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(Received for publication, October 7,
1994; and in revised form, December 5, 1994) From the
Signaling via the fibroblast growth factor receptor 1 (FGFR1, flg) was analyzed in Ba/F3 hematopoietic cells expressing
either wild-type or a mutant FGF receptor (Y766F) unable to activate
phospholipase C-
Growth factor receptor tyrosine kinases play an important role
in the control of cell proliferation, differentiation, and malignant
transformation (for a review, see (1) ). Much has been learned
in recent years about the process through which this family of
receptors transduces their mitogenic signals. Generally, binding of
growth factors to their surface receptors induces receptor
dimerization, activation of protein tyrosine kinase activity and
autophosphorylation (for a review, see (2) ). Consequently,
cellular target proteins, such as PLC- FGFR1 or flg is a receptor tyrosine kinase
which, upon binding of various fibroblast growth factors including
acidic FGF (aFGF), is activated leading to mitogenesis of some cell
types or differentiation of others(6) . One of the target
molecules of FGFR1 is PLC- To further study the function of PLC-
Mammalian expression vectors which direct the synthesis of
wild-type FGFR1 or a mutant FGFR in which tyrosine 766 was replaced by
a phenylalanine residue (Y766F FGF receptor) were transfected into
Ba/F3 cells by electroporation. After limiting dilution and G418
selection, stable clones were selected and expanded, and expression of
FGFR was analyzed by immunoprecipitation/immunoblotting analysis. While
no endogenous FGFR was detected in parental Ba/F3 cells, expression of
FGF receptors was detected in several positive Ba/F3 clones.
Experiments presented below were carried out with clones B/WT (for
cells expressing wild-type FGF receptors) and B/Y766F (for cells
expressing the Y766F FGFR mutant) and were repeated using several other
clones with similar results. Clone B/Y766F and clone B/WT express
90,000 and 65,000 receptors/cell, respectively (Fig. 1A)(28) .
Figure 1:
Expression of wild-type and Y766F FGFR
in Ba/F3 cells (A) and ligand-dependent receptor
autophosphorylation (B). Cells were either
unstimulated(-) or stimulated with 50 ng/ml of FGF for 5 min at
37 °C. Lysates were prepared and immunoprecipitated with anti-FGFR
antibodies. A, half of the immunoprecipitation reaction was
resolved by SDS-PAGE and immunoblotted with anti-FGFR antibodies. B, the other half of the reaction was resolved by SDS-PAGE and
immunoblotted with anti-phosphotyrosine
antibodies.
To characterize cell lines
that express wild-type or Y766F FGF receptors, the cells were
stimulated with aFGF, and receptor autophosphorylation was analyzed.
aFGF induced strong tyrosine autophosphorylation of wild-type FGFR (Fig. 1B). As previously shown, autophosphorylation of
the Y766F receptor mutant was weaker since a major tyrosine
autophosphorylation site was eliminated in this mutant
receptor(8) . In addition, the wild-type receptor stimulated
tyrosine phosphorylation of PLC-
Figure 2:
The Y766F FGFR does not phosphorylate
PLC-
Figure 3:
The Y766F FGFR induces a lower level of
MAP kinase activation in comparison to activation by the wild-type
receptor. Cells were stimulated and lysates prepared as described in Fig. 1. A, same amount of total cell lysate from each
sample was resolved by SDS-PAGE and immunoblotted by
anti-phosphotyrosine antibodies. The upper arrow indicates the
position of FGFR. B, the same lysates used in A were
immunoprecipitated by anti-MAP kinase antibodies, and in vitro kinase assay was carried out using myelin basic protein as the
substrate. The reaction was resolved by SDS-PAGE, and phosphorylated
myelin basic protein was visualized by autoradiography. C,
bands seen in B that correspond to myelin basic protein were
cut and radioactivity quantified by scintillation
counting.
To confirm that the differences
in MAP kinase tyrosine phosphorylation reflected differences in enzyme
activity, a MAP kinase assay was carried out using myelin basic protein
as a substrate. Approximately 3-fold higher phosphorylation of myelin
basic protein was obtained with MAP kinase immunoprecipitated from B/WT
cells as compared to myelin basic protein phosphorylation by MAPK
immunoprecipitated from B/Y766F cells (Fig. 3B). A
similar kinetics of MAP kinase activation was observed in both cell
lines after FGF stimulation. A lower level of MAP kinase activity was
observed at every time point in B/Y766F cells as compared to B/WT cells
(data not shown).
Figure 4:
Wild-type and mutant FGF receptors induce
a similar Sos mobility shift. Cells were starved with growth factor and
stimulated with aFGF for 5 min. Cell lysates were prepared and
immunoprecipitated with anti-Sos1 antibodies. After SDS-PAGE, proteins
were transferred to nitrocellulose and immunoblotted with anti-Sos1
antibodies.
A Ras-GTP assay was carried out as previously
described(25) , and the Ras-bound GTP/GDP ratio was quantified
using a PhosphorImager (Fig. 5). The addition of aFGF to Ba/F3
cells expressing either wild-type or mutant FGFR led to similar
activation of Ras. Therefore, the difference in the abilities of these
two receptors to induce MAP kinase activation could not be accounted
for by their abilities to activate Ras.
Figure 5:
The Y766F FGFR induces normal Ras
activation. Cells were starved and labeled with
[
Figure 6:
Wild-type and Y766F FGF receptors induce
different Raf-1 mobility shift and kinase activity. Cells were
stimulated with aFGF and lysates prepared as described in Fig. 1. A, lysates were immunoprecipitated with
anti-Raf-1 antibodies followed by immunoblotting with anti-Raf-1
antibodies. B, the experiment described in A was
repeated except that the samples were loaded in a different order to
facilitate comparison. C, in vitro Raf-1 kinase assay
using Syntide II as substrates.
Figure 7:
The Y766F FGFR fails to induce PKC
translocation. Starved cells were stimulated with either TPA (100
nM) or aFGF for 5 min. Cytosolic (c) and membrane (m) fractions were prepared as described under
``Materials and Methods.'' These fractions were resolved by
SDS-PAGE and immunoblotted with anti-PKC
antibodies.
We next compared the
pattern of Raf-1 phosphorylation in response to aFGF stimulation of
cells expressing wild-type or mutant FGF receptors utilizing
two-dimensional phosphopeptide mapping analysis. Raf-1 was labeled with
[
Figure 8:
Two-dimensional phosphopeptide mapping of
Raf-1. a, cells were starved in phosphate-free medium for 4 h
followed by [
Figure 9:
Proliferation of B/WT and B/Y766F cells in
the presence of aFGF. B/WT (open squares) and B/Y766F (solid squares) were cultured in the presence of 10 ng/ml FGF
and 10 µg/ml heparin. Cells were counted using a hemocytometer
after addition of trypan blue. The small reduction in aFGF-induced
proliferation of B/Y766F cells was consistently observed in several
independent experiments.
Several important components of intracellular signaling
pathways activated by receptor tyrosine kinases have been identified,
and their functions are being gradually unveiled. A common target for
many receptor tyrosine kinases is PLC- It is likely that multiple pathways exist to
transduce signals from receptor tyrosine kinases to the nucleus. If
multiple pathways do exist, it is important to know whether the
different pathways relay signal to the nucleus independently, or
whether they converge at some point to activate common downstream
elements. The identification of key molecules that receive multiple
signals from the cell surface and relay them to the nucleus is of great
interest. In the present report we used Ba/F3 hematopoietic cells
normally dependent on IL-3 for survival to study signaling via FGF
receptors. Ba/F3 cells expressing Y766F mutant exhibited approximately
3-fold weaker MAP kinase stimulation in response to aFGF treatment as
compared to cells expressing wild-type FGF receptor. aFGF-induced Ras
activation was similar in cells expressing wild-type receptor and the
Y766F receptor mutant, consistent with conclusions derived from
previous reports demonstrating normal Ras response in cells expressing
a PDGF receptor mutant unable to stimulate PI hydrolysis(25) . The Ser/Thr kinase Raf-1 was activated to a lesser extent in cells
expressing the Y766F mutant. Previous studies have shown that PKC can
directly phosphorylate Raf-1 and increase its kinase
activity(42, 43) . A reasonable hypothesis therefore
is that aFGF can activate Raf-1 in a Ras-dependent mechanism and by
PLC- The results presented in this report are consistent with
the view that at least two pathways are stimulated by a single receptor
to activate a common signaling molecule such as Raf, which then
activates MAP kinase and mitogenesis. A model is proposed in Fig. 10to explain the information flow inside the cell upon FGF
receptor activation. In support of this model, a recent report showed
that expression of a dominant interfering mutant of Ras did not
completely abrogate EGF-induced MAP kinase activation in two different
types of cells(16) . Expression of a dominant interfering
mutant of Ras together with inhibition of PKC were required for
complete inhibition of EGF-induced MAPK activation in Swiss 3T3 cells.
It was concluded that EGF stimulation of MAP kinase is mediated by
Ras-dependent and PKC-dependent mechanisms in these cells(16) .
Figure 10:
A model for mitogenic signal transduction
after growth factor stimulation. At least two pathways are utilized to
transduce signals elicited by FGFR1 stimulation, one dependent upon Ras
and a second pathway dependent on PLC-
We have previously shown that PI hydrolysis is not required for
aFGF-induced mitogenic signaling in L6
myoblasts(9, 10) . Similarly, both wild-type and the
Y766F FGFR mutant stimulate neuronal differentiation of PC12
cells(11) . Our observation that Ba/F3 cells expressing
wild-type or the Y766F mutant proliferate in response to aFGF
stimulation with a similar dose response are consistent with the notion
that PI hydrolysis induced by aFGF is not required for the
proliferation of Ba/F3 cells. aFGF-induced Ras-dependent activation of
MAP kinase is apparently sufficient for transducing of a mitogenic
signal although full activation of MAP kinase may require participation
of both the Ras-dependent and PLC- A similar rationale may also apply for the role
of Ras signaling pathway in NGF-induced differentiation of PC12 cells.
Elimination of Shc binding site in NGF receptor reduces NGF-induced
differentiation of PC12 cells. However, total abrogation of
differentiation is observed only following the elimination of both Shc
and PLC-
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5065-5072
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(PLC-) and stimulate phosphatidylinositol
(PI) hydrolysis. Stimulation of cells expressing wild-type or mutant
FGFR with acidic FGF (aFGF) caused similar activation of Ras. However,
an approximately 3-fold reduced activation of Raf-1 and MAP kinase was
observed in aFGF-stimulated cells expressing mutant receptors as
compared to cells expressing wild-type FGF receptors. Comparison of
phosphopeptide maps of Raf-1 immunoprecipitated from the two cell types
activated by either aFGF or the phorbol ester
(12-O-tetradecanoylphorbol-13-acetate) suggests that Raf-1 is
phosphorylated by both Ras-dependent and PLC--dependent
mechanisms. In spite of the differential effect on Raf-1 and MAP kinase
activation, aFGF stimulated similar proliferation of cells expressing
wild-type or mutant receptors indicating that Ras-dependent activation
of Raf-1 and MAP kinase is sufficient for transduction of FGFR
mitogenic signals. Ras may also activate signal transduction pathways
that are complementary or parallel to the MAP kinase pathway to
stimulate cell proliferation.
(phospholipase C) and
GAP
(ras GTPase-activating protein) bind to tyrosine
autophosphorylation sites in the receptor cytoplasmic domain and become
phosphorylated on tyrosine residues (for a review, see (3) ).
Tyrosine autophosphorylation sites serve as binding sites for adaptor
proteins such as Grb2, Shc, and Nck. These proteins bind to activated
receptors through their src homology 2 (SH2) domains. Genetic
and biochemical studies have demonstrated that Grb2 is bound to the Ras
guanine nucleotide-releasing factor Sos, through its src homology 3 (SH3) domains. The binding of Grb2
Sos complex to
tyrosine autophosphorylated EGF receptor results in translocation of
Sos to the plasma membrane in the vicinity of Ras, resulting in
exchange of GDP for GTP and activation of Ras (for a review, see (4) ). This leads to activation of a kinase cascade composed of
Raf, MAP kinase kinase, and MAP kinase (MAPK) (for a review, see (5) ). It is now well established that the Ras signaling
pathway plays an important role in initiation of cell proliferation by
numerous growth factors and lymphokines (for a review, see (3) )., which, upon ligand stimulation, binds
to the receptor and becomes tyrosine phosphorylated and activated,
leading to hydrolysis of phosphatidylinositol (PI) (for a review, see (7) ). We have previously identified Tyr-766 in the cytoplasmic
tail of FGFR1 as the binding site for PLC-(8) .
Elimination of Tyr-766 by site-directed mutagenesis prevents
FGF-induced PI hydrolysis and Ca
release in
transfected cells(9, 10) . However, aFGF is still able
to induce DNA synthesis in L6 cells or differentiation of PC12 cells
expressing the Y766F mutant, indicating that PI hydrolysis is not
essential for FGF-induced mitogenesis of L6 myoblasts and neuronal
differentiation of PC12 cells(9, 10, 11) .
Similar results were obtained with a PDGF receptor mutant that does not
bind PLC- and does not stimulate PI
hydrolysis(12, 13, 14) . However, other
studies suggested that PLC- may play a role in PDGF-induced
mitogenic signaling(15) . These two views are not necessarily
contradictory since receptor tyrosine kinases may induce multiple
signals to activate mitogenesis, one of which may involve PLC-
activation. This possibility is supported by recent studies implicating
MAP kinase as a critical component of mitogenic signaling pathway. MAPK
can be activated by both Ras-dependent and Ras-independent
mechanisms(16) . Moreover, elimination of both the Shc and
PLC- binding sites in the NGF/Trk receptor was shown to be
required for complete inhibition of NGF-induced neuronal
differentiation of PC12 cells(17, 18, 19) . in mitogenic signal
transduction of receptor tyrosine kinases, we transfected wild-type and
the Y766F mutant FGF receptors into an IL-3-dependent hematopoietic
cell line Ba/F3 which does not express endogenous FGF receptors, and
studied signaling via the FGF receptor. We demonstrate that in cells
expressing the Y766F mutant aFGF-stimulated activation of MAPK was
reduced as compared to activation of MAPK in cells expressing wild-type
FGF receptors. However, Ras was activated similarly in the two cell
lines, suggesting that MAPK may be activated through both Ras-dependent
and PLC--dependent pathways. Nevertheless, participation of both
pathways is probably required to achieve full activation of MAP kinase.
We further show that Raf-1 can integrate signals from multiple pathways
initiated by FGF stimulation to activate cellular events required for
mitogenesis.
Antibodies
Rabbit polyclonal anti-flg antibodies used for immunoprecipitation were generated against a
glutathione S-transferase fusion protein containing amino
acids 750-822 of flg. The antibodies for flg immunoblotting was generated against a synthetic peptide derived
from the kinase insert region (amino acids 580-586) of flg. Polyclonal anti-PLC- antibodies were generated
against a synthetic peptide derived from amino acids 1274-1292
(PFEDFRISQEHLADHFDS) in the C terminus of rat PLC-. A synthetic
peptide derived from the CDC25 homology region of human Sos1 (residues
738-757) was used to produce polyclonal anti-Sos1 antibodies.
Polyclonal anti-MAP kinase antibodies were a gift from C. Marshall and
were raised against a C-terminal peptide of ERK2(20) .
Polyclonal anti-Raf-1 antibodies were purchased from Upstate
Biotechnology, Inc., and were raised against a peptide derived from
human Raf-1. Blotting antibodies for PKC was prepared against a peptide
that is common to isoforms 
, 
, and and was
affinity-purified(21) . Monoclonal anti-Ras antibody Y13-259
has been described previously(15) .Cell Lines and Stable Transfection
Ba/F3 cells
were grown in RPMI supplemented with 10% fetal bovine serum together
with 10% Wehi supernatant containing IL-3(22) . Mammalian
expression vectors which direct the synthesis of wild-type or Y766F FGF
receptors were previously described(10) . For expression in
Ba/F3 cells the cDNAs encoding the wild-type or Y766F mutant of FGFR
were cloned into the pAW-neo3 vector which contains a
neomycin-resistance gene(23) . Transfection by electroporation
was carried out according to a previously published
protocol(23) . Stable cell lines were established following
cloning by limiting dilution, G418 selection, and screening for
expression of FGFR by immunoprecipitation/immunoblotting with
polyclonal anti-FGFR1 (flg) antibodies(10) .Stimulation of Cells by Growth Factor and Preparation of
Cell Lysates
Parental Ba/F3 cells and cells that express
wild-type or mutant FGFR1 were grown in medium containing IL-3 (Wehi
supernatant). Cells were washed three times in medium without IL-3 and
starved for 10 h. Stimulation was performed by addition of 50 ng/ml
aFGF and 10 µg/ml heparin for 5 min at 37 °C. Unstimulated and
stimulated cells were lysed with lysis buffer (50 mM Hepes, pH
7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl, 1 mM EGTA, 100 mM sodium fluoride, 10
mM sodium pyrophosphate, 1 mM sodium orthovanadate,
10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged for 10
min at 4 °C to remove nuclei and cell debris.Immunoprecipitation and Immunoblotting
Cell
lysates were incubated with rabbit polyclonal antibodies for 1 h at 4
°C with shaking. Immunocomplexes were bound to protein A-Sepharose
and washed five times with lysis buffer. SDS-PAGE sample buffer was
added to the beads, and the mixture was boiled for 5 min. After
SDS-PAGE analysis proteins were transferred to nitrocellulose filters
that were blocked with Tris-buffered saline-bovine serum albumin and
incubated with polyclonal antibodies for 2 h at room temperature. Bound
antibodies were detected either with 
I-protein A or with
an ECL detection kit.Determination of MAP Kinase Activity
Cell lysates
were immunoprecipitated with polyclonal anti-MAP kinase
antibodies(20) , and the immunocomplexes were immobilized on
protein A-Sepharose beads. The beads were washed extensively with lysis
buffer followed by two washes with a kinase reaction buffer (10 mM Tris, pH 7.4, 10 mM MgCl). The kinase assay was carried
out in 50 µl of kinase buffer containing 0.5 mg/ml myelin basic
protein and 0.1 µCi/ml [-
P]ATP for 30
min at 30 °C. Proteins were analyzed by SDS-PAGE and detected by
autoradiography.Raf-1 Kinase Assay
Full details of the procedure
were previously described(24) . Briefly, rabbit polyclonal
anti-Raf-1 antibodies or preimmune serum was incubated with cell
lysates for 1 h at 4 °C. The immunocomplexes were bound to protein
A-Sepharose beads for an additional 1 h. The beads were washed with 0.5 M LiCl solution, resuspended in 38 µl of buffer (25 mM Tris-HCl, pH 7.5, 10 mM MnCl2, 10 µM ATP, 1
mM dithiothreitol, 25 mM -glycerophosphate), 1
µl of 4 mM Syntide II peptide, and 0.1 µCi of
[-P]ATP, and incubated for 20 min at room
temperature. The reaction mixture was spotted onto Whatman p81 paper
and air-dried. The paper was washed extensively with 0.85% phosphoric
acid and counted in scintillation liquid. Radioactive readings from the
samples with preimmune serum were subtracted from readings obtained for
anti-Raf-1 immunoprecipitates.Ras Assay
Full details of the protocol were
described elsewhere(25) . Cells were starved in phosphate-free
medium for 4 h and labeled for 6 h with 0.5 mCi/ml
[P]orthophosphate. At the end of labeling, cells
were stimulated with 50 ng/ml of aFGF plus heparin for 5 min and lysed
in 50 mM Hepes, pH 7.4, 1% Triton X-100, 100 mM NaCl,
5 mM MgCl
, 1 mg/ml bovine serum albumin, and
protease inhibitors. The lysates were centrifuged to remove the nuclei
and cell debris. The supernatants were transferred to new tubes and
adjusted to 500 mM NaCl, 0.5% sodium deoxycholate, and 0.05%
SDS. Immunoprecipitation was carried out by addition of monoclonal
anti-Ras antibody Y13-259 coupled to CNBr-activated Sepharose 4B and
incubation for 1 h at 4 °C. The beads were then washed extensively
with 50 mM Hepes, pH 7.4, 0.1% Triton X-100, 500 mM NaCl, 0.005% SDS, and 5 mM MgCl. Elution was
carried out by the addition of 20 µl of elution buffer (2 mM EDTA, 2 mM dithiothreitol, 0.5 mM GTP, and 0.5
mM GDP) and incubation for 20 min at 67 °C. The
supernatant was spotted onto a polyethyleneimine cellulose plate, and
chromatography was carried out with 1.0 M KHPO
, pH 3.4. GTP and GDP were visualized
with a UV lamp, and the ratio of GTP versus GDP was determined
by quantitation with a PhosphorImager.PKC Translocation Assay
Starved cells were
stimulated with aFGF and heparin or buffer alone. Preparation of
cytosol or membrane fractions was carried out as described
elsewhere(26) . As a positive control, cells were stimulated
with 100 nM 12-O-tetradecanoylphorbol-13-acetate
(TPA) for 5 min. Cytosol and membrane fractions from 1 
10
cells were resolved by SDS-PAGE followed by immunoblotting with
anti-PKC antibodies according to a published procedure(21) .Raf-1 Phosphopeptide
Mapping
[P]Orthophosphate-labeled cell
lysates were immunoprecipitated with anti-Raf-1 antibodies and resolved
by SDS-PAGE(27) . Radioactive bands corresponding to Raf-1 were
excised from the gel, digested with trypsin as described
before(8) , and eluted. The labeled peptides were separated on
thin layer cellulose plates by electrophoresis at pH 1.9 for 40 min
with 1000 V, followed by ascending chromatography in a buffer
containing 1-butanol/pyridine/acetic acid/water in a ratio of
75:50:15:60. The peptides were visualized with autoradiography and
quantified with a PhosphorImager.Cell Proliferation Study
Cells were cultured
without added growth factor or in the presence of either IL-3 (Wehi
supernatant) or 10 ng/ml aFGF plus 10 µg/ml heparin. Cell numbers
were counted every 24 h after addition of trypan blue with a
hemocytometer. Fresh media were added every 2 days during the course of
the experiment.
while the Y766F receptor failed
to do so (Fig. 2B). We have previously shown that
ligand stimulation induced binding of PLC- via its SH2 domains to
wild-type FGFR leading to PI hydrolysis(8) . However, the Y766F
mutant was unable to bind PLC- and to induce PI hydrolysis and
Ca release in the transfected cells (9, 10, 11, 28) (data not shown).
upon ligand stimulation. Cells were stimulated and lysates
prepared as described in Fig. 1. A, lysates were
immunoprecipitated by anti-PLC- antibodies followed by
immunoblotting by the same antibodies. B, lysates were
immunoprecipitated with anti-PLC- antibodies and immunoblotted
with anti-phosphotyrosine antibodies.
Reduced Activation of MAPK by the Y766F Receptor Mutant as
Compared to the Wild-type Receptor
We first compared the general
pattern of tyrosine-phosphorylated proteins in cells expressing
wild-type or the mutant FGF receptors. Lysates of unstimulated or
stimulated cells were resolved by SDS-PAGE and immunoblotted with
anti-phosphotyrosine antibodies. The most prominent
tyrosine-phosphorylated proteins other than FGF receptors were two
proteins with apparent molecular masses of 42 and 44 kDa. These
proteins were shown to be MAPK using specific anti-MAPK antibodies
(data not shown). The overall pattern of tyrosine-phosphorylated
proteins was similar in Ba/F3 cells expressing wild-type or the Y766F
receptor mutant. However, FGF-induced phosphorylation of MAPK was
significantly reduced in mutant cells as compared to cells expressing
wild-type FGF receptors (Fig. 3A). Quantitation by
PhosphorImager scanning demonstrated that tyrosine phosphorylation of
MAP kinase in B/WT cells was approximately 3-fold stronger in
comparison to that in B/Y766F cells.
Activation of Ras and the Shift of Sos Electrophoretic
Mobility by Wild-type and Y766F FGFRs
MAP kinase plays an
important role as a component of a signaling pathway that relays signal
from the cell surface to the nucleus (for a review, see (5) ).
Additional participants in this pathway include receptor protein
kinases, the adaptor protein Grb2, the guanine nucleotide-releasing
factor Sos, and Ras (for a review, see (4) ). Growth factor
stimulation leads to the exchange of GDP for GTP on Ras (for a review,
see (29) ) and to a shift in the electrophoretic mobility of
Sos due to Ser/Thr phosphorylation by kinases which are dependent upon
Ras activation.Fig. 4shows that stimulation of
Ba/F3 cells expressing wild-type FGF receptors with aFGF induced a
shift in the mobility of Sos (compare lane 4 with lane
3) similar to that seen in L6 cells stimulated with
insulin(31) . A shift in the mobility of Sos was also seen in
aFGF-stimulated Ba/F3 cells expressing the Y766F receptor mutant
(compare lane 6 with lane 5). Hence, aFGF induces a
similar Sos mobility shift in cells expressing either wild-type or the
mutant FGF receptor.
P]orthophosphate as described under
``Materials and Methods.'' Cells were then stimulated with
aFGF, and lysates were immunoprecipitated with the anti-Ras monoclonal
antibody Y13-259. The Ras assay was carried out as described under
``Materials and Methods'' and a PhosphorImager was used to
quantify the ras GTP/GDP ratio.
Reduced Raf-1 Activation in Cells Expressing the Y766F
Receptor Mutant
Since the mutant receptor induced a normal Ras
response but a reduced activation of MAP kinase, it is possible that
activation of MAP kinase by aFGF is achieved by at least two pathways:
one dependent upon Ras and a second dependent upon PLC-.
Biochemical and genetic studies have demonstrated that Ras activation
leads to recruitment and activation of Raf-1, a cellular oncogene with
serine-threonine kinase
activity(32, 33, 34, 35, 36, 37) .
We therefore compared the ability of wild-type and mutant FGF receptors
to activate Raf-1 in response to aFGF stimulation. Growth factor
stimulation leads to phosphorylation of Raf-1 and to a mobility shift,
which correlates with increased kinase activity(38) . aFGF
stimulation did not cause any change in Raf-1 mobility in parental
Ba/F3 cells. However, in cells expressing either wild-type or the Y766F
FGF receptor, a typical mobility shift was observed upon aFGF
stimulation (Fig. 6, A and B). A stronger
shift in the mobility of Raf-1 was observed in cells expressing
wild-type FGFR as compared to cells expressing the Y766F mutant,
suggesting that wild-type and mutant FGF receptors differ in their
abilities to activate Raf-1 kinase. To quantify Raf-1 activation, we
have compared the kinase activity of Raf-1 immunoprecipitated from the
two cell lines using Syntide II as a substrate. The activity of Raf-1
kinase was approximately 3-fold higher in aFGF-stimulated B/WT cells as
compared to aFGF-stimulated B/Y766F cells (Fig. 6C).
Mutant FGFR Fails to Cause PKC Translocation and Induces
a Different Pattern of Raf-1 Phosphorylation
The results
described above suggest that Raf-1 can be activated by Ras-dependent
and -independent pathways. It has been shown recently that Ras is
responsible for translocation of Raf to the plasma membrane which is
then activated by an unknown mechanism(39, 40) .
PLC- activation leads to PI hydrolysis, resulting in production of
inositol triphosphate and diacylglycerol. Diacylglycerol in turn
activates PKC (for a review, see (41) ). It has been shown that in vitro PKC can directly phosphorylate and activate
Raf-1(42, 43) . Therefore, we examined the possibility
that PLC- activation of PKC contributes toward Ras-independent
activation of Raf-1. We assume that the lower activation of Raf-1 and
MAPK is a reflection of the inability of Y766F mutant to activate PKC.
We therefore studied translocation of PKC in response to aFGF
treatment, a process known to correlate with activation of PKC in
response to various stimuli(26) . In unstimulated cells, PKC is
largely localized in the cytosolic fraction (Fig. 7). After
treatment with the phorbol ester TPA, almost all PKC molecules were
found in membrane fraction. A similar translocation was observed in
cells expressing wild-type FGFR upon aFGF stimulation. However, aFGF
was unable to stimulate membrane translocation of PKC in Ba/F3 cells
expressing the Y766F mutant (Fig. 7).
P]orthophosphate in vivo, purified by
immunoprecipitation with anti-Raf-1 antibodies, and digested with
trypsin. A number of phosphopeptides were observed in B/WT cells
stimulated with aFGF (Fig. 8a, Panel D). The
major phosphopeptides are schematically represented in Fig. 8b. By comparing Panel D with C,
it is clear that peptides a, e, and i (group 1 phosphopeptides) were
constitutively phosphorylated, and their level of phosphorylation was
not significantly changed by aFGF stimulation. A significant increase
in phosphorylation was observed for peptides b, c, d, f, g, and h
(group 2 phosphopeptides) after aFGF stimulation. Among group 2
peptides, phosphorylation of peptides b, c, and f was also induced by
TPA, while phosphorylation of peptides d, g, and h was induced only by
aFGF stimulation (compare Panel D and B). aFGF
induced an increase in phosphorylation of peptide b in B/WT cells
(quantitation using a PhosphorImager showed that phosphorylation of
this peptide is increased by approximately 8-fold over the control),
while no significant change was detected in the phosphorylation of this
peptide in B/Y766F cells stimulated with aFGF. However, in both B/WT
and B/Y766F cells, aFGF induced a similar increase in the
phosphorylation of peptides d, g, and h. The phosphorylation of these
peptides was not induced by TPA treatment. Moreover, phosphorylation of
peptides c and f, which is stimulated by both aFGF and TPA treatments,
was similar in Raf-1 immunoprecipitated from either B/WT or B/Y766F
cells. The results of quantitation by PhosphorImager of the relative
intensities of peptides a, b, and d in the three cell lines before and
after stimulation are shown in Fig. 8c.
P]orthophosphate labeling for 6 h.
Cells were then stimulated with TPA (100 mM) or aFGF for 5
min, and lysates were immunoprecipitated by anti-Raf-1 antibodies.
Proteins were resolved by SDS-PAGE, and Raf-1 bands were digested with
trypsin and eluted. Two-dimensional phosphopeptide mapping was carried
out according to the protocol described under ``Materials and
Methods.'' Arrows indicate the position of peptide b. b, schematic representation of the major phosphopeptides in Panel D of a. o is the origin. c,
representation of the relative intensities of phosphopeptides a, b, and
d in a, Panels B, D, and F over
controls as quantified by a PhosphorImager. Solid bar, Ba/F3
cells stimulated by TPA (a, Panel B); shaded
bar, B/WT cells stimulated with aFGF (a, Panel
D); open bar, B/766F cells stimulated with aFGF (a, Panel F).
aFGF Stimulates the Proliferation of Ba/F3 Cells
Expressing Wild-type and Mutant FGF Receptors
Ba/F3 cells
require IL-3 for normal cell proliferation. We have tested the ability
of aFGF to stimulate the proliferation of cells expressing wild-type or
mutant FGF receptors. Cells expressing wild-type FGFR or the Y766F
mutant receptors were maintained in cell culture in the absence of any
growth factors, in the presence of conditioned medium from Wehi cells
containing IL-3, or in the presence of 10 ng/ml of aFGF together with
10 µg/ml heparin. Both cell lines did not survive in the absence of
added growth factors, and both cell lines proliferated in the presence
of IL-3 with a doubling time of approximately 14 h (data not shown). In
the presence of aFGF with heparin, the two cell lines continued to
proliferate with a doubling time of approximately 24 h (Fig. 9).
There was a small reduction in aFGF, but not IL-3, induced
proliferation of B/Y766F cells in comparison to B/WT cells. This was
consistently observed in several independent experiments with two
different cell lines. In addition, similar dose responses of
proliferation were obtained for these two cell lines after aFGF
treatment (data not shown). Cell cycle analysis with bromodeoxyuridine
incorporation assay and propidium iodide labeling followed by flow
cytometry provided a consistent picture; the distribution of the cells
in each phase of the cell cycle was similar for both cell types in
response to aFGF stimulation (data not shown).
. Growth factor-induced
tyrosine phosphorylation enhances the catalytic activity of PLC-
leading to stimulation of PI hydrolysis (for a review, see (2) ). The Y766F FGFR mutant which does not stimulate PI
hydrolysis exhibited normal aFGF-induced mitogenic response in
transfected L6 myoblasts(9, 10) . Similarly, PDGF
receptor mutants which do not activate PI hydrolysis induced a normal
mitogenic response(12, 13, 14) . Moreover,
CSF-1 receptor does not activate PI hydrolysis, yet CSF-1 stimulation
leads to the proliferation of macrophages expressing endogenous CSF-1
receptors and fibroblasts ectopically expressing CSF-1
receptors(30) . It was therefore proposed that PI hydrolysis is
not crucial for growth factor-induced mitogenic
signaling(9, 10, 12, 13, 14, 30) .
Different conclusions were reached from studies with other PDGF
receptor mutants. When five autophosphorylation sites of PDGF receptor
were eliminated, including the site responsible for PLC- binding,
PDGF no longer induced mitogenic response in HepG2 cells(15) .
However, when the PLC- binding site was restored in the background
of the other four mutated tyrosine phosphorylation sites, the mitogenic
capacity of PDGF receptor was rescued. It was thus proposed that
PLC- activation may play a role in PDGF-induced DNA
synthesis(15) .-dependent activation of PKC which may directly activate Raf.
We do not have a proof for direct PKC phosphorylation of Raf-1 in
living cells. However, comparison of two-dimensional phosphopeptide
maps of Raf-1 phosphorylation in response to aFGF or TPA in the two
different cell types provides some support to this notion. Clearly, one
of the TPA-induced phosphopeptides from Raf-1 is phosphorylated in
aFGF-stimulated wild-type cells, but not in aFGF-stimulated Y766F
cells. However, other peptides that are phosphorylated in response to
aFGF and not by TPA are phosphorylated to a similar extent in the two
cell lines upon aFGF stimulation. These results are consistent with a
mechanism in which Raf-1 is subject to phosphorylation by kinase(s)
that are dependent upon Ras activation and by kinase(s) that are
dependent upon PLC- activation, such as PKC. We do not know why
phosphopeptides c and f (Fig. 8a, Panel D)
that are phosphorylated in response to either TPA or aFGF stimulation
of cells expressing wild-type receptor are also phosphorylated in the
Y766F cells to similar levels by aFGF treatment. One possible
explanation is that these two phosphopeptides contain phosphorylation
sites that serve as targets for both PKC and the Ras-dependent
kinase(s).
. Raf-1 integrates signals
from these two pathways to activate MAP kinase and mitogenesis. The
Ras-dependent pathway is sufficient for aFGF-induced proliferation of
Ba/F3 cells overexpressing FGF receptor.
-dependent pathways. That may
explain why we observed a small but reproducible reduction in the
growth responses induced by the mutant receptor. Nevertheless, it seems
that blocking one signaling pathway in a multipathway signal
transduction system does not result in a significant difference in the
final response if the remaining pathway(s) can deliver a sufficiently
strong signal. This explanation may also be relevant to studies with
various PDGF receptor mutants(15) . For example, the mitogenic
response of PDGF-receptor is abrogated by simultaneous elimination of
five tyrosine autophosphorylation sites(15) . Restoration of
the tyrosine autophosphorylation site responsible for PLC- binding
may lead to the activation of MAP kinase or other pathways that relay
mitogenic response. binding sites (18, 19) . Hence, both Shc
which is required for Ras-dependent pathway and the PLC--dependent
pathway are essential for NGF-induced differentiation of PC12
cells(18, 19) . Thus, the model presented in Fig. 10may apply for signaling via various receptor tyrosine
kinases expressed in different types of cells.
))
We thank C. Marshall for anti-MAP kinase antibodies,
A. Weiss for pAW-neo3 vector, S. Kranczer and D. Yannoukakos for their
help in preparing figures, M. Ren for aid in the PKC translocation
assay, T. Spivak for useful comments on the manuscript, and members of
our laboratory for stimulating discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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