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Mammalian Sprouty Proteins Inhibit Cell Growth and Differentiation by Preventing Ras Activation

2001; Elsevier BV; Volume: 276; Issue: 49 Linguagem: Inglês

10.1074/jbc.m108234200

1083-351X

Isabelle Groß, Bhramdeo Bassit, Miriam Benezra, Jonathan D. Licht,

Hippo pathway signaling and YAP/TAZ

Sprouty was genetically identified as an antagonist of fibroblast growth factor signaling during tracheal branching in Drosophila. In this study, we provide a functional characterization of mammalian Sprouty1 and Sprouty2. Sprouty1 and Sprouty2 inhibited events downstream of multiple receptor tyrosine kinases and regulated both cell proliferation and differentiation. Using NIH3T3 cell lines conditionally expressing Sprouty1 or Sprouty2, we found that these proteins specifically inhibit the Ras/Raf/MAP kinase pathway by preventing Ras activation. In contrast, activation of the phosphatidylinositol 3-kinase pathway was not affected by Sprouty1 or Sprouty2. We further showed that Sprouty1 and Sprouty2 do no prevent the formation of a SNT·Grb2·Sos complex upon fibroblast growth factor stimulation, yet block Ras activation. Taken together, these results establish mammalian Sprouty proteins as important negative regulators of growth factor signaling and suggest that Sprouty proteins act downstream of the Grb2·Sos complex to selectively uncouple growth factor signals from Ras activation and the MAP Kinase pathway. Sprouty was genetically identified as an antagonist of fibroblast growth factor signaling during tracheal branching in Drosophila. In this study, we provide a functional characterization of mammalian Sprouty1 and Sprouty2. Sprouty1 and Sprouty2 inhibited events downstream of multiple receptor tyrosine kinases and regulated both cell proliferation and differentiation. Using NIH3T3 cell lines conditionally expressing Sprouty1 or Sprouty2, we found that these proteins specifically inhibit the Ras/Raf/MAP kinase pathway by preventing Ras activation. In contrast, activation of the phosphatidylinositol 3-kinase pathway was not affected by Sprouty1 or Sprouty2. We further showed that Sprouty1 and Sprouty2 do no prevent the formation of a SNT·Grb2·Sos complex upon fibroblast growth factor stimulation, yet block Ras activation. Taken together, these results establish mammalian Sprouty proteins as important negative regulators of growth factor signaling and suggest that Sprouty proteins act downstream of the Grb2·Sos complex to selectively uncouple growth factor signals from Ras activation and the MAP Kinase pathway. fibroblast growth factor receptor tyrosine kinases nucleotide(s) platelet-derived growth factor calf serum nerve growth factor green fluorescent protein polyacrylamide gel electrophoresis glutathioneS-transferase phosphatidylinositol 3-kinase mitogen-activated protein kinase Ras-binding domain GTPase activating protein Normal development requires precise spatial and temporal regulation of signal transduction pathways involved in cell growth and differentiation. Negative control of growth factor response is achieved both by restriction of the incoming signal itself and induction of counter regulatory mechanisms affecting the propagation of the signal. The expression of many inhibitors are induced by the pathway they eventually antagonize, providing the potential for a tight autoregulation (for a review, see Ref. 1Perrimon N. McMahon A.P. Cell. 1999; 97: 13-16Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Recently, sprouty(spry) was identified by genetic studies as such an inhibitor (2Hacohen N. Kramer S. Sutherland D. Hiromi Y. Krasnow M.A. Cell. 1998; 92: 253-263Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar). Spry was originally described as an antagonist of Breathless FGF1 receptor signaling during tracheal branching in Drosophila Loss of function mutations of spry led to excessive FGF signaling and ectopic branching, whereas engineered overexpression of spry blocked the branching (2Hacohen N. Kramer S. Sutherland D. Hiromi Y. Krasnow M.A. Cell. 1998; 92: 253-263Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar). As other groups reported genetic interactions between spry and several different receptor tyrosine kinases (RTK) in multiple contexts, it became clear that spry was a general inhibitor of RTK signaling during Drosophiladevelopment (3Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 4Kramer S. Okabe M. Hacohen N. Krasnow M.A. Hiromi Y. Development. 1999; 126: 2515-2525Crossref PubMed Google Scholar, 5Reich A. Sapir A. Shilo B. Development. 1999; 126: 4139-4147Crossref PubMed Google Scholar, 6Taguchi A. Sawamoto K. Okano H. Genetics. 2000; 154: 1639-1648PubMed Google Scholar). Through a data base search, three human genes were identified with sequence similarity to Drosophila spry (2Hacohen N. Kramer S. Sutherland D. Hiromi Y. Krasnow M.A. Cell. 1998; 92: 253-263Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar) and a fourth family member was described in the mouse (7de Maximy A.A. Nakatake Y. Moncada S. Itoh N. Thiery J.P. Bellusci S. Mech. Dev. 1999; 81: 213-216Crossref PubMed Scopus (159) Google Scholar). Mammalianspry genes are expressed in highly restricted patterns in the embryo during early development and in many adult tissues (7de Maximy A.A. Nakatake Y. Moncada S. Itoh N. Thiery J.P. Bellusci S. Mech. Dev. 1999; 81: 213-216Crossref PubMed Scopus (159) Google Scholar, 8Minowada G. Jarvis L.A. Chi C.L. Neubuser A. Sun X. Hacohen N. Krasnow M.A. Martin G.R. Development. 1999; 126: 4465-4475Crossref PubMed Google Scholar, 9Chambers D. Mason I. Mech. Dev. 2000; 91: 361-364Crossref PubMed Scopus (110) Google Scholar). In most tissues, the different family members appear to be co-regulated and their expression shows a close correlation with known sites of FGF signaling. Mammalian Spry proteins may be key regulators of several developmental processes, including lung branching morphogenesis, midbrain and anterior hindbrain patterning, and limb chondrocyte differentiation (8Minowada G. Jarvis L.A. Chi C.L. Neubuser A. Sun X. Hacohen N. Krasnow M.A. Martin G.R. Development. 1999; 126: 4465-4475Crossref PubMed Google Scholar, 9Chambers D. Mason I. Mech. Dev. 2000; 91: 361-364Crossref PubMed Scopus (110) Google Scholar, 10Tefft J.D. Lee M. Smith S. Leinwand M. Zhao J. Bringas Jr., P. Crowe D.L. Warburton D. Curr. Biol. 1999; 9: 219-222Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Genetic and biochemical analysis performed by Casci et al.(3Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar) suggested that Drosophila Spry negatively regulates the Ras pathway, but the molecular mechanism of this inhibitory activity was not determined (5Reich A. Sapir A. Shilo B. Development. 1999; 126: 4139-4147Crossref PubMed Google Scholar). All Spry proteins share a unique, highly conserved, cysteine-rich C-terminal domain. This domain was shown to be necessary for the membrane translocation of Spry by a yet unknown mechanism (2Hacohen N. Kramer S. Sutherland D. Hiromi Y. Krasnow M.A. Cell. 1998; 92: 253-263Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar, 3Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 11Lim J. Wong E.S. Ong S.H. Yusoff P. Low B.C. Guy G.R. J. Biol. Chem. 2000; 275: 32837-32845Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The N-terminal portion of the Spry proteins is less conserved as it exhibits only 25–37% identity among the different mouse family members. These sequence differences could be responsible for functional divergence among the Spry proteins. In particular, the size difference between Drosophila and much smaller mammalian Spry N-terminal regions is intriguing. Our laboratory has studied the Wilms Tumor 1(WT1) gene, a tumor suppressor gene involved in embryonic kidney development, for several years. We performed a representative difference analysis screen to isolate transcriptional target genes of WT1. One of the genes identified was mousespry1. 2I. Gross, D. Morrison, K. Georgas, M. English, S. Hosono, M. Wei, D. Hyink, P. Wilson, B. Tycko. M. Little, and J. Licht, manuscript in preparation. To model the role of mammalian Spry during development and tumorigenesis, we established stable inducible Spry1 and Spry2 NIH3T3 cell lines. We demonstrated that Spry1 and Spry2 antagonized growth factor signaling by specifically inhibiting the Ras/Raf/MAP kinase pathway. We methodically examined the inhibitory effect of Spry on the different components of the signal transduction cascade and identified the activation of Ras as the target of Spry activity. We showed that Spry1 and Spry2 can inhibit both proliferation of NIH3T3 cells and differentiation of PC12 cells. These results suggest that Spry proteins, by limiting RTK signaling, play an important role in development and growth control. The nucleotide (nt) positions for murinespry1 and spry2 are as listed under AF176903 andAF176905, respectively. The mouse spry1 cDNA was reconstituted by ligation of a StuI polymerase chain reaction fragment (nt 402–1007)2 to an EST fragment (GenBankTMAA591484, nt 962–2489) in the pSPORT1 vector (Life Technologies). Nt 481–1469 of spry1 were subclonedEcoRI-XbaI in-frame with the Flag tag in the pcF2H vector (gift of D. Sassoon (12Relaix F. Wei X.J. Wu X. Sassoon D.A. Nat. Genet. 1998; 18: 287-291Crossref PubMed Scopus (130) Google Scholar)). Mouse spry2 cDNA was isolated by reverse transcriptase-polymerase chain reaction using primers to human spry2 (nt 391–411, nt 1330–1350 of GenBankTMAF039843) and RNA extracted from mouse podocytes (13Mundel P. Reiser J. Zuniga Mejia Borja A. Pavenstadt H. Davidson G.R. Kriz W. Zeller R. Exp. Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (768) Google Scholar). The EcoRI-XbaI fragment was subcloned in the pcF2H vector. Flag-Spry1 or Flag-Spry2 were used as polymerase chain reaction templates to subclone spry1 andspry2 into the pCEV29 vector (14Chan A.M. Miki T. Meyers K.A. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7558-7562Crossref PubMed Scopus (86) Google Scholar). For the Tet-off Spry expression system, Flag-Spry1/2 fragments were subcloned into the pTRE vector (CLONTECH). Flag-tagged spry1 (nt 1–1469) or spry2 (nt 1–1245) were subcloned into the MIGRI vector (15Pear W.S. Miller J.P. Xu L. Pui J.C. Soffer B. Quackenbush R.C. Pendergast A.M. Bronson R. Aster J.C. Scott M.L. Baltimore D. Blood. 1998; 92: 3780-3792Crossref PubMed Google Scholar) to obtain Spry1/2-IRES-GFP. The Myc-Grb2 construct was made by linking nt 79–729 of human grb2 (GenBankTMNM-002086) to the Myc tag within pcDNA 3–1/Myc-His (Invitrogen). SRE-Luc (R. Prywes) contains nt −355 to −297 of the murine c-fos promoter (16Wang Y. Falasca M. Schlessinger J. Malstrom S. Tsichlis P. Settleman J. Hu W. Lim B. Prywes R. Cell Growth Differ. 1998; 9: 513-522PubMed Google Scholar). NF-κB-Luc (A. Chan) contains 3 NF-κB elements in the pGL2 Luciferase (CLONTECH) (17Kimmelman A. Tolkacheva T. Lorenzi M.V. Osada M. Chan A.M. Oncogene. 1997; 15: 2675-2685Crossref PubMed Scopus (78) Google Scholar). All novel constructs were sequenced. NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and transfected with LipofectAMINE Plus (Life Technologies). PC12 cells were grown on tissue culture dishes coated with poly-l-lysine (0.001%, Sigma) in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 10% horse serum. Tet-off NIH3T3 cells (gift of I. Gelman (18Shockett P. Difilippantonio M. Hellman N. Schatz D.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6522-6526Crossref PubMed Scopus (343) Google Scholar)) were cultured in histidine-deficient Dulbecco's modified Eagle's high glucose completed with 0.5 mm l-histidinol (Sigma), 4 mm sodium bicarbonate, 2 mm l-glutamine, 10% calf serum, and 0.5 μg/ml tetracycline. Spry1 and Spry2 Tet-off NIH3T3 cell lines were established by transfection of the Tet-off cells with pTRE-Spry1 or pTRE-Spry2 or an empty vector and selection in 0.5 μg/ml G418-sulfate (Roche Molecular Biochemicals). 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and were transfected with Superfect (Qiagen Inc.). All growth factors (mouse NGF 2.5 S, recombinant human PDGF-BB, recombinant murine tumor necrosis factor-α, recombinant human epidermal growth factor, and recombinant human bFGF) were from Life Technologies. Total RNA was extracted using TRIzol (Life Technologies), fractionated on a formaldehyde-agarose gel (20 μg/lane), and transferred to Hybond-N (Amersham Pharmacia Biotech). cDNA fragments (spry2 nt 529–799, spry1 nt 1345–1652, and c-fos, from R. Kraus, nt 754–1265 of GenBankTMX06769) were 32P-labeled using the RediprimeTM II kit (Amersham Pharmacia Biotech) and purified with microSpinTM G-50 columns (Amersham Pharmacia Biotech). All hybridizations were performed under standard conditions. The blots were quantified by phosphoimaging using ImageQuant software (Molecular Dynamics). 106 NIH3T3 cells (100 mm plates) were transfected by pCEV29, pCEV29-Spry1, or pCEV29-Spry2. After 3 days, cells were diluted (1/25) and selected with G418 (0.5 μg/ml) for 2 to 3 weeks. Colonies were stained with Giemsa and counted on 5 plates for each vector. To measure DNA synthesis, control or Spry1/2 inducible cells (70,000 cell/well of 24-well plates) were starved for 24 h (0.3% CS with tetracycline) before stimulation with 10% CS for 24 h in the presence or absence of tetracycline. During the last 4 h, cells were labeled with 1 μCi/ml [methyl-3H]thymidine (PerkinElmer Life Sciences). After 2 phosphate-buffered saline washes, cells were fixed with methanol and lysed with 0.25% SDS, 0.25m NaOH. The lysates were neutralized with 0.2 mHCl and mixed with scintillation liquid (ScintiverseTM, Fisher). [3H]Thymidine incorporation was measured using a Beckman LS 6500 scintillation counter. Apoptosis was examined with fluorescence microscopy using annexin V-fluorescein isothiocyanate labeling (Annexin V-FLUOS, Roche) in NIH3T3 cells transfected for 24 h with control, spry1, spry2, orbax expression vectors (1 μg) along with a vector expressing a red fluorescent protein (100 ng, DsRed,CLONTECH). Proliferating PC12 cells were transfected with bicistronic IRES-GFP plasmids using LipofectAMINE 2000 Reagent (Life Technologies). After 48 h, differentiation was induced by dilution of the cells (1/15) and treatment with NGF (50 ng/ml) or bFGF (20 ng/ml) in Dulbecco's modified Eagle's medium containing 1% horse serum, 2 mm l-glutamine. After 3 days, cells were observed with phase-contrast microscopy to check the general efficiency of the differentiation. The differentiation of the transfected cells was examined with fluorescence microscopy to visualize GFP expression. The efficiency of differentiation of the transfected cells was quantified by counting the cells exhibiting neurite outgrowth (twice the diameter of the cell) among the GFP positive cells. Cells were lysed (20 mmHEPES, pH 7.5, 10 mm EGTA, pH 8, 40 mmβ-glycerophosphate, 1% Nonidet P-40, 2.5 mmMgCl2, 2 mm sodium orthovanadate, and one tablet of CompleteTM protease inhibitors (Roche Molecular Biochemicals) for 50 ml). 20–50 μg of proteins were separated by SDS-PAGE and transferred to Immobilon-P nylon membranes (Millipore). The following primary antibodies were used: FLAG M2 mouse monoclonal (Sigma); phospho-p44/42 MAP kinase (Thr202/Tyr204) E10 mouse monoclonal (NEB); Erk2 (K-23) rabbit polyclonal (Santa Cruz); phospho-Akt (Ser473) rabbit polyclonal (New England Biolabs); Akt rabbit polyclonal (New England Biolabs); phospho-Gsk-3β (Ser9) rabbit polyclonal (New England Biolabs); phospho-Mek1/2 (Ser217/221) rabbit polyclonal (New England Biolabs); Mek1 (12-B) rabbit polyclonal (Santa Cruz); c-Raf-1 mouse monoclonal (Transduction Laboratories); Ras clone Ras10 mouse monoclonal (Upstate Biotechnology); Tyr(P) clone 4G10 mouse monoclonal (Upstate Biotechnology); FRS2 (H-91) rabbit polyclonal (Santa Cruz); c-Myc clone 9E10 mouse monoclonal (Santa Cruz); GST (Z-5) rabbit polyclonal (Santa Cruz); AU5 mouse monoclonal (Babco); Sos 1 (C-23) rabbit polyclonal (Santa Cruz); Grb2 mouse monoclonal (Transduction Laboratories). For the detection, we used one of the following conjugated antisera: peroxidase goat anti-rabbit IgG (H+L) (Roche Molecular Biochemicals) at 1/7000; peroxidase goat anti-mouse IgG (H+L) (Roche Molecular Biochemicals) at 1/7000; Finally, the membranes were developed using ECL (Amersham Pharmacia Biotech). The assay was performed with a Raf-1 Immunoprecipitation Kinase Assay kit purchased from Upstate Biotechnology as indicated by the manufacturer. Raf-1, RBD, and agarose was purchased from Upstate Biotechnology and the assay was performed as recommended with 0.5–1 mg of cellular lysate and 10 μl of GST-Raf-1 RBD, agarose conjugate (10 μg) at 4 °C for 30 min. The proteins bound were subjected to SDS-PAGE and immunoblot analysis as described above. Cells were lysed (50 mmTris, pH 7.5, 1% Nonidet P-40, 150 mm NaCl, 5 mm EDTA, and one tablet of CompleteTM protease inhibitors (Roche Molecular Biochemicals) for 50 ml) and precleared by centrifugation. Lysates (0.5–2 mg) were incubated with 1–2 μg of the precipitating antibody overnight at 4 °C with gentle rocking. During the last hour, 50 μl of Protein G- or Protein A-agarose beads (50% slurry, Roche Molecular Biochemicals) were added. The beads were collected by centrifugation, washed 3 times with 1 ml of lysis buffer, and boiled in 50 μl of Laemmli sample buffer. The immunoprecipitates were fractionated by SDS-PAGE and analyzed by immunoblot as described above. To characterize mammalian Spry, we chose NIH3T3 cells which have been extensively utilized as a model for cell proliferation, oncogenesis, and growth factor signaling. We first examined the expression of spry1 and spry2 in proliferating NIH3T3 cells (Fig. 1 A). Northern blot analysis revealed a major transcript of about 2.5 kilobases for both genes but endogenous spry2 expression was always significantly higher than spry1 expression, which was barely detectable in NIH3T3 cells. We next stimulated NIH3T3 cells with FGF after serum starvation and examined the spry1 andspry2 expression patterns. As presented in Fig.1 B, FGF clearly stimulated spry2 expression. This 3-fold up-regulation was transient (1 to 5 h), occurred withoutde novo protein synthesis (data not shown) and was maximal 2 h after treatment. Similar results were obtained with PDGF (data not shown). Surprisingly, in the same cells, expression ofspry1 was down-regulated (about 50%) by FGF and PDGF (Fig.1 B and data not shown). These results show that endogenousspry1 and spry2 are differentially regulated by growth factors in NIH3T3, suggesting that they may play specific roles in these cells. In addition, as endogenous spry2 andspry1 expression are rapidly modified upon growth factor treatment, NIH3T3 cells appear to constitute an appropriate model to study the effect of Spry1 and Spry2 on growth factor signaling. As growth factors stimulate proliferation of fibroblasts, we examined the effect of spry overexpression on cell growth using a colony suppression assay. NIH3T3 cells transfected by spry1 orspry2 formed about 50% fewer colonies upon selection than the cells transfected with a control vector (Fig.2, A and B). This indicates that Spry1 and Spry2 inhibit either cell growth or induce apoptosis. Using annexin V labeling (early marker for apoptosis (19Bossy-Wetzel E. Green D.R. Methods Enzymol. 2000; 322: 15-18Crossref PubMed Google Scholar)), we did not detect significant levels of apoptosis in NIH3T3 cells transiently transfected with spry1 or spry2 (Fig.2 C). To determine how Spry1 and Spry2 inhibit cell growth, we established NIH3T3 stable cell lines in which expression of mousespry1 or spry2 is induced by the removal of tetracycline from the culture medium (Fig. 2 D). Using [3H]thymidine incorporation assays, we compared the DNA synthesis induced by serum in the absence or presence of Spry1 or Spry2. Fig. 2 E shows that Spry1, as well as Spry2, markedly reduced (50 to 80%) the level of DNA synthesis induced by serum. Together, these data indicate that expression of Spry proteins reduces cell growth by limiting DNA synthesis, and not by inducing cell death. We next examined the ability of Spry1 and Spry2 to inhibit differentiation induced by growth factors. We chose as a model the PC12 pheochromocytoma cells, in which neurite outgrowth can be induced by NGF or FGF via the Ras pathway (20Hagag N. Halegoua S. Viola M. Nature. 1986; 319: 680-682Crossref PubMed Scopus (302) Google Scholar, 21Szeberenyi J. Cai H. Cooper G.M. Mol. Cell. Biol. 1990; 10: 5324-5332Crossref PubMed Scopus (276) Google Scholar, 22Wood K.W. Sarnecki C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (662) Google Scholar). Proliferating PC12 cells were transiently transfected with spry1 or spry2bicistronic GFP vectors, which co-express Spry1 or Spry2 and the GFP and thus allowed the identification of the transfected cells. Two days after transfection, cells were treated by NGF or FGF to trigger differentiation. After 3 days, most of the cells had developed neurites (Fig. 3 A). When the transfected cells were specifically visualized by immunofluorescence, we saw a striking difference between the control and the Spry1 or Spry2 expressing cells (Fig. 3 B). Indeed, at least 80% of the cells expressing Spry1 or Spry2 were unable to differentiate upon FGF or NGF treatment compared with the control transfected cells (Fig.3 C). The neurites present in spry-transfected cells were shorter and showed reduced branching compared with the control cells. This shows that Spry1 and Spry2 can inhibit differentiation induced by growth factors. However, endogenous expression of neither spry1 nor spry2 could be detected in PC12 cells by Northern blot, even upon FGF, NGF, or epidermal growth factor treatment (data not shown). This suggests that Spry1 and Spry2 are not usually involved in the differentiation or the growth of PC12 cells yet the Spry proteins could affect the consequences of signaling through the NGF/FGF receptors. It is possible that other members of the spry family may be up-regulated by growth factors and control this neuronal differentiation. Nevertheless, this result led us to focus on the NIH3T3 cells for further experiments. Since we had shown that Spry1 and Spry2 could reduce proliferation of NIH3T3 cells, we used this cell line to determine the molecular mechanism by which Spry proteins exert their inhibitory activity. We first examined their effect on nuclear targets of growth factor signaling. NIH3T3 cells were transfected with a serum response element-luciferase reporter gene which is as a model for growth factor regulation of transcription (reviewed in Refs. 23Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1854) Google Scholar and 24Treisman R. EMBO J. 1995; 14: 4905-4913Crossref PubMed Scopus (347) Google Scholar). Fig.4 A shows that the expression of the reporter gene is stimulated upon growth factor treatment but in the presence of Spry proteins, this stimulation is blunted, especially in the case of Spry2. When this assay was repeated with a NF-κB response element-luciferase reporter gene, which can be stimulated by growth factor treatment through the Ras/Raf/MAP kinase pathway (25Devary Y. Rosette C. DiDonato J.A. Karin M. Science. 1993; 261: 1442-1445Crossref PubMed Scopus (577) Google Scholar), expression was also inhibited by Spry proteins (Fig. 4 B). In contrast, the stimulation of this reporter gene by tumor necrosis factor-α treatment, which is mediated by the TRAF/TRAD pathway, independently of Ras (26Perona R. Montaner S. Saniger L. Sanchez-Perez I. Bravo R. Lacal J.C. Genes Dev. 1997; 11: 463-475Crossref PubMed Scopus (536) Google Scholar), was not significantly affected by the Spry proteins. Fig. 4 C indicates that the spryconstructs were indeed expressed in the conditions used for the reporter assays. To reinforce the biological significance of these results, we examined the expression of the endogenous c-fos gene in the Spry1 and Spry2 inducible cell lines. c-fos is an immediate early gene whose expression is induced by growth factors and is necessary for progression through the G1 phase of the cell cycle and subsequent cell proliferation (23Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1854) Google Scholar, 24Treisman R. EMBO J. 1995; 14: 4905-4913Crossref PubMed Scopus (347) Google Scholar). As expected, c-fosexpression was rapidly induced upon serum or FGF treatment (Fig.4 D). However, in the presence of Spry1 or Spry2, c-fos expression was significantly blocked. Compared with the control NIH3T3 Tet-off cell line, c-fos expression stimulated by serum was reduced by about 30%. More strikingly, c-fos expression induced by FGF was decreased by 70 (Spry1) to 80% (Spry2) (Fig. 4 D). Thus, this set of experiments shows that mammalian Spry1 and Spry2 are able to inhibit the transcriptional events mediated by growth factor signaling and the induction of a gene required for DNA synthesis and cell division. We used the Spry inducible cell lines to examine the effect of Spry1 and Spry2 on the Ras/Raf/MAP kinase and the phosphatidylinositol 3-kinase (PI 3-kinase) pathways, which are two major pathways mediating growth factor signaling in NIH3T3 cells (for review, see Ref. 27Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (920) Google Scholar). In the presence of Spry1 or Spry2, FGF or PDGF-mediated activation of the Erk1/2 MAP kinases, as visualized by phospho-specific antibodies, was strikingly inhibited (Fig.5, A and B). No such effect was found upon tetracycline withdrawal in the control cell line (Fig. 5, A and B). A time course of FGF stimulation showed that in the presence of Spry2 or Spry1, the stimulation of Erk1/2 was also delayed, with the peak of phosphorylated MAP kinases accumulation occurring at 10 min rather than 3 min after stimulation (Fig. 5 C and data not shown for Spry1). Furthermore, MAP kinase activation was less sustained. In contrast, PDGF-mediated activation of the key protein of the PI 3-kinase pathway, the serine/threonine kinase Akt, was not inhibited in the presence of Spry proteins (Fig. 5 D). Moreover, activation of a downstream target of Akt, the serine/threonine Gsk-3β, was also not affected (data not shown). A time course of PDGF stimulation indicated that Spry proteins did not inhibit Akt activation at any specific time point (Fig. 5 E). These results show that mammalian Spry1 and Spry2 do not inhibit the PI 3-kinase pathway but specifically inhibit the Erk1/2 MAP kinase pathway. They also imply that in the presence of Spry proteins, some signals downstream of the RTK can be propagated. We next determined the effect of Spry proteins on the stepwise activation of the different components of the MAP kinase pathway upon growth factor treatment. We examined the activation of the dual-specificity MAP kinase kinases Mek1/2 which phosphorylate Erk1/2, stimulating their activity in response to growth factor treatment. As seen in Fig.6 A, Mek1/2 phosphorylation was dramatically reduced in the presence of Spry1 or Spry2, indicating that the signal was blocked upstream of Mek1/2, possibly at the level of the MAP kinase kinase kinase Raf-1. Therefore, we directly examined Raf-1 kinase activity. Fig. 6 B shows that in the presence of Spry2, the ability of immunoprecipitated Raf-1 from FGF-stimulated cells to phosphorylate a recombinant GST-Mek1 protein was greatly reduced. This indicates that Raf-1 is inactive in the presence of Spry proteins and suggests that Spry act upstream of Raf-1. The steps leading to Raf-1 activation are not fully understood but require binding to Ras (for a review, see Ref. 27Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (920) Google Scholar). Therefore, we examined the binding of Raf-1 to Ras in the Spry cell lines (28Taylor S.J. Shalloway D. Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Fig.6 C shows that the amount of endogenous Ha-Ras bound by recombinant GST-Raf-1 Ras-binding domain (RBD) fusion protein upon FGF treatment was greatly reduced in the presence of Spry1 or Spry2. This could be due either to a direct inhibition of the Ras/Raf-1 interaction or to an inhibition of Ras activation, since only activated, GTP-bound Ras, can bind Raf-1. To discriminate between these two possibilities, we examined the binding of a constitutively active form of Ras (Ha-Ras R12) to Raf-1 in the presence of Spry1 or Spry2. We performed the same GST-Raf-1/Ras binding assay with lysates of NIH3T3 cells transfected with Ras and Spry1 or Spry2 expression vectors. As presented in Fig.6 D, expression of Spry1 or Spry2 reduced the binding of wild type Ha-Ras (induced by FGF) to GST-Raf-1, but had no significant effect on the binding of the constitutively active Ha-Ras R12. This suggests that Spry proteins do not interfere directly with the binding of Ras to Raf-1, but rather inhibit the activation of Ras. Finally, we examined the phosphorylation of the adaptor FRS2/SNT-1, which is the primary substrate of the activated FGF receptor (29Wang J.K. Xu H. Li H.C. Goldfarb M. Oncogene. 1996; 13: 721-729PubMed Google Scholar, 30Kouhara H. Hadari Y.R. Spivak-Kroizman T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar) and thus reflects the activity of the RTK. Fig. 6 Egraphically shows that the ability of the FGF receptor to mediate phosphorylation of FRS2/SNT-1 after FGF stimulation was not inhibited in the presence of Spry proteins while at the same time, the activation of the Erk1/2 MAP k