©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reduced Activation of RAF-1 and MAP Kinase by a Fibroblast Growth Factor Receptor Mutant Deficient in Stimulation of Phosphatidylinositol Hydrolysis (*)

(Received for publication, October 7, 1994; and in revised form, December 5, 1994)

Jiaoti Huang (§) Moosa Mohammadi Gerard A. Rodrigues (¶) Joseph Schlessinger (**)

From the Department of Pharmacology, New York University Medical Center, New York, New York 10016

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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^1 (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 Grb2bulletSos 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.


MATERIALS AND METHODS

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 alpha, beta, 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 beta-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(2), 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(2). 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 KH(2)PO(4), 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 times 10^6 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.


RESULTS

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.



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.


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).

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.^2Fig. 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.


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.



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).


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.



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).


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).



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).


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.




DISCUSSION

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.


FOOTNOTES

*
This work was supported in part by a grant from Sugen (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Fellow of the Leukemia Society of America.

Supported by a long term fellowship from the International Human Frontier Science Program Organization (HFSPO).

**
To whom correspondence should be addressed: Dept. of Pharmacology, New York University Medical Center, 550 First Ave., New York, NY. Tel.: 212-263-7111; Fax: 212-263-7133.

(^1)
The abbreviations used are: GAP, GTPase-activating protein; FGF, fibroblast growth factor; FGFR, FGF receptor; aFGF, acidic FGF; PLC, phospholipase C; PI, phosphatidylinositol; MAP, microtubule-associated protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; MAPK, MAP kinase; PDGF, platelet-derived growth factor; NGF, nerve growth factor; EGF, epidermal growth factor; IL, interleukin; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis.

(^2)
K. Degenhardt, N. Li, N. W. Gale, M. Mohammadi, J. Schlessinger, and D. Bar-Sagi, manuscript submitted for publication.


ACKNOWLEDGEMENTS

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.


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