From the 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
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ABSTRACT |
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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.
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 C 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.
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.
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.
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).
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.
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.
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.
INTRODUCTION
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EXPERIMENTAL PROCEDURES
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(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.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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RESULTS
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ABSTRACT
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DISCUSSION
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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 Ig 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.
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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.
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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).
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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).
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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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.
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