against Raf-1 was a kind gift from Dr. Ulf Rapp (Frederick Cancer. Research Facility ..... Reid, H. H., Wilks, A. F., and Bernard, 0. (1990) Proc. Natl. Acad. Sci.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269, No. 28, Issue of July 15, pp. 18320-18326, 1994 Printed in U.S.A.
Signal Transduction by Fibroblast Growth Factor Receptor-4 (FGFR-4) COMPARISON WITH FGFR-1* (Received for publication, March 16, 1994)
Satu VainikkaS, Vladimir JoukovSO, Stefan Wennstromn, Mathias BergmanS, Pier Giuseppe Peliccill, and Kari AlitaloS** From the SMolecularICancer Biology Laboratory, Department of Pathology, University of Helsinki, Haartmanink. 3, 00290 Helsinki 29, Finland, the llLudwig Institute for Cancer Research, Uppsala Branch, Box 595, S-75124 Uppsala, Sweden, and the IVstituto Clinica Medica, Policlinico Monteluce, University of Perugia, 06100 Perugia, Italy
We have studied the signal transduction pathways of coreceptor. To date, four members of the human FGFreceptor fibroblast growth factor receptor-4 (FGFR-4) and gene family have been characterized: FGFR-1 ( f i g ) (15, 16), FGFR-1, which showed virtually identical acidic fibro- FGFR-2 ( b e k ) (16, 171, FGFR-3 (18),and FGFR-4 (19). Related blast growth factor binding profilesas well as tyrosine receptors binding FGFs havealso been described from chicken, autophosphorylation upon activationin transfected L6 mouse, Xenopus, and Drosophila (20-24). I t has been demonrat myoblasts and NIH3T3 mouse fibroblasts.A promi- strated that each one of these receptors is capable of binding nently tyrosyl-phosphorylated doublet of polypeptides and responding to more than one type of FGF, although the of 85 kDa coprecipitated with activated FGFR-4 from affinities for different FGFs are specific for each receptor type both cell lines studied,but these polypeptideswere (16, 18, 19, 21, 25-28). Furthermore, the complexity of FGFR notdetecteduponimmunoprecipitation of activated biology is increased by alternative splicing that generates reFGFR-1. Furthermore, FGFR-4 induced only a weakty- ceptor isoforms varying in their extraor intracellular domains rosy1 phosphorylation of phospholipase C-7 and no de- (29-31). Binding of growth factor to the extracellular domain of tectable tyrosyl phosphorylation of the SHC adaptor the FGFR leads to receptor dimerization followed by receptor proteins in contrast to FGFR-1. No phosphorylation of autophosphorylation andtyrosine phosphorylation of subRas GTPase-activating protein, p64 SypRTPlD tyrosine strates (32). phosphatase, or association of the GRB2 adaptor proSeveral studies have shown that FGF stimulation leadsto tein SH2 domain with these receptors was detected. Un- the phosphorylation of a set of cellular polypeptides in FGFRlike FGFR-1, FGFR-4 induced only a barely detectable transfected cell lines (26, 33-35). However, littleis known phosphorylation of the cellular serinekhreonine kinase about thepossible specificity of signal transduction by the difRaf-1 and a weaker tyrosyl phosphorylation of mitogenferent FGFRs. Several substrates bind to activated receptor activated protein kinases than FGFR-1. Despite these tyrosine kinases through association between Src homology 2 differences, stimulation of both receptors resulted in in(SH2) domainsandautophosphorylated tyrosine residues. creased DNA synthesis. These include phospholipase C--y(PLC-y), Ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3-kinase, pp60"~""(36-38), the docking or adaptor proteinsGRB2/Sem-5, Fibroblast growth factors (FGFs)' are a family of heparin- SHC, Nck (39-44), and phosphotyrosyl phosphatase Syp/ binding polypeptide mitogens having a role in a wide array of PTPlD (45,46). Thus far, only one ofthese proteins,PLC--y,has biological processes such as cell growth, differentiation, angiobeen identified as a substrate of FGFR-1 (471, and its direct (1-4).At present the genesis, tissue repair, and transformation association with the receptor has been demonstrated (48). A FGF familyconsists of nine members: acidic FGF (aFGF, mutated FGFR-1 in which Tyr766 is replaced with phenylalaFGF-1, 5), basic FGF (bFGF, FGF-2, 6), Int-2 (FGF-3, 7), K- nine is unable to activate PLC-y or t o increase phosphatidylFGF/Hst-1 (FGF-4, 8, 91, FGF-5 (101, FGF-6 (111,keratinocyte inositol turnover. This receptor is, however, still capable of growth factor,(FGF-7, 12), androgen-inducedgrowthfactor transducing a mitogenic signal (34,481, indicating thatPLC--y (13), and glia-activating factor (FGF-9, 141, sharing 30-80% may not be required for FGF-induced mitogenic responses. amino acid sequence identity with each other. The possible association of other signal transducing compoFGFs are known to mediate their biological effects by bind- nents with FGFRs involved in activation of downstream siging t o high affinity cell surface receptors, which consist of a naling molecules is still largely unknown. It has been shown receptor tyrosine kinase and a heparan sulfate proteoglycan that bFGF stimulation of fibroblasts leads to hyperphosphorylation of the Raf-1 serinekhreonine kinase. This finding sug* This workwas supported by The FinnishAcademy, the University of Helsinki, the Sigrid Juselius Foundation, the Finnish Cancer Research gests thatRaf-1 has a role as one of the downstream signaling Organizations and by the Research and Science Foundation of Farmos. components for FGFR (49).In agreement with this idea, it has The costs of publication of this article were defrayed in part by the been demonstrated thatRaf-1 is needed for FGF-induced mespayment of page charges. Thisarticle must thereforebe hereby marked oderm induction inXenopus embryos (50). We have previously "advertisement" in accordance with 18 U.S.C.Section 1734 solelv to shown that after activation of receptors overlapping but differindicate this fact. ent sets of polypeptides are phosphorylated in FGFR-1 and I Supported by the Centre for International Mobility (CIMO). ** TO w h o m correspondence should be addressed. -Fax: 358-0-434- FGFR-4 transfected NIH 3T3 cells (26). Here we have further 6448. analyzed and compared the signal transduction pathways of The abbreviations used are: FGFs, fibroblastgrowth factors, aFGF, the two receptorsusingtransfectedrat L6 myoblasts and acidic FGF; bFGF, basic FGF; GST, glutathione S-transferase; FGFR, We showdifferences as well as fibroblast growth factor receptor; DMEM, Dulbecco's modified Eagle's mouseNIH3T3fibroblasts. similarities in the cascade of protein phosphorylation set forth medium; PLC-7, phospholipase C-y; PBS, phosphate-buffered saline.
18320
FGFR-4 Signal fiansduction by aFGF-stimulation of FGFR-1 or FGFR-4. Despite the observed differences, activation of both receptor types led to increased cellular DNA synthesis.
l2
18321
1 R1,
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"
EXPERIMENTAL PROCEDURES Plasmids-The expression vectors for FGFR-1 and FGFR-4 contain 8the correspondingcDNAs under SV40 and Mo-MuLV Moloney murine leukemia virus 3'U3 long terminal repeat promoters, respectively (26). For production of the GST-PLC-y SH2 fusion protein, the cDNA fragment containing the PLC-y SH2 domain corresponding to base pairs 2030-2632 (GeneBank accession number: M344667) (47) wasamplified by the polymerase chain reaction using the PLC-y cDNA containing plasmid (pMJ30, a kind gift from Dr. Asher Zilberstein) a s a template and primers5'-CCGGATCCCGAC'N"I'CAGAGCCT-3' (forward) and5'GCGAATTCGGGG'M'GACCATCTC-3' (reverse). The polymerase chain reaction product was digested with BamHI EcoRI and and inserted into the corresponding sitesof pGEX-lht (Pharmacia). Cell Culture and I).ansfections-L6 rat myoblasts, which lack endog2 0 I enous FGF receptors,as well a s FGFR-1- and FGFR-4-transfected NIH 0 0.63 1.88 1.25 nM 3T3 cells (26) were grown in DMEM containing 10% calf serum. L6 FIG. 1. Saturation binding and cross-linking of radiolabeled cells weretransfectedusingthe lipofection method (DOTAP,Boehand FGFR-4(R4)expressingL6 cells. The aFGF to FGFR-1 (RI) ringer) and clones positive for wild type and mutant FGFR-4 were cells were incubated for 2 h with different concentrations of iodinated selected using SDS-PAGE and Western blottingof cell lysates followed aFGF, and bound radioactivity was measured. For cross-linking reacby detectionwith FGFR-4-specific antisera.FGFR-1 clones were tions (inset), thecells were incubated for 1.5 h, followed by addition of screened by immunoprecipitation (described below) after aFGF stimu- disuccinimidyl suberate, and cell lysates were analyzedby SDS-PAGE lation, followedby SDS-PAGE and immunoblotting using antibodies (6%). The molecular weights of the cross-linked receptor-ligand comagainst phosphotyrosine. plexes are indicated. Neomycin-resistantL6 cells were used as controls Antisera-FGFR-4, FGFR-1, and SHC antisera have been described ( C ) . previously (25,26 and40, respectively). Thepolyclonal antiserum sp63 Phosphoinositol-3 Kinase Assay-Serum-starved cells were stimuagainst Raf-1 was a kind gift from Dr.Ulf Rapp (Frederick Cancer lated, lysed, and the lysatessubjected to receptor immunoprecipitation Research Facility, Frederick, MD). Monoclonal antibodiesagainst PLC-y were obtained from AMs Biotechnology, monoclonal antibodies a s described above. The immune complexes were washed and a phosagainst phosphotyrosine and mitogen-activated protein kinase from phoinositol-3 kinase assay was performed essentially as described by Fukui and Hanafusa (53).Briefly, the immunocomplexes were washed UBI, and PY20 monoclonal antiphosphotyrosine from Zymed. three times with PBS, 1% Nonidet P-40, once with PBS, once with 0.1 F G F Receptor Cross-linking and Binding Analyses-Recombinant aFGF produced in Sf9 insect cells (51) (a kind gift from Dr. Ralf Pet- M Tris-HCI, pH 7.5, 0.5 M LiC1, once with distilled water, and once with tersson, Ludwig Institute for Cancer Research, Stockholm) was iodi- 20 mM Tris-HC1, pH 7.5, 100 mM NaCI, 1mM EDTA a t 0 "C. The beads were suspended in 50 p1 of sonicated 20 mM Tris-HCI, pH 7.5, 100 mM nated by the chloramine T method (52), and labeled aFGF was purified NaCI, 0m 5. M EGTA, and 0.2 mg/ml phosphatidylinositol (Sigma) and using a heparin-Sepharose column. Thecross-linkingandbinding analysesweredone a s described previously (26). Briefly, confluent preincubated a t room temperature for 10 min. 20 pCiof [Y-~~PIATP monolayers of transfected L6 cells were incubated with 25 ng of labeled (Amersham) and10 mM MgCI, were added and further incubatedfor 10 aFGF for 90 min in binding buffer (DMEM containing 0.1% gelatin and min. Reactions were stopped by the addition of chloroform, methanol, 50 mM HEPES, pH 7.5). Bound aFGF was cross-linked to the cell sur- 11.6 M HCI (50:100:1), phospholipids were extracted with chloroform, face receptors by incubating the dishesat +4 "C with 0.3 mM disuccin- and the organic phase washed with methanol,1M HCI (1:l). Reaction imidyl suberate in PBS for 20 min. The cells were washed,lysed in hot products were dried inuucuo, dissolved in chloroform, spotted on Silica lysis buffer (3.3% SDS and 0.125 M Ms-HCI, pH 6.8), centrifuged, and Gel-60 plates (Merck) impregnated with 1% potassium oxalate, and aliquots of the supernatants were analyzed by SDS-PAGE. For the resolved by chromatographyinchloroform/methano~28%ammonia/ binding analyses, the cells grown in 24-well dishes were incubated with water (43:38:5:7) for 45 min. The phosphorylated products were deincreasing amountsof iodinated aFGF in binding buffer for 2 h, washed tected by autoradiography. Expression, Purification, and Afinity Precipitation of GST-PLC-y three times with the same buffer, and lysed with 0.3 M NaOH. The SH2 Fusion Proteins-The PLC-y SH2-GST fusion protein and GST solubilized radioactivity was measured ina gamma counter. Immunoprecipitation and Immunoblotting-Cells were starvedover- protein were produced inbacteriaand purified usingglutathionenight inserum-free DMEM containing 0.2% bovine serum albumin and Sepharose affinity chromatography (Pharmacia). The GST-PLCy SH2 then stimulated for the indicated times with 50 ng/ml of aFGF. For protein bound to glutathione-Sepharose was then incubated for 3 h at immunocomplex analyses, the cells were washed with 20 m HEPES, 4 "C with lysatesof non-stimulated and stimulatedFGFR-4 expressing pH 7.5,150mM NaCI, 1m Na,VO,, scrapped from the plates, andlysed cells. The precipitates were washed twice with lysis buffer and once on ice for 15min inimmunoprecipitation buffer (20 mM HEPES, 150mM with 20 mM HEPES, 150 mM NaCl before analysis by SDS-PAGE and NaCI, 10% glycerol, 1% Triton X-100, 1 mM Na3V04, and 5.5 pg/ml Western blotting. Thymidine Incorporation-Transfected L6 cells were seeded in 96aprotinin). For antiphosphotyrosine, FGFR-4, and PLC-y immunopremedium cipitations, the cells were washed with PBS, lysed in RIPA buffer (10 well plates (5 x lo3cells/well), and after overnight growth the mM Tris-HC1, pH 7.5, 50 mM NaCI, 0.5% deoxycholate, 0.5% Nonidet was changed to DMEM containing 0.5% fetal calf serum. After 2 days, P-40,0.1% SDS, 1mM Na,VO,, 5.5 pg/ml aprotinin) and sonicated. The the cells received new medium containing 0, 0.5,5 , 10, 50, 100, or 500 lysates were centrifuged for 10 min, and the supernatants were incu- ng/ml aFGF and,as a control, 10%fetal calf serum. After stimulation for bated for 2 h to overnight on ice with specific antisera againstFGFR-4, 20 h, cells were incubated with 1pCi of L3H1thymidinefor 4 h at 37 "C. cell a FGFR-1, PLC-y, SHC, or phosphotyrosine. ProteinA-Sepharose C M B The cells were washed with PBS, trypsinized, and harvested using of incorporated thymidine was meas(Pharmacia) was used to collect the immune complexes, which were harvester (Nunc), and the amount analyzed by Western blotting with antibodies against phosphotyrosine,ured in a liquid scintillation counter. FGFR-4, FGFR-1, or the mitogen-activated protein kinase. For analysis RESULTS of total cellular proteins, the cells were lysed in hot lysis buffer(3.3% SDS, 0.125 M Ms-HCI, pH 6.8,5.5pg/ml aprotinin, and1mM Na3V0,), Binding of aFGF to FGFR-4 and FGFR-1 Expressing L6 sonicated, and centrifuged. The protein concentrations were measuredCells-Fig. 1shows saturation binding of radiolabeled aFGF to by the BCA method (Pierce), and equal amountsof protein were anaFGFR-4 and FGFR-1 expressing L6 cells and to neomycinlyzed by SDS-PAGE and Westernblottingusing specific antisera against Raf-1. Stripping of the filters was donefor 30 min at 50 "C in resistant control cells. As can be seen from the graph,both cell of receptors on their surface. stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris- lines express about equal amounts Scatchard analyses of binding experiments indicated that the HCI, pH 6.7) with occasional agitation.
4L
18322
FGFR-4 Signal Dansduction
Kd value for recombinant aFGF binding to both receptors was about 300 PM (not shown), as previously described for transfected NIH 3T3 cells (26). When aFGF was cross-linked to its FGFR.1 mFRr4 receptors, prominent bands of 160 and 125 were detected in SDS-PAGE (Fig. 1, inset) corresponding to the expected molecular weights of FGFR-1 and FGFR-4 complexed with aFGF, respectively. These values areconsistent with our previous re0 2 6 10 20 min sults (19,26). All further experiments were carried outwith cell aFGF 0 2 5 10 20 clones expressing comparable amounts of the FGFreceptors on their surface. Association of Tyrosyl Phosphoproteins with Activated FGFR-4 and FGFR-1-In order to analyze the kinetics of receptor autophosphorylation, FGFR-4 and FGFR-1 expressing L6 cells were stimulated for 2, 5, 10, and 20 min with50 ng/ml aFGF. After stimulation, cells were lysed, and the receptors were immunoprecipitated withthe corresponding antisera. The immune complexes were separated by SDS-PAGE, and phosphoproteins were analyzedby Western blottingusing antiphosphotyrosine antibodies. As can be seen from Fig. 2 4 , autophosphorylation of both receptors can be detected already at 2 min of stimulation, with a maximum at 5-10 min. In addition, a tyrosyl-phosphorylated 85-kDa protein doublet coprecipitates with FGFR-4 at all time points analyzed after stimulation. Receptor tyrosine phosphorylation and complex formation a F G F - + + + + + with potential substrates was furthercharacterized by stimuWB: apTYl. aR-4 aPTyr lating transfected L6 and NIH3T3 cells for 5 min with the ligand and using nondissociating or dissociating conditions for immunoprecipitation. Besides tyrosyl-phosphorylated proteins corresponding to the molecular mass of FGFR-4 and FGFR-1, the mostprominent phosphoprotein coprecipitatingwith FGFR-4 from L6 and NIH 3T3 cells was doublet of about 85 kDa apparentmolecular mass (Fig. 2, B and C). This phosphoprotein doublet was not detected when immunoprecipitation was done in dissociating conditions (RZPA buffer in B ) . The possibility that thecoprecipitating band representsa degradation product of FGFR-4 was studied by stripping the filters and reprobing them with receptor-specific antisera (B, lanes aR4). This analysis showed that the 85-kDa doublet is not a degradation product of the receptor. In addition tothis major polypeptide doublet, a 150-kDa polypeptide coprecipitated with activated FGFR-4 from NIH 3T3 cells, and polypeptides of 180, 105, and 75 kDa coprecipitated with activated FGFR-1 (Fig. 2C). Neither polypeptide of the doublet detected in FGFR-4 imaFGF + + munoprecipitates appeared to be the p85 adaptor subunit of FIG. 2. Qrosyl phosphorylation of FGF'R-associated proteins. phosphoinositol-3 kinase, since no reactivity was seen in WestA, L6 cells expressing FGFR-1 or FGFR-4 were stimulated withaFGF ern blotting using p85-specific antiserum (data not shown). for the indicated times and anti-receptor immunoprecipitates were anaAlthough some sort of phosphatidylinositol kinase activity lyzed by Western blotting using antibodies against phosphotyrosine to could be detected in FGFR-4 immunoprecipitates from L6 and estimate the kinetics of receptor autophosphorylation. In B, FGFR-I, NIH3T3 cells, this activity was notligand inducible in contrast FGFR-4, and theY754F mutant were stimulatedfor 5 min and analyzed as in A. The filter was then stripped and the FGFR-4 lanes were reto theactivity seen inplatelet-derived growth factorreceptor-P probed with receptor-specific antisera (lanes d 4 ) . Inthe case of immunoprecipitates (Fig. 3, A and B). In addition, the FGFR-4 FGFR-4, immunoprecipitation was also performed in theRIPA buffer. associated activity was not inhibited by the phosphoinositol-3 In C, FGFR-1 and FGFR-4 expressing NIH3T3 cells were stimulatedfor in The molecular masses of FGFR-1 (RI,150 kinase inhibitor wortmannin (54, data not shown). Neither sort 5 min and analyzed as A. m a ) , FGFR-4 (R4,llO kDa) and major coprecipitated polypeptides are of phosphatidylinositol kinase activity was detected in FGFR-1 given. immunoprecipitates (Fig. 3B). Further immunoprecipitation and blotting experiments indicated that the 85-kDa phosphotyrosyl doublet was not ~80185cortactin, p80 ezrin, or a p91- which lack endogenous FGFRs. Antiphosphotyrosine immunorelated component of the interferon-stimulatedgene factor blotting analysis of PLC-y immunoprecipitates from L6 cells (data not shown). showed that PLC-y is considerably more weakly phosphoBinding and Phosphorylation of PLC-"We have previously rylated by FGFR-4 than by FGFR-1 (Fig. 4A).We also analyzed shown that thephosphorylation of PLC-y is considerably stron- similar immunoprecipitates after 10 and 20 min of FGFR-4 ger in FGFR-1 expressing NIH3T3 cells than in FGFR-4 ex- induction and no further increase of PLC-y phosphorylation was detected (data not shown). pressing cells (26). Because endogenous FGFRs presentin The difference in PLC-y phosphorylation was somewhat unNIH3T3 cells are capable of contributing to PLC-y phosphorylthe consensus sequence ation, a more detailed analysis was carried out in L6 cells, expected because FGFR-4 has
A
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R1l -
-
-
-
-
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-
nansduction FGFR-4 Signal
18323
FIG.3. Analysis of phosphoinositol-3 kinase activity after FGFR-4 and FGFR-1 stimulation. In A, FGFR-4 immunoprecipitates from receptor expressing and control L6 cells treated for 5 min with or without aFGF were analyzed for phosphoinositol-3 kinase activity. In B, ligandstimulation of phosphoinositol-3 kinase activity was measured FGFR-1, in platelet-derived growth factor receptor-& andFGFR-4immunoprecipitates from transfected NIH3T3 cells, and as a control, in FGFR-4 immunoprecipitates from non-transfected NIH3T3 cells. Ligand
-
+
-
+
L Ligand
-
+ -
+
-
+
-
+ -
+
A 7 7 ”
. I
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150-
PLC-y-
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aFGF+
B
-
GST
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-
SH2
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t
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FGFR-4 nFGF - + + FIG.4. ’&rosy1 phosphorylation of PLC-y by wild type and mutant receptors (A) and binding of FGFR-4 to the PLC-y SH2 domain ( B ) .Lysates from untreated and aFGF-treated (5min) L6 cells were subjected to immunoprecipitation of PLC-y (150 kDa) followed by Western blotting using antibodies against phosphotyrosine (A). In B , affinity precipitation withGST or a GST-PLCy SH2 fusion protein was followed by Western blotting using antibodies against FGFR-4.
-
52 46 -
aFCF
-
+
-
+
FIG.5. Tyrosyl phosphorylation of SHC proteins after aFGF stimulation. SHC proteins were immunoprecipitated from L6 cells stimulated for 5 min with aFGF and analyzed for tyrosine phosphorylation a s above. Note the band withthe electrophoreticmobility of 150 kDa coprecipitating with the phosphorylated SHC polypeptides of 46 and 52 kDa.
phosphorylated in FGFR-1 expressing cells, but not in FGFR-4 expressing cells (Fig. 5). Moreover, a phosphorylated component of the size of 150 kDa was detected in SHC immunoprecipitates from stimulated FGFR-1 expressing cells. No phosphorylation was detected either when SHC was immunoprecipitated from FGFR-4 expressing cells after 10 and 20 min of stimulation. Interaction with GRB2-GRB2 has been shown to bind diY(754)LDL for phosphotyrosyl-mediated binding of the PLC-y rectly to the epidermal growth factor receptor and to mediate SH2 domain, corresponding to the major autophosphorylation its interaction with SOS, a Ras GTP/GDP exchange factor. In site Y(766)LDL in the FGFR-1 (33, 34). When the PLC-y SH2 contrast toplatelet-derived growth factor receptor+, no direct domain was expressed as a GST fusion protein, and used in interaction of GRB2 with FGFR-1 or FGFR-4 was detected in affinityprecipitation analysis of unstimulatedand aFGF- affinity precipitates using a GST-GRB2 SH2 fusion protein stimulated cell lysates, it was found t o bind activated FGFR-4 (data not shown). This is consistent with the fact that neither (Fig. 4B).The weak phosphorylation of PLC-y by FGFR-4 was receptor has the consensus binding site for GRB2. Analysis of Raf-1and Mitogen-activated Protein Kinase dependent on tyrosyl phosphorylation of the Ty1;754 residue (see Fig. 2B), since mutation of this into a phenyalalanine residue Phosphorylation-The phosphorylation of the serinekhreonine completely abolished PLC-7 phosphorylation (Fig. 4A). On the kinase Raf-1 and mitogen-activated protein kinase were asbasis of these results, it appears that the tyrosine kinase ac- sessed by electrophoretic mobility shift and tyrosine phosphotivity of FGFR-4 in comparison with FGFR-1 is weaker, at least rylation analyses, respectively. For mobility shift analysis of Raf-1 hyperphosphorylation (see Ref. 56), the transfected L6 toward this substrate. Phosphorylation of SHC-SHC proteins are recently identi- cells were stimulated for different time periods with aFGF and fied SH2 domain containing proteins that associate with cer- subjected to Western blotting using a Raf-1-specific antiserum. tain receptors and become phosphorylated upon activation of In Fig. 6, a stimulation-dependent mobility shift of Raf-1 is protein tyrosine kinase (40). In addition, SHC has been re- seen in FGFR-1 expressing cells. In contrast, only a barely, ported to bind GRB2, a SH2- and SH3-containing adaptor pro- detectable shift occurred after 5 min in samples from FGFR-4tein, in a phosphorylation-dependent manner, suggesting that transfected cells, while no mobility shift of Raf-1 occurred in SHC functions in the Rasactivation pathway ( 5 5 ) . In order to control L6 cells. determine thepotential role of SHC in FGFR-1- and FGFR-4For phosphorylation analysis of mitogen-activated protein mediated signal transduction, SHC tyrosine phosphorylation kinase, thetransfected L6 cells were stimulated, lysed, immuwas analyzed after ligand stimulation. After 5 min of stimula- noprecipitated using phosphotyrosine antibodies, and blotted tion, the 46 and 52kDa formsof SHC were prominently tyrosyl with antisera againstmitogen-activated protein kinase. Fig. 7
FGFR-4 Signal Dansduction
18324 FGFR.4
."R 0
aFGF 0 2 5 10 20 0 2 5 10 20min. FIG.6. Analysis of Raf-1 polypeptides following FGFR-1 and FGFR-4 stimulation. Total cell lysates from unstimulated and stimulated cells were analyzed by Western blotting using Raf-1-specific antibodies. Note a mobility shift occurring a t 5 min of stimulation in FGFR-1 expressing cells, indicating a hyperphosphorylation of Raf-1 polypeptides. The mobility shift in the FGFR-4 expressing cells is much smaller.
Y
B
a
." s
P
m b
aFGF [ng/mll
FIG.8. DNA synthesis after aFGF stimulation. Cells were serum starved for 48 h and stimulatedfor 20 h with the indicatedconcentrations of aFGF, labeled for 4 h with tritiated thymidine, and subsequently analyzed for radioactivity incorporated into DNA as detailed under "Experimental Procedures." The average values obtained from four different experimentsare shown.
44-1
421
L
aFGF
-
+
-
+
-
+
doublet of 85 kDa coprecipitated with FGFR-4 from ligandstimulated cells, but this polypeptide could not be detected in precipitates were analyzed for the presence of mitogen-activated pro- FGFR-1 immunoprecipitates. A similar polypeptide doublet of tein kinase by Western immunoblotting. Note a slight increase of the about 85 kDa wasalso coprecipitated with FGFR-4 from trans42- and 44-kDa mitogen-activated protein kinase polypeptides in im- fected NIH 3T3 cells, suggesting that this putative substrate is munoprecipitates from FGFR-4 expressing cells stimulated for 5 min not restricted to only one cell type. with aFGF and a more marked increase in the stimulated FGFR-1 expressing cells. The strong upper band present in all lanes represents The phosphoinositol-3 kinase subunit p85 is a well-characunspecific detection of the immunoglobulin heavy chain by the antibod- terized substrate for tyrosine kinases. It directly associates ies used. with phosphorylated tyrosine residues of several tyrosine kinase receptors (36-38). It has previously been shown that shows that a major protein with a molecular mass of 42 kDa bFGF is able to induce 2-3-fold increase of phosphoinositol-3 and a minor one of 44 kDa become phosphorylated in FGFR-1 kinase activation in PC12 pheochromytoma cells (58).However, and FGFR-4 expressing cells after stimulation. These proteins several lines of evidence indicate that the phosphoprotein coare most likely the pp42kDmUpklerk2 a n d ~ p 4 4 k D " ' ~ P ~How' ~ ~ ~ ' . precipitated with FGFR-4 is notp85. No activation-dependent ever, in FGFR-4 expressing cells, tyrosyl phosphorylation of phosphatidylinositol kinase activity was associated witheither these polypeptides was much weaker than inFGFR-1 express- of the FGFRs, although there was some constitutive activity ing cells. Again, no phosphorylation of any of these components present in theFGFR-4 immunoprecipitates. However, this acwas observed in control cells. Taken together these results sug- tivity was not inhibitedby the phosphoinositol-3 kinase inhibigest that both FGFR-1 and FGFR-4 can activate Raf-1 and tor wortmannin. Furthermore, no association between FGFR-4 mitogen-activated protein kinase, although withdifferent efi- and p85 could be detected by in vitro kinase assays and Westciencies. ern blotting. From these experiments, we conclude that the Thymidine Incorporation-Because of the observed differ- constitutive phosphatidylinositol kinase activity associated ences in ligand-induced phosphorylation of proteins after with FGFR-4 does not originate from a known phosphoinosiFGFR-4 and FGFR-1 activation, we explored possible differ- tol-3 kinase. ences in the stimulation of DNA synthesis via the two recepWe have previously shown that PLC-y is more extensively tors. Serum-starved FGFR-4 and FGFR-1 expressing L6 cells phosphorylated by FGFR-1 than by FGFR-4 in NIH3T3 cells and control cells were pulse-labeled with [3Hlthymidine for 4 h (26). This was also the case in transfected L6 cells, although after a 20-h incubation with various concentrations of recom- here we have shown, using a PLC-y SH2 fusion protein, that binant aFGF. In this analysis the FGFR-4- and FGFR-l-trans- activated FGFR-4 binds to the PLC-y SH2 domain. Furtherfected cell lines showed similar 50% effective doses (ED,, val- more, we here show that interaction between FGFR-4 and ues) for aFGF induced DNA synthesis (Fig. 8). aFGF PLC-y is mediated by a conserved phosphotyrosyl residue, stimulation of control cells did not lead to a significant increase which corresponds to the siteknown to interactwith PLC-y in in thymidine incorporation. FGFR-1. The reason for the weaker phosphorylation of PLC-y by FGFR-4 remains tobe determined. The overall role of PLC-y DISCUSSION in mediating the transductionof mitogenic signals by tyrosine In this study we have analyzed and compared the signal kinase receptors has been under investigation. For FGFR-1, transduction pathways of two related tyrosine kinase recep- activation of PLC-y is not needed for a full mitogenic response tors, FGFR-4 and FGFR-1. In the FGFR family, consisting of (33,34). In agreement with this data, our present results show four FGF receptors, FGFR-4 and FGFR-1 share the leastover- that despite differences in PLC-y phosphorylation, both recepall amino acid sequence identity with each other (56%) (57). A tors were able to transduce mitogenic signals. major question regarding thebiological roles of the four differIt has been recently shown that SHC, a SH2 containing ent FGFreceptors and nine different FGFs is thespecificity of adaptor protein, is tyrosyl phosphorylated by several tyrosine signal transduction within this complex system. Using kinases (40). Phosphorylation of SHC results in itsassociation FGFR-4- and FGFR-1-transfected L6 rat myoblasts, we here with the GRB2-SOS complex, and this association has been show differences as well as similarities in the recruitment of proposed to activate Rasby increasing Ras guaninenucleotide signal transduction components by these two receptors. A yet exchange (59). While the p46 and p52 forms of SHC were uncharacterizedstrongly tyrosyl-phosphorylated polypeptide strongly phosphorylatedafter aFGFstimulation of FGFR-1, we
FIG.7. Qrosine phosphorylationof mitogen-activated protein kinase after receptor stimulation. Antiphosphotyrosine immuno-
FGFR-4 Signal Dansduction
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were not able t o detect SHC phosphorylation in immunoprecipitates of activated FGFR-4 expressing cells. However, it cannot be excluded that FGFR-4 is also capableof phosphorylating SHC to an extent undetectableby our methods. The activation of Raf-1 and the subsequent steps of signal transduction havebeen under extensive investigation, and several groups have demonstrated an interaction between Rasand Raf-1 in cells and in vitro (60-63). However, it has also been shown that upstream kinases, like protein kinase C and Src family members, are capable of phosphorylating Raf-1 at specific sites independently of Ras (56). Raf-1 activation leads to activation of the mitogen-activatedprotein kinasedextracellular signal-related kinases, which are needed for phosphorylation and activation of mitogen-activated protein kinase. On the other hand,a Raf-1-independent pathway of mitogen-activated protein kinase activation involving a mitogen-activated protein kinase/ERK kinase has been reported (64). We show here that Raf-1 and mitogen-activated protein kinase are phosphorylated by FGFR-4 and FGFR-1 in a ligandinduced manner. However, differences were observed in the extent of phosphorylation of these kinases by FGFR-4 and FGFR-1. A shift of electrophoretic mobility, indicating hyperphosphorylation of Raf-1 (see Ref. 561, was detected in analysis of FGFR-1 transfectants after aFGF stimulation. Strong phosphorylation of the p42 and p44 mitogen-activated protein kinases was also observed. In contrast, only a barely detectable mobility shift of Raf-1 occurred in aFGF-stimulated FGFR-4 transfectants, and in agreement with this,p42 and p44 mitogen-activated protein kinase were only weakly phosphorylated. Since activation of PLC--y has been shown to stimulateprotein kinase C, which in turn causes hyperphosphorylation and activation of Raf-l(56), itis possible that thedifference in PLC--y phosphorylation is responsible, at least partly, for the differences seen in the phosphorylation of Raf-1 and mitogen-activated protein kinase. Despite the detected differences of substrate phosphorylation by FGFR-4 and FGFR-1, both receptors stimulated DNA synthesis inL6 cells. This is in contrast to results obtained by lymphoid cells, Wang et al.(65) using transfected BaF3 murine where FGFR-1 but not FGFR-4 mediated proliferation signals. On the other hand, FGFR-4 mediates mitogenic signals for aFGF also in Vero monkey kidney cells and U20S human osteosarcoma cells.' Thus, although both receptors are able to induce DNA synthesis, the signal transduction pathways activated by FGFR-1 and FGFR-4 are somewhat different. These differences are under furtherinvestigation.
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