(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- (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.
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- (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) ).
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-, 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) .
To further study the function of PLC- 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.
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- 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).
Figure 2:
The Y766F FGFR does not phosphorylate
PLC- 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.
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
[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.
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
[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.
Figure 8:
Two-dimensional phosphopeptide mapping of
Raf-1. a, cells were starved in phosphate-free medium for 4 h
followed by [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).
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-. 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) .
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--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).
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-. 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.
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--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.
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- 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.