c-Cbl Associates Directly with the C-terminal Tail of the Receptor for the Macrophage Colony-stimulating Factor, c-Fms, and Down-modulates This Receptor but Not the Viral Oncogene v-Fms
2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês
10.1074/jbc.m109214200
ISSN1083-351X
AutoresAnnalisa Mancini, Alexandra Koch, Regina Wilms, Teruko Tamura,
Tópico(s)interferon and immune responses
ResumoThe receptor for the macrophage colony-stimulating factor (CSF-1, also termed M-CSF), the tyrosine kinase c-Fms, was originally determined to be the oncogene product of the McDonough strain of feline sarcoma virus, v-Fms. The structural difference between c-Fms and v-Fms amounts to only five point mutations in the extracellular domain, two mutations in the cytoplasmic domain, and the replacement of 50 amino acids by 14 unrelated amino acids at the C-terminal tail. Here, we have identified c-Cbl as the direct binding partner for c-Fms. c-Cbl binds to phosphotyrosine residue 977 at the C-terminal end of feline c-Fms, which is absent in v-Fms. The replacement of the C-terminal end of v-Fms by the corresponding part of c-Fms (vc-Fms) restored the binding potential. As a result, vc-Fms reduced the transforming potency of v-Fms. The overexpression of Cbl did not influence the v-Fms-transformed phenotype, although c-Cbl forms a complex with v-Fms indirectly. In contrast, the expression of Cbl drastically reduced the vc-Fms-transformed phenotype and the activation of Erk and enhanced Fms ubiquitination via phosphotyrosine residue 977. Furthermore, the replacement of tyrosine 977 into phenylalanine in feline c-Fms and vc-Fms reduced the Cbl-dependent ubiquitination. These data suggest that an indirect association of c-Cbl via multimeric complex induced a different signaling pathway from the pathway induced by c-Cbl direct interaction. The receptor for the macrophage colony-stimulating factor (CSF-1, also termed M-CSF), the tyrosine kinase c-Fms, was originally determined to be the oncogene product of the McDonough strain of feline sarcoma virus, v-Fms. The structural difference between c-Fms and v-Fms amounts to only five point mutations in the extracellular domain, two mutations in the cytoplasmic domain, and the replacement of 50 amino acids by 14 unrelated amino acids at the C-terminal tail. Here, we have identified c-Cbl as the direct binding partner for c-Fms. c-Cbl binds to phosphotyrosine residue 977 at the C-terminal end of feline c-Fms, which is absent in v-Fms. The replacement of the C-terminal end of v-Fms by the corresponding part of c-Fms (vc-Fms) restored the binding potential. As a result, vc-Fms reduced the transforming potency of v-Fms. The overexpression of Cbl did not influence the v-Fms-transformed phenotype, although c-Cbl forms a complex with v-Fms indirectly. In contrast, the expression of Cbl drastically reduced the vc-Fms-transformed phenotype and the activation of Erk and enhanced Fms ubiquitination via phosphotyrosine residue 977. Furthermore, the replacement of tyrosine 977 into phenylalanine in feline c-Fms and vc-Fms reduced the Cbl-dependent ubiquitination. These data suggest that an indirect association of c-Cbl via multimeric complex induced a different signaling pathway from the pathway induced by c-Cbl direct interaction. Retroviral oncogenes have been intensively investigated to further understanding of neoplastic conversion and tumor development in animals (1.Bishop J.M. Cell. 1991; 64: 235-248Abstract Full Text PDF PubMed Scopus (1388) Google Scholar). A number of receptor tyrosine kinases were originally identified as retroviral oncogene products, including v-Fms, v-erbB, or v-Kit of which the cellular counterparts are receptors for macrophage colony-stimulating factor (CSF-1 1The abbreviations used are: CSFcolony-stimulating factorSHSrc homologyPTBphosphotyrosine bindingPIphosphatidylinositolAMLacute myeloblastic leukemiaGSTglutathione S-transferase or M-CSF), epidermal growth factor, or stem cell factor, respectively. In normal cells, the life span and enzymatical activity of protooncogene-products are tightly regulated. Through mutation and/or deletion of the receptor tyrosine kinase, they escaped from cellular regulatory mechanisms and activated constitutively without ligand binding. colony-stimulating factor Src homology phosphotyrosine binding phosphatidylinositol acute myeloblastic leukemia glutathione S-transferase In the normal state, binding of growth factors causes dimerization of the receptors and activation of their inherent receptor tyrosine kinases leading to autophosphorylation of the cytoplasmic domains at multiple tyrosine residues. The newly formed phosphotyrosines constitute binding sites for Src homology 2 (SH2) domain- or phosphotyrosine binding (PTB) domain-containing cytoplasmic proteins, which are thought to participate in the control of mitogenic or differentiation pathways, cell metabolism, and/or cell morphology. After triggering the signal transduction pathways, the receptors are internalized and either recycled again to the cell surface or degraded. The receptor for the CSF-1, the tyrosine kinase c-Fms, was originally determined to be the oncogene product of the McDonough strain of feline sarcoma virus, v-Fms. The Fms tyrosine kinase was reported to interact with several SH2 domain-containing proteins, including the growth factor receptor bound protein 2 (Grb2; Refs. 2.van der Geer P. Hunter T. EMBO J. 1993; 12: 5161-5172Crossref PubMed Scopus (124) Google Scholar, 3.Mancini A. Niedenthal R. Joos H. Koch A. Trouliaris S. Niemann H. Tamura T. Oncogene. 1997; 15: 1565-1572Crossref PubMed Scopus (45) Google Scholar), STAT1 (4.Novak U. Nice E. Hamilton J.A. Paradiso L. Oncogene. 1996; 13: 2607-2613PubMed Google Scholar), the p85 subunit of phosphatidylinositol (PI) 3-kinase (5.Reedijk M. Liu X. van der Geer P. Letwin K. Waterfield M.D. Hunter T. Pawson T. EMBO J. 1992; 11: 1365-1372Crossref PubMed Scopus (179) Google Scholar), the p120RasGTPase activating protein (6.Trouliaris S. Smola U. Chang J.-H. Parsons S.J. Niemann H. Tamura T. J. Virol. 1995; 69: 6010-6020Crossref PubMed Google Scholar), phospholipase C-γ (7.Bourette R.B. Myles G.M. Choi J.-L. Rohrschneider R. EMBO J. 1997; 16: 5880-5893Crossref PubMed Scopus (91) Google Scholar), c-Src (8.Courtneidge S.A. Dhand R. Pilat D. Twamley G.M. Waterfield M.D. Roussel M.F. EMBO J. 1993; 12: 943-950Crossref PubMed Scopus (204) Google Scholar), Mona (9.Bourette R.P. Arnaud S. Myles G.M. Blanchet J-P. Rohrschneider L.R. Mouchiroud G. EMBO J. 1998; 17: 7273-7281Crossref PubMed Scopus (90) Google Scholar), Fms-interacting protein (10.Tamura T. Mancini A. Joos H. Koch A. Hakim C. Dumanski J. Weidner K.M. Niemann H. Oncogene. 1999; 18: 6488-6495Crossref PubMed Scopus (42) Google Scholar), and p55, a polypeptide of yet unknown function (11.Joos H. Trouliaris S. Helftenbein G. Niemann H. Tamura T. J. Biol. Chem. 1996; 271: 24476-24481Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Expression of the viral fms gene in mammalian fibroblasts leads to cell transformation and to formation of fibrosarcoma in vivo. The structural difference between c-Fms and v-Fms consists of only five point mutations in the extracellular domain, two mutations in the cytoplasmic domain, and the replacement of 50 amino acids by 14 unrelated amino acids at the C-terminal tail (12.Woolford J. McAuliffe A. Rohrschneider L.R. Cell. 1988; 55: 965-977Abstract Full Text PDF PubMed Scopus (108) Google Scholar). Roussel et al., (13.Roussel M.F. Downing J.R. Rettenmier C.W. Sherr C.J. Cell. 1988; 55: 979-988Abstract Full Text PDF PubMed Scopus (172) Google Scholar) showed that a single point mutation at position 301 was solely responsible for a conversion of the human c-fms gene product into a transforming protein. However, Woolford et al. (12.Woolford J. McAuliffe A. Rohrschneider L.R. Cell. 1988; 55: 965-977Abstract Full Text PDF PubMed Scopus (108) Google Scholar) showed that in addition a second mutation involving residue 374 and the exchange of the C-terminal domain were required for an effective transforming potency. The mutation of residues 301 and 374 in the extracellular domain leads to receptor dimerization without a ligand. There is only one tyrosine residue, tyrosine 977, in the feline c-Fms-specific C-terminal tail. Mutation of tyrosine residue 969 of human c-Fms (corresponding to tyrosine 977 of feline c-Fms) slightly enhanced the transforming potency in fibroblasts (14.Roussel M.F. Shurtleff S.A. Downing J.R. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6738-6742Crossref PubMed Scopus (95) Google Scholar) or gained cytokine independence for growth in the interleukin-3-dependent murine hematopoietic cell line, FDC-P1 (15.McGlynn H. Baker A.H. Padua R.A. Leuk. Res. 1998; 22: 365-372Crossref PubMed Scopus (12) Google Scholar). Interestingly, the same point mutation has been detected in a number of hematological disorders. The C-terminal mutation has also been observed in children suffering from secondary acute myeloblastic leukemia (AML) or myelodysplasia (16.Ridge S.A. Worwood M. Oscier D. Jacobs A. Padua R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1377-1380Crossref PubMed Scopus (220) Google Scholar, 17.Baker A.H. Ball S. McGynn H. Whittaker J.A. Burnett A.K. Padua R.A. Leukemia. 1995; 9: 155-158PubMed Google Scholar). However, the role of the C-terminal at the molecular level was poorly understood. To elucidate the molecular principles underlying the up-regulation of oncogenic potency via mutation of the C-terminal tail, we employed a yeast two-hybrid screening protocol based on the tyrosine-phosphorylated cytoplasmic domain of c-Fms and v-Fms as baits. We identified c-Cbl as a binding partner of c-Fms. c-Cbl binds to c-Fms specifically at tyrosine 977, which is absent in v-Fms. Using cells expressing a chimera receptor of v-Fms and c-Fms, we show that the overexpression of c-Cbl suppressed the transforming potential of chimera Fms by the enhancement of the receptor ubiquitination but not that of v-Fms. Furthermore, the replacement of tyrosine residue 977 into phenylalanine avoided the quick ubiquitination of Fms molecules. These data indicate that the observed negative regulatory function of the C-terminal tail is mediated by a Cbl binding site. The construction of LexA fusion genes encoding the cytoplasmic domains of c-Fms, v-Fms, and vc-Fms downstream of LexA using BTM116 vector, the expression in Saccharomyces cerevisiae strain YRN974, and the qualitative and quantitative evaluations of various two-hybrid protein/protein interactions were described previously (3.Mancini A. Niedenthal R. Joos H. Koch A. Trouliaris S. Niemann H. Tamura T. Oncogene. 1997; 15: 1565-1572Crossref PubMed Scopus (45) Google Scholar, 10.Tamura T. Mancini A. Joos H. Koch A. Hakim C. Dumanski J. Weidner K.M. Niemann H. Oncogene. 1999; 18: 6488-6495Crossref PubMed Scopus (42) Google Scholar). GST-Cbl-(amino acids 1–350) and Fms-fusion proteins were generated in the pGex system (Amersham Biosciences). For expression in mammalian cells, the felinec-fms, v-fms, and its chimeravc-fms-cDNA were cloned into pcDNA3 (Invitrogen, Carlsbad, CA). NIH3T3 and HEK293 cells expressing thev-fms, c-fms, or vc-fms genes were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. CSF-1 (Sigma) was added at a concentration of 2000 units/ml. Rat sera against feline c-Fms and v-Fms were used as described previously (22.Tamura T. Simon E. Niemann H. Snoek G.T. Bauer H. Mol. Cell. Biol. 1986; 6: 4745-4748Crossref PubMed Scopus (18) Google Scholar). Monoclonal antibodies against phosphotyrosine (4G10) and against c-Cbl, ubiquitin, Grb2, and PI 3-kinase were from Upstate Biotechnology Incorporated (Lake Placid, NY) or from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibodies against ubiquitin and Fms were from Santa Cruz Biotechnology, and Erk1/2 and phospho-Erk1/2 antibodies were from Promega (Madison, WI). For tyrosine kinase assays, Fms-specific immune complexes were incubated for 20 min at room temperature with 3 μCi of γ-[32P]ATP (Amersham Biosciences) in the presence of 10 mm MnCl2, and were analyzed by SDS-PAGE. For co-immunoprecipitation studies, 5 × 106cells were lysed with the lysis buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EGTA, 1% trasylol, 100 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 200 μm sodium orthovanadate, 10 mm sodium pyrophosphate, and 100 mm sodium fluoride). For identification by immunoblotting, proteins were transferred onto Polablot polyvinylidene difluoride membrane or nitrocellulose membrane (Macherey-Nagel) by a semi-dry blotting technique. Bound antibody was visualized by incubation of blots in 3 ml of 20 mm Tris-HCl, pH 7.6, containing 137 mm NaCl and 2 μCi of 125I-labeled anti-species-specific immunoglobulin G (Amersham Biosciences) or horseradish peroxidase-conjugated immunoglobulin G (Santa Cruz) followed by applying with BM chemiluminescence detection kit (Roche Diagnostics). Bound radioactivity was quantified using a model BAS1500 bioimaging analyzer (Fuji Photo Film Co., Kanagawa, Japan). Strain TKX-1 (TKX, Stratagene, La Jolla, CA) was used for the isolation of phosphorylated GST-Fms fusion proteins. Phosphorylated GST fusion proteins were produced as recommended by the manufacturer. The corresponding nonphosphorylated molecular species were isolated from E. coli strain DH5α. Purified GST fusion proteins were bound for 1 h at 4 °C to glutathione-agarose beads (40 μl of slurry; Amersham Biosciences) suspended in the lysis buffer. Precharged beads were incubated overnight at 4 °C with 32P-labeled Fms, as generated byin vitro autophosphorylation or obtained from Fms-expressing HEK293 cell lysates in a total volume of 2 ml of binding buffer. Beads were washed five times with binding buffer, and pellets were analyzed by SDS-PAGE. Using a yeast two-hybrid technique, we have isolated cDNA encoding several signaling proteins such as PI 3-kinase, Grb2, Grb10, phospholipase C-γ, c-Src, c-Abl, Crk, Fms-interacting protein, and c-Cbl, which specifically interact with the cytoplasmic domain of c-Fms (10.Tamura T. Mancini A. Joos H. Koch A. Hakim C. Dumanski J. Weidner K.M. Niemann H. Oncogene. 1999; 18: 6488-6495Crossref PubMed Scopus (42) Google Scholar). To determine whether one of these associates with c-Fms or v-Fms specifically, we again employed the yeast two-hybrid system using green fluorescent protein as a reporter gene product (3.Mancini A. Niedenthal R. Joos H. Koch A. Trouliaris S. Niemann H. Tamura T. Oncogene. 1997; 15: 1565-1572Crossref PubMed Scopus (45) Google Scholar). All of the proteins bound to both v-Fms and c-Fms equally well except the PTB domain of c-Cbl (amino acid residues 1–350). c-Cbl bound to c-Fms but not to v-Fms in this assay system (Fig. 1 A). Since the major difference of the cytoplasmic domains of v-Fms and c-Fms is the C-terminal tail, we replaced the last 14 amino acids of v-Fms by 50 amino acids derived from the C-terminal tail of c-Fms (vc-Fms) (Fig. 1 B). As shown in Fig. 1, the resultant vc-Fms was indeed able to bind to c-Cbl. As expected, the kinase-negative vc-Fms mutant (vc-FmsK613M) did not associate with c-Cbl, indicating this association is tyrosine phosphorylation-dependent. Furthermore, mutation of tyrosine 720, which was previously suggested as a direct or indirect binding site of c-Cbl (18.Ota J. Sato K. Kimura F. Wakimoto N. Nakamura Y. Nagata N. Suzu S. Yamada M. Shimamura S. Motoyoshi K. FEBS Letters. 2000; 466: 96-100Crossref PubMed Scopus (8) Google Scholar), did not influence the Fms interaction with c-Cbl in this assay system, indicating that Tyr-720 is not a direct binding site for c-Cbl. To demonstrate the protein/protein interaction between c-Cbl and c-Fms in different systems, we first performed a Cbl-specific co-immunoprecipitation study. v-Fms, c-Fms, and vc-Fms were expressed in HEK293 cells, and cell extracts were supplied for an in vitro kinase reaction of Fms-specific immune complexes. Autophosphorylated v-Fms, c-Fms, and vc-Fms polypeptides were liberated from these complexes by a low pH shock treatment (Fig. 2 A). The same counts of radiolabeled individual Fms molecules were incubated with HEK293 cell lysates, and were immunoprecipitated with anti Cbl antibody. In this assay, all three, v-, c-, and vc-Fms, were co-immunoprecipitated with c-Cbl molecules (Fig. 2 B). In addition, c-Cbl was co-precipitated by anti-Fms antibody with all three autophosphorylated Fms polypeptides. After incubation of autophosphorylated Fms molecules with alkaline phosphatase, the interaction of c-Cbl with Fms was completely abolished (Fig. 2 B). Thus, in the presence of cellular proteins, both c- and v-Fms associate with c-Cbl. This is not surprising because it has been well documented that CSF-1 stimulation induces the formation of a multiprotein complex including c-Fms, c-Cbl, CrkII, PI 3-kinase, and Grb2 (19.Husson H. Mograbi B. Schmid-Antomarchi H. Fischer S. Rossi B. Oncogene. 1997; 14: 2331-2338Crossref PubMed Scopus (33) Google Scholar). Here, both Grb2 and PI 3-kinase bind to v-Fms as well. In addition, c-Cbl forms a stable complex via its proline-rich sequence with the SH3 domain of PI 3-kinase and Grb2 (20.Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar, 21.Odai H. Sasaki K. Iwamatsu A. Hanazono Y. Tanaka T. Mitani K. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 10800-10805Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Therefore, we utilized the GST fusion protein binding assay using GST-Cbl (amino acid residues 1–350), which is lacking the proline-rich domain. In agreement with data obtained from the yeast two-hybrid assay, both32P-labeled autophosphorylated c-Fms and vc-Fms bound to the GST-Cbl, but v-Fms did not (Fig. 3), suggesting that c-Cbl associates directly with the C-terminal of the c-Fms molecule. However, in addition, c-Cbl binds to v-Fms indirectly through multiprotein complex formation.Figure 3c-Cbl interacts with c-Fms and chimera vc-Fms, but not v-Fms in the GST-Cbl-binding assay. GST-Cbl-PTB (amino acids 1–350) or GST alone, were produced in bacterial strains DH5α. v-Fms, c-Fms, and vc-Fms genes were expressed in HEK293 cells, and cell extracts were supplied for in vitro kinase reaction in the presence of γ-[32P]ATP. GST-fusion protein or GST (5 μg each) were bound to GT-agarose beads and incubated with 32P-labeled v-Fms, c-Fms, and vc-Fms. Binding materials were analyzed by SDS-PAGE and autoradiography. As a control, aliquots were analyzed by SDS-PAGE and staining with Coomassie Brilliant Blue.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To study whether the c-Cbl/c-Fms interaction relied on the presence of particular phosphotyrosine residues of Fms, we employed a set of GST-Fms fusion proteins including GST-CT-v-Fms (amino acids 904–944), and GST-CT-c-Fms (amino acids 904–980) (Fig. 4 A). The recombinant tyrosine-phosphorylated GST-Fms polypeptides were isolated fromE. coli strain TKX that expresses an active Elk tyrosine kinase (11.Joos H. Trouliaris S. Helftenbein G. Niemann H. Tamura T. J. Biol. Chem. 1996; 271: 24476-24481Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). As demonstrated by Western blotting using anti-phosphotyrosine, these proteins were indeed phosphorylated on tyrosine (Fig. 4 B, anti-Try (anti-pY)). GST Fms fusion proteins were incubated with HEK293 cell extracts, and bound proteins were analyzed by c-Cbl- and Grb2-specific immunoblotting. In agreement with previous data (3.Mancini A. Niedenthal R. Joos H. Koch A. Trouliaris S. Niemann H. Tamura T. Oncogene. 1997; 15: 1565-1572Crossref PubMed Scopus (45) Google Scholar), Grb2 bound to both GST-CT-c-Fms and GST-CT-v-Fms that contain the Grb2 binding site, phosphotyrosine 921, while c-Cbl bound to GST-CT-c-Fms exclusively. GST-CT-c-Fms contains two tyrosine residues, tyrosine 921 and tyrosine 977, while GST-CT-v-Fms contains only phosphotyrosine 921 (Fig. 4 A). Therefore, we have replaced the unique tyrosine residue in c-Fms, tyrosine 977, by phenylalanine (GST-CT-c-Fms977F). This GST fusion protein was still able to bind to Grb2; however, c-Cbl was almost undetectable (Fig. 4 B). Furthermore, no binding of c-Cbl and Grb2 to unphosphorylated GST fusion proteins were observed (Fig. 4). These data indicate that phosphotyrosine 977 indeed provides the binding site for c-Cbl. One important characteristic which distinguishes normal cells from transformed cells is the ability of the latter to form colonies in soft agar. To determine whether the replacement of the C-terminal tail influences the transforming potency of v-Fms, 5 × 105 NIH3T3 cells were transfected with 10 μg of pcDNA3 containing feline c-Fms, v-Fms, and chimera vc-Fms. Fig. 5 shows that v-Fms transfectants formed colonies in 0.5% soft agar, but c-Fms transfectants were not able to form colonies. In agreement with previous data (12.Woolford J. McAuliffe A. Rohrschneider L.R. Cell. 1988; 55: 965-977Abstract Full Text PDF PubMed Scopus (108) Google Scholar), vc-Fms transfectants formed colonies; however, sizes of colonies were drastically reduced. To determine whether the observed reduction of v-Fms-transforming potency by the C-terminal replacement is due to a direct interaction with c-Cbl, we introduced the exogenous c-Cbl at different concentrations into vc-Fms- and v-Fms-transformed cells. For this experiment, we further established cell lines that express vc-Fms or v-Fms. Established cell lines were transfected with 1 and 9 μg of pcDNA3 containing c-Cbl cDNA and tested again for formation of colonies in soft agar. In the absence of exogenous c-Cbl, v-Fms-transformed cells formed colonies with an average size of 60 μm in diameter, while vc-Fms-transformed cells formed colonies with an average size of 35 μm in diameter within 6 days (Fig. 6). Sizes of colonies of vc-Fms expressing cells were drastically reduced by overexpression of exogenous c-Cbl after transfection even with 1 μg of pcDNA3 containing c-Cbl cDNA. In contrast, c-Cbl expression (Fig. 6 A, 1 μg) did not change, if rather enhanced, the size of colonies after the same treatment of v-Fms-transformed cells. These results indicate that the direct interaction with c-Cbl to the Fms molecule may down-modulate the Fms signal. In addition, although wild-type v-Fms interacts with c-Cbl indirectly via other signaling molecules such as PI 3-kinase and Grb2, c-Cbl did not affect transforming potency of v-Fms, suggesting an indirect association of c-Cbl via a multimeric complex that induced a different signaling pathway from the pathway induced by c-Cbl direct interaction. Since it has been well documented that c-Cbl plays a role in protein ubiquitination, the question arises as to whether the reduced transforming potency of vc-Fms is a result of enhanced ubiquitination. Therefore, we compared first the ubiquitination of v-Fms and vc-Fms upon stimulation with CSF-1. Sister cultures of 1 × 106 cells each derived from v- or vc-Fms-transformed 3T3 cell lines were labeled with [3H]leucin for 16 h and were stimulated with CSF-1 for 3, 10, or 30 min. Cell extracts from each preparation were supplied for Fms-specific immunoprecipitation. In agreement with previous data (22.Tamura T. Simon E. Niemann H. Snoek G.T. Bauer H. Mol. Cell. Biol. 1986; 6: 4745-4748Crossref PubMed Scopus (18) Google Scholar), about 10% of v-Fms molecules were detected as a mature glycoprotein, gp140v-Fms that is expressed at the cell surface. Although similar amounts of immature glycoprotein gp120v-Fms and gp130vc-Fms were detected from both cell lines, less of the mature glycoprotein of vc-Fms, gp150vc-Fms, was detected than gp140v-Fms (Fig. 7 A). The same aliquots of Fms-specific immunoprecipitates were analyzed by ubiquitin-specific immunoblot. vc-Fms was ubiquitinated about 2-fold more than v-Fms; however, no significant difference was observed in the presence or absence of CSF-1. It is noteworthy that vc-Fms was dimerized and activated constitutively without CSF-1 via mutations in the extracellular domain (12.Woolford J. McAuliffe A. Rohrschneider L.R. Cell. 1988; 55: 965-977Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 13.Roussel M.F. Downing J.R. Rettenmier C.W. Sherr C.J. Cell. 1988; 55: 979-988Abstract Full Text PDF PubMed Scopus (172) Google Scholar) and might be ubiquitinated constantly to some extent. As control, cell extracts from the same preparation were analyzed by Western blot using anti-Erk and activated Erk antibodies. In both cell lines, Erk1/2was activated slightly without ligand; however, CSF-1 stimulation induced an activation of Erk within 3 min in both cell lines, whereby the level of phosphorylation decreased within 10 min. To determine whether c-Cbl molecules indeed influenced ubiquitination via phospho-Tyr-977, we generated the mutant vc-Fms whose tyrosine 977 was replaced by phenylalanine (vc-Y977F-Fms). Five μg of each vc-Fms and vc-Y977F-Fms cDNA were transfected into 2 × 105 HEK293 cells with and without c-Cbl cDNA. After serum starvation for 16 h, cells were stimulated with CSF-1 for 3 or 30 min at 37 °C. Aliquots of cell extracts were immunoprecipitated by Fms-specific antibodies and were analyzed by Fms- or ubiquitin-specific immunoblotting. The following results were obtained. Firstly, c-Cbl over-expression down-modulated Erk activation induced by CSF-1 in vc-Fms-transformed cells. Secondly, c-Cbl over-expression up-regulated the vc-Fms ubiquitination. Thirdly, the mutation of tyrosine 977 in vc-Fms enhanced the CSF-1-mediated Erk activation. Fourthly, over-expression of c-Cbl in vc-Y977F-Fms cells has a lesser effect on the activation of Erk than in vc-Fms cells, and no effect on vc-Y977F-Fms ubiquitination. We then examined the c-Cbl-dependent ubiquitination of their cellular counterpart, the feline c-Fms, the kinase of which is inactive in the absence of CSF-1. Furthermore, we replaced tyrosine 977 in feline c-Fms by phenylalanine (c-Y977F-Fms). Five μg of c-Fms and c-Y977F-Fms cDNA were transfected into HEK293 cells with and without c-Cbl cDNA. After serum starvation for 16 h, cells were stimulated with CSF-1 for 3 or 30 min for 37 °C, and cell extracts were then immunoprecipitated using a Fms-specific antibody. In agreement with the previous data using the mouse c-Fms-expressing cells (23.Lee P.S.W. Wang Y. Dominguez M.G. Yeung Y-G. Murphy M.A. Bowtell D.D. Stanley E.R. EMBO J. 1999; 13: 3616-3628Crossref Scopus (253) Google Scholar), the feline c-Fms was ubiquitinated in the presence of the exogenous c-Cbl (Fig. 8); however, this ubiquitination is drastically reduced by replacement of tyrosine 977 into phenylalanine in feline c-Fms molecules, indicating that tyrosine 977, our newly found c-Cbl binding site, plays a key role for the c-Cbl-mediated ubiquitination.Figure 8The replacement of tyrosine residue 977 by phenylalanine in the feline c-Fms reduced the ubiquitination. Five μg of pcDNA3 containing c-Fms or c-Y977F-Fms cDNA were transfected into 2 × 105 of HEK293 together with or without 5 μg of c-Cbl cDNA. After 36 h of transfection, cells were serum-starved for 16 h and were then stimulated with CSF-1 for 3 or 30 min at 37 °C. Cell extracts were divided into two aliquots and precipitated by anti-Fms rat antibody (IP:Fms) followed by ubiquitin (WB:anti-Ub) or Fms-specific immunoblot (WB:anti-Fms), or cell lysates (WCL) were analyzed by c-Cbl, phosphoErk, or Erk-specific immunoblot (WB).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Employing a yeast two-hybrid technique, we detected c-Cbl as a direct interacting partner of c-Fms tyrosine kinase. We show here c-Cbl binds to phosphotyrosine 977 at the C-terminal tail of feline c-Fms, which is absent in the v-Fms molecule. Oncogenic v-Fms, which was reconstituted the Cbl binding site, did not transform cells in the presence of the high level of c-Cbl, indicating that the direct binding of c-Cbl plays a key role in Fms tyrosine kinase-mediated cell transformation. Furthermore, the exogenous c-Cbl expression in vc-Y977F-Fms cells did not influence Fms ubiquitination of and Erk activation mediated by CSF-1. Interestingly, v-Fms formed a complex with c-Cbl indirectly; however, expression of c-Cbl did not alter transforming potency of v-Fms, indicating that c-Cbl may mediate two different signaling pathways dependent upon distinct multimeric complexes. The protooncogne c-cbl product is the 120-kDa protein containing an unconventional PTB domain, a ring finger, a proline rich-domain, and a leucine zipper-like domain. This molecule was originally identified as a retroviral oncogene product, v-Cbl (24.Blake T.J. Shapiro M. Moese III H.C. Langdon W.Y. Oncogene. 1991; 6: 653-657PubMed Google Scholar), that contains only the PTB domain of c-Cbl. Recently, it was demonstrated that the protooncogene product c-Cbl acts as a ubiquitin ligase, E3 ubiquitin conjugate enzyme, (25.Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.-C. Science. 1999; 286: 309-312Crossref PubMed Scopus (915) Google Scholar, 26.Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geigr B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (720) Google Scholar, 27.Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar) and leads to the increased rate of ubiquitination and degradation of several receptor tyrosine kinases, including the receptor for epidermal growth factor, platelet-derived growth factor, and CSF-1 (23.Lee P.S.W. Wang Y. Dominguez M.G. Yeung Y-G. Murphy M.A. Bowtell D.D. Stanley E.R. EMBO J. 1999; 13: 3616-3628Crossref Scopus (253) Google Scholar, 26.Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geigr B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (720) Google Scholar, 27.Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 28.Miyake S. Lupher M.L. Druker B. Band H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7927-7932Crossref PubMed Scopus (237) Google Scholar, 29.Miyake S. Mullane-Robinson K.P. Lill N.L. Douillard P. Band H. J. Biol. Chem. 1999; 274: 16619-16628Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). We show here for the first time that c-Fms binds to c-Cbl directly via phosphotyrosine 977. It has been suggested that Cbl is associated with phosphotyrosine 723 in human c-Fms (corresponding to the tyrosine residue 720 in v-Fms and feline c-Fms). Since this tyrosine residue was identified as a PI 3-kinase binding site, this interaction may be via PI 3-kinase (18.Ota J. Sato K. Kimura F. Wakimoto N. Nakamura Y. Nagata N. Suzu S. Yamada M. Shimamura S. Motoyoshi K. FEBS Letters. 2000; 466: 96-100Crossref PubMed Scopus (8) Google Scholar). The authors showed that c-Cbl molecules were less immunoprecipitated by a mutant human c-Fms in which tyrosine 723 was changed to phenylalanine. In agreement with this work, we observed that the phosphorylated v-Fms formed a complex with c-Cbl in the presence of other signaling molecules, indicating that c-Cbl binds also indirectly via a multiprotein complex. Furthermore, it has been demonstrated that PI 3-kinse or Grb2 forms a complex with c-Cbl upon stimulation with CSF-1 (30.Wang Y. Yeung Y.-G. Stanley E.R. J. Cell. Biochem. 1999; 72: 119-134Crossref PubMed Scopus (79) Google Scholar). Taken together these data suggest that the Fms/c-Cbl interaction may mediate two different signaling pathways dependent upon distinct multimeric complexes; direct c-Cbl/Fms association leads to the down-modulation of receptor signaling, whereas the indirect association via a multiprotein complex leads instead to the up-regulation of receptor signaling. Along these same lines, Grishinet al. (31.Grishin A. Sinha S. Roginskaya V. Boyer M.J. Gomez-Cambronero J. Zup S. Kurosaki T. Romero G. Corey S.J. Oncogene. 2000; 19: 97-105Crossref PubMed Scopus (49) Google Scholar) reported that the mutation of the PI 3-kinase binding site, tyrosine 731, in the Cbl molecule down-regulated Lyn tyrosine kinase-mediated thymidine incorporation, suggesting that the association of Cbl with PI 3-kinase plays a key role in proliferative signaling pathways. It has also been shown that the interaction of PI 3-kinase with c-Cbl enhanced PI 3-kinase activity (32.Ueno H. Sasaki K. Honda H. Nakamoto T. Yamagata T. Miyagiwa K. Mitani K. Yazaki Y. Hirai H. Blood. 1998; 91: 46-53Crossref PubMed Google Scholar), suggesting that c-Cbl may act as positive regulator for signaling by the activation of receptor tyrosine kinase. In addition, c-Cbl also up-regulates Fc receptor-mediated platelet activation (33.Saci A. Pain S. Rendu F. Bachelot-Loza C. J. Biol. Chem. 1999; 274: 1898-1904Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) or CD16-mediated signaling (34.Cerboni C. Gismondi A. Palmieri G. Piccoli M. Frati L. Santoni A. Eur. J. Immunol. 1998; 28: 1005-1015Crossref PubMed Scopus (26) Google Scholar) via distinct multiprotein complexes. On the other hand, it is clear that the direct binding of c-Cbl to phosphotyrosine 977 of Fms leads to the down-modulation of Fms and its downstream signal cascades. Interestingly, the ubiquitination of platelet-derived growth factor β receptor, which is closely related to Fms tyrosine kinase, was drastically reduced via deletion of 98 amino acids at the C-terminal end or replacement of tyrosine residues 1009 and 1021 by phenylalanine at the C-terminal domain. Furthermore, the ubiquitination-deficient receptors possessed an amplified mitogenic activity (35.Mori S. Heldin C-H. Claesson-Welsh L. J. Biol. Chem. 1993; 268: 577-583Abstract Full Text PDF PubMed Google Scholar). The C-terminal tail of Neu receptor tyrosine kinase is also required for c-Cbl-mediated ubiquitination (36.Levkowitz G. Oved S. Klapper L.N. Harari D. Lavi S. Sela M. Yarden Y. J. Biol. Chem. 2000; 275: 35532-35539Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and mutations that impair the property of c-Cbl to induce the ubiquitination of the epidermal growth factor receptor has oncogenic properties (25.Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.-C. Science. 1999; 286: 309-312Crossref PubMed Scopus (915) Google Scholar), suggesting that C-terminal tail has a function as a regulatory domain for many receptor tyrosine kinases. These data also reveal that receptor ubiquitination is one of the important control mechanisms for anti-cell transformation. Interestingly, impairing the function of TSG101/Vsp23, a protein that contains an inactive E2 ubiquitin-conjugase domain, perturbs endosomal trafficking and induces cell transformation (37.Babst M. Odorizzi G. Estepa E.J. Emr S.D. Traffic. 2000; 1: 248-258Crossref PubMed Scopus (356) Google Scholar). In addition to the Cbl binding site, the C-terminal tail of c-Fms contains a PEST-like domain of 33 amino acids (amino acid residues 925–957 of feline c-Fms) with 18 serine residues, five glutamate residues, two proline residues, and one threonine residue. Hence, Oberg et al. (38.Oberg C. Li J. Pauley A. Wolf E. Gurney M. Lendahl U. J. Biol. Chem. 2001; 276: 35847-35853Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar) reported that Notch 1 is ubiquitinated by mSel-10 and that ubiquitination requires the presence of the Notch 1 C-terminal domain, including the PEST domain. The role of this PEST-like domain of c-Fms, however, still remains to be studied. It is noteworthy that mutation of the human c-fms gene at codon 969 (corresponding to the feline 977 tyrosine residue) has been detected in a number of hematological disorders, AML, B-cell lymphoma and children suffering from secondary AML or myelodysplasia (16.Ridge S.A. Worwood M. Oscier D. Jacobs A. Padua R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1377-1380Crossref PubMed Scopus (220) Google Scholar, 17.Baker A.H. Ball S. McGynn H. Whittaker J.A. Burnett A.K. Padua R.A. Leukemia. 1995; 9: 155-158PubMed Google Scholar). These observations taken together, indicate that this mutation may represent a general mechanism by which oncogenic potential is achieved by escaping the c-Cbl, negative regulation of tyrosine kinase signaling. We thank Karsten Heidrich for fluorescenceactivated cell sorter analyses, Bruce. C. Boschek for critically reading the manuscript, Wallace Longdon for providing c-Cbl cDNA, and Morag Park for helpful discussion.
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