Sef Inhibits Fibroblast Growth Factor Signaling by Inhibiting FGFR1 Tyrosine Phosphorylation and Subsequent ERK Activation*

Dmitry KovalenkoDagger §, Xuehui YangDagger §, Robert J. NadeauDagger , Lauren K. HarkinsDagger , and Robert FrieselDagger ||

From the Dagger  Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine 04074-7205 and the  Cooperative Graduate Program in Molecular Genetics and Cell Biology, University of Maine, Orono, Maine 04469

Received for publication, October 31, 2002, and in revised form, February 24, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Signaling through fibroblast growth factor receptors (FGFRs) is essential for many cellular processes including proliferation and migration as well as differentiation events such as angiogenesis, osteogenesis, and chondrogenesis. Recently, genetic screens in Drosophila and gene expression screens in zebrafish have resulted in the identification of several feedback inhibitors of FGF signaling. One of these, Sef (similar expression to fgf genes), encodes a transmembrane protein that belongs to the FGF synexpression group. Here we show that like zebrafish Sef (zSef), mouse Sef (mSef) interacts with FGFR1 and that the cytoplasmic domain of mSef mediates this interaction. Overexpression of mSef in NIH3T3 cells results in a decrease in FGF-induced cell proliferation associated with a decrease in Tyr phosphorylation of FGFR1 and FRS2. As a consequence, there is a reduction in the phosphorylation of Raf-1 at Ser338, MEK1/2 at Ser217 and Ser221, and ERK1/2 at Thr202 and Tyr204. Furthermore, mSef inhibits ERK activation mediated by a constitutively activated FGFR1 but not by a constitutively active Ras and decreases FGF but not PDGF-mediated activation of Akt. These results indicate that Sef exerts its inhibitory effects at the level of FGFR and upstream of Ras providing an additional level of negative regulation of FGF signaling.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The fibroblast growth factors (FGFs)1 comprise a family of polypeptides currently consisting of 23 members (1). FGFs play important roles in the regulation of cell proliferation, migration, differentiation, and embryonic pattern formation. FGFs elicit their effects on cells by forming a ternary complex that includes the ligand, a high affinity tyrosine kinase receptor, and a heparan-sulfate proteoglycan (2, 3). Upon ligand binding, FGF receptors dimerize, resulting in autophosphorylation on several tyrosine residues that serve as docking sites for SH2 domain containing polypeptides such as phospholipase Cgamma (2, 4) and Crk (3). The PTB-domain containing adaptor protein FRS2 (SNT1) binds to the FGFR in a phosphotyrosine-independent manner and is tyrosine-phosphorylated upon activation of FGFRs (3, 5). Once FRS2 is tyrosine-phosphorylated, it binds the SH2 domain containing adaptor protein Grb2 as well as the protein-tyrosine phosphatase SHP2 (3, 5). Grb2 recruits SOS to the plasma membrane where it participates in activation of Ras and subsequently ERK1/2. FRS2 also binds Gab1 indirectly via Grb2 resulting in activation of PI 3-kinase and Akt (6, 7). These molecular interactions and biochemical events provide part of the basis for regulation of FGF signaling during development and in the adult.

Targeted deletion of members of the FGF family as well as FGFRs in the mouse shows that FGF signaling is essential for cell proliferation and survival in the preimplantation mouse embryo, as well as for cell migration during gastrulation (1, 2, 8). At later stages of development FGF signaling plays a role in development of the brain, limb buds, and lung as well as many other tissues and organs. Recently, FGF signaling was shown to be negatively regulated by members of the Sprouty family of genes, which antagonize Ras/Raf/ERK signaling induced by receptor tyrosine kinases (RTKs) (9-12). The precise site of action of Spry remains unclear, but the most recent evidence points upstream of Ras and downstream of the RTKs (12, 13). Based upon similarities in spatial and temporal patterns of expression as well as functioning in a common pathway, FGF8, FGF3 and Spry have been placed in the FGF synexpression group (11, 14). Another member of the FGF synexpression group called Sef (similar expression to fgf genes) has recently been identified in Zebrafish and mouse (15-18). Sef is a transmembrane protein whose embryonic expression pattern is similar to that of other members of the FGF synexpression group, and its expression is regulated by FGF. Furthermore, overexpression studies in zebrafish and Xenopus show that Sef inhibits FGF signaling (15, 18). The mechanism of the inhibitory function of Sef is unclear. It has been reported that Sef co-immunoprecipitates with FGFR1 and FGFR2 (15), but it has also been reported that Sef functions at or downstream of MEK (18). To establish the functional significance of the physical association of Sef with FGFRs, we overexpressed mouse Sef (mSef) in NIH3T3, COS7, and HEK293 cells and examined the effects of mSef on FGF signaling. Here we demonstrate that overexpression of mSef inhibits tyrosine phosphorylation of FGFR1 and reduces FGF-induced cell proliferation. These effects are accompanied by a decrease in the phosphorylation of FRS2 and a reduction in the activation of components of the Ras/Raf/MEK/ERK pathway as well as of Akt. Thus, Sef functions by binding to and inhibiting the activation of FGFR1 at the cell membrane.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular Cloning and Expression Vectors-- mSef was cloned by PCR using primers designed to amplify the open reading frame using the published sequence (GenBankTM accession number AF459444) and cloned into pcDNA3.1/V5-His TOPO vector (Invitrogen). The sequence of mSef was confirmed using an ABI 310 automated DNA sequencer. The deletion mutants mSefECTM (amino acids 1-325) and mSefIC (amino acids 301-738) were constructed by PCR in a similar manner. The Sef transmembrane domain was replaced by the PDGFR transmembrane domain by cloning the Sef extracellular domain into the pDisplay vector (Invitrogen) followed by subsequent subcloning into pcDNA3.1/V5-His TOPO (mSefEC). For the preparation of mSef or GFP (hrGFP, Stratagene) expressing adenoviruses (AdmSef, AdGFP) a Cre-lox-based system was employed (19) (gift from P. Robbins, University of Pittsburgh). Plasmids encoding FGFRs were described previously (20). ERK2-HA was from S. Gutkind (National Institutes of Health) and RasQ61L was from Upstate Biotechnology, Inc. (pUSE H-RasL61).

Cell Lines, Transfections, and Adenoviral Transduction-- COS7 and HEK293 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (growth medium). NIH3T3 cells (ATCC) were grown in DMEM containing 10% bovine calf serum (CS). For transient transfection experiments, cells were plated at 60-70% confluence in either 10-cm culture dishes or six-well plates and grown overnight in growth medium. Transfections were carried out in 0.1% CS or Opti-MEM (Invitrogen) serum-free medium with the Genejuice reagent (Novagen). Eighteen to twenty-four hours after transfection, cells were stimulated with FGF2, PDGF-BB (R & D Systems) or EGF (Upstate Biotechnology, Inc.) for the indicated times. NIH3T3 cells were transduced with AdGFP or AdmSef (104 viral particles/cell) complexed with LipofectAMINE (Invitrogen) as described previously (21). Infection efficiency was monitored by fluorescence microscopy of AdGFP-infected cells and was usually greater than 80%.

Immunoprecipitation and Immunoblotting-- Cells that were either transiently transfected with the indicated plasmids or transduced with adenoviruses and treated as described above were lysed in HNTG buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl, 1.0 mM EGTA, 0.1 mM NaVO4) as described (22). Lysates were either subjected directly to immunoblot analysis or immunoprecipitated overnight with the indicated antibodies. Immune complexes were captured on protein A/G-agarose (Santa Cruz) washed extensively in HNTG buffer, eluted with 2× SDS sample buffer, separated on 8 or 10% SDS-PAGE, and transferred to Hybond-P (Amersham Biosciences) followed by immunoblotting as previously described (22, 23). Ras activation was assessed using Raf-1 RBD (Upstate Biotechnology, Inc.) according to the manufacturer's protocol. Bound antibodies were visualized by chemiluminescence (ECL reagent, Amersham Biosciences). The intensity of the bands was determined by scanning with a Canon scanner (model N1220) and analysis with NIH image software. Antibodies used in this study were rabbit anti-FGFR FB817 (22-25); anti-FGFR1 monoclonal antibody 5G11 (23, 26), anti-V5 (Invitrogen); anti-HA, anti-His, anti-FRS2 (H-91), and normal rabbit IgG (Santa Cruz); anti-pMEK1/2, anti-ERK1, and anti-Akt (Cell Signaling); anti-pERK1/2 (Sigma, M8159); anti-phosphotyrosine 4G10, anti-pRaf-S338, and anti-ERK1/2 (Upstate Biotechnology, Inc.); and anti-pAkt (S473) (BIOSOURCE).

[3H]Thymidine Incorporation Assay-- NIH3T3 cells were plated in 12-well plates (Costar) at 2 × 104 cells/ml, transduced with the indicated adenoviruses, and made quiescent in DMEM containing 0.1% CS for 36 h. Cells were stimulated with 10 ng/ml FGF2 and incubated with [3H]thymidine (ICN) (1 µCi/ml) for 24 h, followed by fixing with 5% trichloroacetic acid and solubilization in 0.5 M NaOH prior to scintillation counting.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mouse Sef Interacts with FGFR1-- It was reported previously that zSef co-immunoprecipitated with FGFRs from lysates of transfected COS7 cells (15). In an effort to determine whether mSef associates with FGFR1 and to elucidate which structural domains of the mSef protein mediate the interaction, we have prepared full-length and deletion mutants of mSef (Fig. 1A) and co-transfected them with FGFR1 constructs (Fig. 1, B-D). Full-length mSef and mSefIC co-immunoprecipitated with FGFR1 but not mSefECTM or mSefEC. mSef co-immunoprecipitated with both unstimulated and FGF-stimulated FGFR1 with equal efficiency (Fig. 1B). mSef also associated with a constitutively activated form of FGFR1 (FGFR1-K562E) (Fig. 1C) as well as a kinase inactive form of FGFR1 (FGFR1-KD) (Fig. 1D). These data indicate that the cytoplasmic domain of mSef mediates, at least in part, the association between FGFR1 and mSef. In addition, these data indicate that the association between mSef and FGFR1 is independent of FGFR1 tyrosine kinase activity.


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Fig. 1.   mSef binds to FGFR1. A, mSef expression constructs. Blue indicates the signal sequence, yellow the transmembrane domain, red the PDGFR transmembrane domain substitution, and brown the Igkappa signal sequence substitution. B, V5/His-tagged mSef or mSef deletion constructs were transiently expressed in COS7 cells with wild-type FGFR1 under serum-free conditions for 24 h and then treated with 100 ng/ml FGF1 and 10 units/ml heparin for 10 min as indicated. Whole cell lysates were either immunoblotted directly with anti-V5 (Sef) or immunoprecipitated with anti-FGFR antibodies followed by immunoblotting with anti-V5, anti-His, or anti-FGFR antibodies. C, COS7 cells were transiently transfected with the indicated mSef constructs and a constitutively activated FGFR1 (K562E) as described above, immunoprecipitated, and immunoblotted with the indicated antibodies. D, COS7 cells were transiently transfected with mSef constructs and a kinase-dead (KD) FGFR1 (K420A/C289R), immunoprecipitated, and immunoblotted as indicated.

Overexpression of mSef Inhibits FGF-induced NIH3T3 Cell Proliferation and ERK Activation-- It has been shown that in zebrafish embryos, overexpression of zSef inhibits MAPK activation, although the mechanism for this inhibition was not determined (18). To explore the ability of mSef overexpression to inhibit FGF-induced ERK activation, NIH3T3 cells were transduced with AdGFP or AdmSef, and after serum starvation, were stimulated with FGF2 for 10 min. Activation of ERK1/2 was determined by immunoblotting with antibodies that specifically recognize the phosphorylated (activated) form of ERK1/2 (p42/44 MAPK) (Fig. 2A). Adenovirus-mediated expression of mSef but not GFP resulted in a reduction in the levels of activated ERK1/2, whereas overall levels of ERK1/2 were unaffected (Fig. 2A). Adenovirus-mediated expression of mSef was confirmed by immunoblotting for the V5 epitope tag on the C terminus of mSef. Immunoblotting with antibodies that recognize the phosphorylated (activated) forms of MEK1/2 reveals that mSef inhibited FGF-induced MEK activation (Fig. 2A). In addition, immunoblotting with antibodies that recognize the Ser338 phosphorylation site of activated Raf-1 reveals a reduction in FGF-induced activation of Raf-1 by mSef (Fig. 2A). Densitometric analysis of immunoblots of three independent experiments showed that FGF-induced phosphorylation of ERK1/2, MEK1/2, and Raf-1 was reduced by an average of 86 ± 3%, 61 ± 5%, and 59 ± 9%, respectively, by mSef overexpression. These data indicate that the site of action of mSef is likely to be upstream of Raf-1. The mitogenic action of the FGFs is mediated in part by the activation of ERK1/2 (27). To determine whether inhibition of ERK activation by mSef correlates with inhibition of FGF-mediated cell proliferation, we transduced NIH3T3 cells with either AdGFP or AdmSef followed by stimulation with FGF2 and tritiated-thymidine incorporation assay. Transduction with AdmSef resulted in a decrease in FGF-stimulated DNA synthesis consistent with a reduction in ERK1/2 phosphorylation (Fig. 2B).


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Fig. 2.   mSef inhibits FGF-induced ERK pathway activation and proliferation in NIH3T3 cells. A, NIH3T3 cells were transduced with either AdGFP or AdmSef followed by serum starvation for 36 h. Cells were stimulated with FGF2 (10 ng/ml) for 10 min followed by subjecting cell lysates to immunoblotting with antibodies to pRafS338, pMEK1/2, and pERK1/2. To show expression of mSef, blots were re-blotted with anti-V5. To show equivalence of loading, cell lysates were blotted with a pan ERK1/2 antibody. The intensity of each band was determined by scanning and quantitation using NIH Image software. After normalizing to control lanes, the mSef-mediated changes in phosphorylation are expressed as the mean ± S.D. (n = 3) (see "Results"). B, NIH3T3 cells were transduced with either AdGFP or AdmSef and made quiescent by incubating in 0.1% CS for 36 h followed by stimulation with FGF2 (10 ng/ml) for 24 h, in the presence of 1 µCi/ml [3H]thymidine followed scintillation counting. The results are the mean ± S.D. performed in triplicate and correspond to the -fold induction of [3H]thymidine incorporation relative to unstimulated cells. The data are representative of two independent experiments.

mSef Inhibits FGF Signaling Upstream of Ras-- To further establish whether mSef mediated inhibition of the Raf/MEK/ERK pathway occurs at the level of Ras or upstream of it, we assayed for ERK1/2 phosphorylation in NIH3T3 cells transduced with either AdGFP or AdmSef followed by stimulation with FGF2, PDGF-BB, EGF, or calf serum. Overexpression of mSef does not inhibit ERK1/2 activation by PDGF-BB, EGF, or calf serum in NIH3T3 cells (Fig. 3A), indicating that signaling from Ras is intact in mSef overexpressing cells. In addition, co-transfection of HEK293 cells with ERK2-HA and either a constitutively activated form of FGFR1 (C289R) (19) or an activated form of Ras (RasQ61L) with or without mSef demonstrates that mSef effectively decreases ERK phosphorylation induced by a constitutively active FGFR1 but not by activated RasQ61L (Fig. 3B). Furthermore, employing RBD-GST pull-downs, we show that adenoviral-mediated overexpression of mSef inhibits FGF-induced activation of Ras in NIH3T3 cells (Fig. 3C). Since FGF and PDGF have been shown to activate the PI 3-kinase/Akt signaling cascade albeit by different mechanisms (7, 28), we sought to determine whether mSef inhibits FGF-induced Akt activation. Immunoblot analysis was performed using anti-phospho-Akt antibodies that specifically recognize activated Akt. Lysates from NIH3T3 cells transduced with either AdGFP or AdmSef and stimulated with either FGF2 or PDGF-BB were immunoblotted with anti-phospho-Akt or anti-Akt antibodies (Fig. 3D). AdmSef inhibited FGF-induced, but not PDGF-induced, activation of Akt. These results indicate that mSef inhibits multiple FGF-induced signaling pathways and that this inhibition occurs upstream of Ras.


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Fig. 3.   mSef inhibits FGFR1-induced but not RasQ61L-induced ERK activation. A, NIH3T3 cells were transduced with either AdGFP or AdmSef followed by serum starvation for 36 h. Transduced cells were stimulated with FGF2 (10 ng/ml), PDGF-BB (10 ng/ml), EGF (50 ng/ml), or 10% calf serum for 10 min followed by lysis and immunoblotting for pERK1/2, pan-ERK, and the V5 epitope (Sef). B, HEK293 cells were transiently transfected with ERK2-HA (0.5 µg) with either an activated FGFR1 (C289R) (0.5 µg) or an activated Ras (Q61L) (5.0 ng), with or without mSef (4.0 µg). Cells were serum-starved for 24 h followed by cell lysis, immunoprecipitation with anti-HA antibodies, and immunoblotting with pERK1/2 and anti-HA antibodies. Cell lysates were immunoblotted for FGFR and V5 (Sef) to demonstrate the level of expression. C, NIH3T3 were transduced with either AdGFP or AdmSef followed by serum starvation for 36 h. Transduced cells were stimulated with FGF2 (10 ng/ml) for 10 min followed by lysis and pull-down with GST-RBD according the manufacturer's protocol. GST-RBD pull-downs were immunoblotted for Ras. Whole cell lysates were immunoblotted for anti-Ras and V5 (Sef). D, NIH3T3 cells were transduced with either AdGFP or AdmSef followed by serum starvation for 36 h. Transduced cells were stimulated with FGF2 (10 ng/ml) for 10 min followed by lysis and immunoblotting with antibodies to pAkt (pS473), Akt, and V5 (Sef). E, HEK293 cells were transiently transfected with ERK2-HA (0.5 µg) and FGFR1 (0.5 µg) with either mSef (4.0 µg) or mSefIC (4.0 µg) followed by serum starvation for 24 h. Cells were stimulated with FGF2 (10 ng/ml) for 10 min. Cell lysates were prepared and subjected to immunoprecipitation with anti-HA antibodies followed by immunoblotting for pERK1/2 (ERK2-HA), anti-HA (ERK2-HA), V5 (Sef), and FGFR1. The intensity of each band was determined by scanning and quantitation using NIH Image software. After normalizing for expression of ERK2-HA in each sample, mSef-mediated inhibition of ERK2-HA phosphorylation was 93 ± 2% (mean ± S.D.), whereas mSefIC expression resulted in a 69 ± 13% decrease in ERK1/2 phosphorylation (n = 6).

Since mSefIC mediates binding to FGFR1, we next sought to determine whether mSefIC was sufficient to inhibit FGF-induced ERK phosphorylation. HEK293 cells were co-transfected with ERK2-HA, FGFR1 with or without mSef or mSefIC (Fig. 3E). mSefIC was able to inhibit FGF-induced ERK2-HA phosphorylation, but to a lesser extent when compared with full-length mSef. These results indicate that while mSefIC is sufficient to interact with FGFR1, the inhibitory effect of mSefIC on FGF signaling is reduced.

mSef Inhibits FGFR1 Tyrosine Phosphorylation-- Since mSef co-immunoprecipitates with FGFR1, we next sought to determine whether mSef affects FGFR1 tyrosine phosphorylation. Lysates of NIH3T3 cells transduced with either AdGFP or AdmSef and treated with or without FGF2 were subjected to immunoprecipitation with FGFR antibodies followed by immunoblotting with anti-phosphotyrosine, anti-FGFR, and V5 (Sef) antibodies. These data indicate that adenovirus-mediated expression of mSef reduced FGF-induced tyrosine phosphorylation of FGFR without affecting the overall level of FGFR expression (Fig. 4A). Densitometric analysis of immunoblots from three independent experiments indicates that overexpression of mSef mediates a greater than 70% decrease in ligand-induced FGFR1 tyrosine phosphorylation (Fig. 4B). Since FRS2 is a major substrate of FGFR1, we immunoprecipitated FRS2 from lysates of NIH3T3 cells transduced with AdmSef or AdGFP with or without FGF2 stimulation. Overexpression of mSef in NIH3T3 cells results in a decrease in FGF-induced tyrosine phosphorylation of FRS2 (Fig. 4C). Densitometric analysis of immunoblots indicates that mSef reduces FRS2 tyrosine phosphorylation by almost 70% (Fig. 4D). These data strongly indicate that the interaction of mSef with FGFR1 mediates a reduction in FGF-induced tyrosine phosphorylation of FRS2. Taken together these data indicate that mSef binds to FGFR1 and mediates a reduction in tyrosine phosphorylation of FGFR1 and its immediate downstream target, FRS2, thereby modulating multiple FGF-mediated signaling pathways.


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Fig. 4.   mSef inhibits FGFR1 and FRS2 tyrosine phosphorylation. A, NIH3T3 cells were transduced with AdGFP or AdmSef followed by serum starvation for 36 h. Cells were then stimulated with FGF2 (10 ng/ml) for 10 min, and cell lysates were prepared and subjected to immunoprecipitation with anti-FGFR antibodies (FB817) or normal rabbit IgG (nrIgG) as a control followed by immunoblotting with anti-phosphotyrosine antibodies (4G10). Blots were stripped and re-blotted with anti-FGFR and V5 antibodies (Sef). B, intensity of FGFR1 tyrosine phosphorylation was determined by scanning and quantification with NIH Image software. Overexpression of mSef resulted in a 72 ± 6% (mean ± S.D.) decrease in FGFR1 tyrosine phosphorylation (n = 3). C, NIH3T3 cells were transduced and stimulated with FGF2 as described above. Cell lysates were lysates subjected to immunoprecipitation with anti-FRS2 antibodies and immunoblotting with anti-phosphotyrosine antibodies. Blots were stripped and reprobed with FRS2 antibodies. Cell lysates were also immunoblotted with anti-V5 to demonstrate level of mSef expression. D, intensity of FRS2 tyrosine phosphorylation was determined as described above. Overexpression of mSef resulted in 70 ± 15% (mean ± S.D.) decrease in FRS2 tyrosine phosphorylation (n = 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

zSef was shown to inhibit FGF signaling in zebrafish and Xenopus embryos (15, 18), at least in part by inhibiting activation of MAPK (ERK1/2); however, the mechanism by which this occurs is not understood. It has also been reported that zSef associated with FGFR1and FGFR2 in co-immunoprecipitation assays (15). In the present study we sought to establish a functional link between these two observations. To begin to address potential mechanisms for Sef function in FGF signaling, we have examined its effects on FGF-mediated cell proliferation in vitro. We show that overexpression of mSef in NIH3T3 cells results in inhibition of FGF-induced cell proliferation. This inhibition is mediated at least in part by a reduction in ERK1/2 activation. Immunoblot analysis with antibodies that recognize the activated forms of Raf-1 and MEK reveals that activation of these effectors is also reduced by forced expression of mSef and indicates that mSef most likely acts upstream of Raf-1. Overexpression of mSef in the presence of a constitutively activated FGFR1 resulted in a reduction in ERK activation, whereas in the presence of an activated Ras, mSef had no effect on ERK activation. Indeed, overexpression of mSef in NIH3T3 cells results in an inhibition of FGF-induced activation of Ras. The unanticipated result that mSef overexpression did not affect ERK activation by PDGF, EGF, or CS suggests that Ras signaling is intact in mSef overexpressing cells and that mSef functions upstream of Ras specifically at the level of the FGFR. Further support for the specificity of mSef for FGF-mediated signaling was demonstrated by a reduction in activation of Akt by FGF but not by PDGF. The inhibition of Raf/MEK/ERK by mSef is often incomplete but is consistent with the level of inhibition of tyrosine phosphorylation of FGFR1 and FRS2. Failure of mSef to completely inhibit all FGFR1 signaling indicates that either mSef is not a high affinity antagonist and/or that mSef may require another protein to exert complete inhibition of signaling through FGFR1.

Since mSef interacts with FGFR1, we examined the effect of mSef on FGF-induced FGFR1 tyrosine phosphorylation. Overexpression of mSef reduces FGF-induced tyrosine phosphorylation of FGFR1 suggesting that mSef inhibits, at least in part, receptor activation. Although the cytoplasmic domain of mSef is necessary for efficient binding to FGFR1, its inhibitory effect on ERK1/2 activation was reduced when compared with full-length mSef. These data suggest a mechanism that requires the extracellular domain of mSef for full inhibitory activity of FGFR-mediated cell signaling. One possibility is that mSef interferes with receptor dimerization and subsequent transphosphorylation. Another potential mechanism might involve modification of FGFR interaction with its ligands by mSef resulting in attenuation of FGFR-mediated signal transduction.

FGF-mediated tyrosine phosphorylation of FRS2 is decreased in mSef overexpressing cells. Inhibition of FRS2 tyrosine phosphorylation is consistent with mSef inhibiting multiple FGF signaling pathways. This suggests that the observed inhibition of activation of the ERK pathway is due to reduced recruitment of Grb2 to FRS2. Furthermore, the mSef-mediated reduction in Akt activation is also most likely due to reduced recruitment of Grb2 to FRS2, since Grb2 mediates binding of Gab1 to FRS2, which results in activation of PI 3-kinase and Akt (7). These results also address the issue of the specificity of mSef for FGFR signaling. FGF-mediated activation of both ERK and Akt requires tyrosine phosphorylation of FRS2 and subsequent binding of Grb2 (7), whereas Grb2 and PI 3-kinase bind directly to tyrosine-phosphorylated PDGFR to activate the ERK pathway and Akt (28). These differences may explain in part the ability of mSef to inhibit FGF-mediated ERK and Akt activation but not PDGF-mediated ERK and Akt activation.

In a previous report, data were presented to indicate that in zebrafish embryos, overexpression of zSef was able to inhibit MAPK activation induced by FGF8 as well as constitutively activated Ras, Raf, and MEK, indicating that Sef acts downstream or at the level of MEK (18). Our data indicate that mSef acts at a point upstream of Ras at the level of FGFR1 and not at the level of MEK. It is unlikely that these differences are due to difference in zSef and mSef, since zSef also inhibits FGFR tyrosine phosphorylation (data not shown). The differences could arise from differences in assays using zebrafish embryos or cell culture systems. It is also possible that the function of endogenous Sef may differ from that of overexpressed Sef. For example, if Sef acts as a scaffolding protein, overexpressed Sef may compete for a limited pool of interacting proteins, thus preventing assembly of functional signaling complexes. Further work in this area will reveal the reason for these differences.

    ACKNOWLEDGEMENTS

We thank P. Robbins, S. Gutkind, I. Daar, A. Yoshimura, and Viktoria Andreeva for reagents. We thank T. Maciag, J. Verdi, C. Vary, I. Dawid, and M. Tsang for helpful discussions and comments during the course of this work.

    FOOTNOTES

* This work supported by National Institutes of Health Grants DE13248, HL65301, and RR15555 (to R. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

|| To whom correspondence should be addressed: Center for Molecular Medicine, Maine Medical Center Research Inst., 81 Research Dr., Scarborough, ME 04074-7205. Tel.: 207-885-8147; Fax: 207-885-8179; E-mail: friesr@mmc.org.

Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.C200606200

    ABBREVIATIONS

The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; SH, Src homology; PI, phosphatidylinositol; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RTK, receptor tyrosine kinase; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; CS, calf serum; EGF, epidermal growth factor; HA, hemagglutinin; MAPK, mitogen-activated protein kinase; RBD, Ras binding domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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