transforming homolog of c-Fes found in the chicken sarcoma virus, FSV (46Shibuya M. Hanafusa H. Cell. 1982; 30: 787-795Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 47Hanafusa T. Wang L.-H. Anderson S.M. Karess R.E. Hayward W.S. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3009-3013Crossref PubMed Scopus (71) Google Scholar). Myr-Fes is a form of human c-Fes that is activated by N-terminal addition of the v-Src myristylation signal sequence, which results in membrane targeting (22Greer P. Haigh J. Mbamalu G. Khoo W. Bernstein A. Pawson T. Mol. Cell. Biol. 1994; 14: 6755-6763Crossref PubMed Scopus (93) Google Scholar). To investigate whether Ras, Rac, and Cdc42 are involved in fibroblast transformation by fps/fes oncogenes, we inhibited the function of these small G proteins in Rat-2 cells with dominant-inhibitory mutants in which the Ser residue at position 17 was changed to Asn (N17 mutants). These mutants bind preferentially to GDP and act as dominant inhibitors of endogenous small G protein function presumably by blocking their access to exchange factors (39Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1053) Google Scholar, 48Cai H. Szeberenyi J. Cooper G.M. Mol. Cell. Biol. 1990; 10: 5314-5323Crossref PubMed Scopus (180) Google Scholar, 49Qiu R.-G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (809) Google Scholar). Populations of Rat-2 fibroblasts stably expressing each of these mutants were infected with v-Fps and Myr-Fes retroviruses and assayed for soft agar colony formation as a measure of transformation. As a control, we also examined transformation by v-Src in these cell lines, because previous work has shown that dominant-negative mutants of Ras and Rac interfere with transformation by this oncogenic tyrosine kinase (10Minden A. Lin A. Claret F.-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1441) Google Scholar). As shown in Fig. 1 A, we observed that v-Src, v-Fps, and Myr-Fes consistently produced fewer transformed colonies from cells expressing the dominant-negative small G proteins as compared with control cells. Control immunoblots demonstrate equivalent expression of v-Src, v-Fps, or Myr-Fes in all four cell lines (Fig. 1 B). These results indicate that fibroblast transformation by oncogenically activated Fps/Fes tyrosine kinases as well as v-Src requires the activation of Ras and Rho family GTPases. To verify that the dominant-negative small GTPases did not induce a general suppression of cellular proliferation, the growth rates of the Rat-2 cell populations stably expressing each of the mutants were compared with that of the control cells expressing only the drug-selection marker. As shown in Fig.2 A, Rat-2 cells stably expressing dominant-negative Ras, Rac, and Cdc42 grew at the same rate and to the same saturation density as the vector control cells. Expression of the dominant-negative small G proteins was verified by immunoprecipitation followed by immunoblotting (Fig.2 B). As an alternative experimental approach to investigate the effect of dominant-negative small G proteins on Fps/Fes-mediated transformation, soft agar colony-forming assays were performed on Rat-2 fibroblasts following co-infection with recombinant retroviruses carrying the transforming oncogenes and the dominant-negative GTPase mutants. As shown in Fig. 3, this approach also led to strong suppression of transforming activity. These results are in good agreement with those observed using the Rat-2 cell populations stably expressing the dominant-negative mutants (Fig. 1). Taken together, these data support the conclusion that Ras, Rac, and Cdc42 play essential roles in transformation signaling downstream from oncogenic Fps/Fes tyrosine kinases as well as v-Src. Activation of many tyrosine kinase families is linked to stimulation of ERK activity (see Introduction). To determine whether the ERK pathway is activated by Fps/Fes kinases, we measured ERK activity in v-Fps- and Myr-Fes-transformed Rat-2 cell lines derived from single transformed soft-agar colonies. We also examined ERK activation in a Rat-2 cell line stably expressing c-Fes and in control G418-resistant Rat-2 cells. Endogenous ERK1 and ERK2 were immunoprecipitated from clarified cell lysates and an in vitro kinase assay was conducted with MBP and [γ-32P]ATP as co-substrates. As shown in Fig.4, ERK activity was increased 6–7-fold in the v-Fps- and Myr-Fes-transformed cells. In contrast, stable expression of c-Fes, which is nontransforming, resulted in only a 2-fold increase in ERK activity. These results suggest that transformation by activated forms of Fps/Fes kinases may require sustained ERK activation. In contrast, no activation of ERK was observed in the v-Src transformants, suggesting that maintenance of the v-Src-transformed phenotype does not require constitutive MAPK activation. The surprising result that transformation by v-Src was not associated with constitutive ERK activation led us to investigate the effects of v-Src and Fps/Fes kinases on this pathway under transient expression conditions. For these experiments, we employed the human embryonic kidney cell line, 293T (43Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2274) Google Scholar), and the in vitro ERK assay described above. As shown in Fig. 4, both v-Src and v-Fps stimulated ERK activity in this system. However, both c-Fes and Myr-Fes were unable to stimulate ERK activity in 293T cells. This lack of activation may reflect the tight regulation of both c-Fes and Myr-Fes tyrosine kinase activity in this cell line, despite the high level of protein expression (42Rogers J.A. Read R.D. Li J. Peters K.L. Smithgall T.E. J. Biol. Chem. 1996; 271: 17519-17525Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar).2 A nearly identical pattern of ERK activation was also observed in Rat-2 cells transiently expressing v-Fps, Myr-Fes, c-Fes, and v-Src, validating the use of 293T cells as a model system for the investigation of small G protein/MAPK signaling following transient expression of these tyrosine kinases (data not shown). Recent studies have shown that JNK is activated downstream of Rac and Cdc42 (see Introduction). To determine whether the JNK pathway is activated by v-Fps and Myr-Fes, JNK activity was measured in the v-Src-, v-Fps-, and Myr-Fes-transformed Rat-2 cells. Endogenous JNK was precipitated from cell lysates with an immobilized GST-Jun fusion protein and kinase activity of substrate-bound JNK was assayed by addition of [γ-32P]ATP. As shown in Fig.5, JNK activity was elevated in the v-Src-, v-Fps-, and Myr-Fes- transformed Rat-2 cells, suggesting that constitutive JNK activation may contribute to transformation by these oncogenes. In contrast, no increase in JNK activity was observed in nontransformed Rat-2 cells stably expressing c-Fes. Thus, JNK activation correlates with the transforming activity of these tyrosine kinase oncogenes. Endogenous JNK activity was also measured in 293T cells transiently expressing v-Src, v-Fps, Myr-Fes, and c-Fes. As shown in Fig. 5, expression of v-Src and v-Fps readily stimulated JNK activity in 293T cells. In contrast to Rat-2 cells, JNK activity was only modestly increased by Myr-Fes in 293T cells. Similar differences were observed for Myr-Fes-induced activation of ERK in Rat-2 versus 293T cells (Fig. 4), and are consistent with the hypothesis that cells responding with activation of these pathways are selected for during transformation. c-Fes also slightly activated JNK over background in 293T cells, suggesting that JNK may be more sensitive than ERK to the low level of Fes tyrosine kinase activity resulting from the overexpression of Fes in this cell line (42Rogers J.A. Read R.D. Li J. Peters K.L. Smithgall T.E. J. Biol. Chem. 1996; 271: 17519-17525Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Given that ERKs are often activated downstream of Ras, we tested whether dominant-negative Ras selectively blocks ERK activation in response to transforming signals from v-Fps and Myr-Fes. Populations of Rat-2 cells stab" @default.
• W2040969916 created "2016-06-24" @default.
• W2040969916 creator A5048630922 @default.
• W2040969916 creator A5051930564 @default.
• W2040969916 date "1998-05-01" @default.
• W2040969916 modified "2023-10-16" @default.
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