Tyrosine 766 in the fibroblast growth factor receptor - Semantic Scholar

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Journal of Cell Science 113, 643-651 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS0270

Tyrosine 766 in the fibroblast growth factor receptor-1 is required for FGFstimulation of phospholipase C, phospholipase D, phospholipase A2, phosphoinositide 3-kinase and cytoskeletal reorganisation in porcine aortic endothelial cells Michael J. Cross1,*, Matthew N. Hodgkin2, Sally Roberts2, Eva Landgren1, Michael J. O. Wakelam2 and Lena Claesson-Welsh1 1Department of Genetics and Pathology, Rudbeck Laboratory, S-75185 Uppsala, Sweden 2Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK

*Author for correspondence at Department of Genetics and Pathology, Rudbeck Laboratory, SE-75185 Uppsala, Sweden (e-mail: [email protected])

Accepted 30 November 1999; published on WWW 31 January 2000

SUMMARY Fibroblast growth factor-mediated signalling was studied in porcine aortic endothelial cells expressing either wildtype fibroblast growth factor receptor-1 or a mutant receptor (Y766F) unable to bind phospholipase C-γ. Stimulation of cells expressing the wild-type receptor resulted in activation of phospholipases C, D and A2 and increased phosphoinositide 3-kinase activity. Stimulation of the wild-type receptor also resulted in stress fibre formation and a cellular shape change. Cells expressing the Y766F mutant receptor failed to stimulate phospholipase C, D and A2 as well as phosphoinositide 3-kinase. Furthermore, no stress fibre formation or shape change

was observed. Both the wild-type and Y766F receptor mutant activated MAP kinase and elicited proliferative responses in the porcine aortic endothelial cells. Thus, fibroblast growth factor receptor-1 mediated activation of phospholipases C, D and A2 and phosphoinositide 3-kinase was dependent on tyrosine 766. Furthermore, whilst tyrosine 766 was not required for a proliferative response, it was required for fibroblast growth factor receptor-1 mediated cytoskeletal reorganisation.

INTRODUCTION

phosphoinositide 3-kinase (PI 3-kinase) and Ras-GTPase activating protein (Ras-GAP), do not appear to bind directly to FGF receptors (Vainikka et al., 1994; Wennström et al., 1992; Molloy et al., 1989). Recently, a novel FGFR substrate, termed FRS2, has been identified (Kouhara et al., 1997). This molecule is tyrosine phosphorylated in response to FGF and recruits Grb2 and the tyrosine phosphatase SHP-2, leading to activation of the MAP kinase cascade. However, unlike PLC-γ, the interaction with FRS2 is not dependent on receptor autophosphorylation at a specific site; instead, interaction occurs via the juxtamembrane region of the receptor and a PTB domain present in the FRS2 molecule (Xu et al., 1998). Activation of PLC-γ results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), generating inositol 1,4,5 trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 mobilises calcium from internal stores whilst DAG activates protein kinase C (PKC; Divecha and Irvine, 1995). The physiological role of FGF-stimulated PLC-γ activation has been addressed by analysing a number of different cell types expressing the Y766F receptor mutant. Such studies have shown that PLC-γ activation is not obligatory for FGFR-1 mediated mitogenesis (Peters et al., 1992;

Fibroblast growth factors (FGFs) are a family of heparinbinding polypeptides which regulate a variety of biological processes such as growth, differentiation, chemotaxis and angiogenesis. The mammalian FGF gene family comprises 18 members, of which FGF-1 (acidic FGF) and FGF-2 (basic FGF), have been the most extensively studied. Four transmembrane receptor tyrosine kinases, FGFR-1 (flg), FGFR-2 (bek), FGFR-3 and FGFR-4, specifically bind FGF. Ligation of the extracellular domain of the FGF receptor causes receptor dimerisation, activation of the intrinsic tyrosine kinase activity and autophosphorylation (Ullrich and Schlessinger, 1990). A number of tyrosine residues within the FGFR-1 have been shown to be autophosphorylated and therefore serve as potential binding sites for SH2 domain-containing proteins (Mohammadi et al., 1996). To date, only phospholipase C-γ (PLC-γ), which binds to tyrosine 766 (Mohammadi et al., 1991), and Crk, which binds to tyrosine 463 (Larsson et al., 1999), have been identified as binding directly to the FGFR-1, via their SH2 domains. Other proteins known to interact with many different receptor tyrosine kinases, such as Grb2,

Key words: Fibroblast growth factor, Phospholipase, Cytoskeleton

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Mohammadi et al., 1992), chemotaxis (Clyman et al., 1994; Landgren et al., 1998) and neurite outgrowth (SpivakKroizman et al., 1994). Whilst previous studies have focused on the physiological role of PLC-γ in FGFR-1 transduced cellular responses, the roles of phospholipase D (PLD) or phospholipase A2 (PLA2) have not been investigated. Indeed, FGF-2 has been shown to stimulate PLD (Ahmed et al., 1994), PLA2 (Virdee et al., 1994; Sa et al., 1994) and PI 3-kinase activity (Jackson et al., 1992) in different cell types. We have examined the ability of the FGFR-1 Y766F mutant to stimulate phospholipase and PI 3kinase activity in transfected PAE cells. Furthermore, we have studied the effect of this mutation on cytoskeletal reorganisation and mitogenesis and demonstrate that FGFR-1 stimulated PLC, PLD, PLA2 and PI 3-kinase activity, as well as cytoskeletal reorganisation, are dependent upon phosphorylation of tyrosine 766. MATERIALS AND METHODS Materials Cell culture media and sera were obtained from Gibco-BRL. FGF-2 was purchased from Advanced Protein Products. Radiochemicals were obtained from Amersham International. The rhodamineconjugated phalloidin was from Molecular Probes. The PKC inhibitor GF 109203X was purchased from Calbiochem; human thrombin and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. The phospho-specific p42/p44 MAP kinase antibody was purchased from New England Biolabs and the p42 MAP kinase antibody was purchased from Santa Cruz Biotechnology. Cell culture Porcine aortic endothelial (PAE) cells were maintained in Ham’s F12 medium containing Glutamax-1, supplemented with 10% (v/v) foetal calf serum (FCS) at 37°C in humidified air:CO2 (19:1). For phospholipase assays, cells were seeded at a density of 4×104/ml in 24-well plates, grown for 48 hours, quiesced and labelled in medium containing 1% (v/v) foetal calf serum for 24 hours. cDNA contructs and transfection cDNA for FGFR-1 (Wennström et al., 1991) was subcloned into the pAlter vector (Promega corporation) and site-directed mutogenesis was performed using the Altered Sites in vitro Mutogenesis System (Promega Corporation) to change tyrosine 766 in the FGFR-1 to phenylalanine. The wild-type and FGFR-1 Y766F cDNAs were inserted into pcDNA1/neo (Invitrogen), transfected into PAE cells by electroporation (Claesson-Welsh et al., 1988) and selected in 0.4 mg/ml Geneticin. Clones were picked after 2 weeks and examined for receptor expression by labelling with [35S]methionine, followed by immunoprecipitation with an anti-FGFR-1 antiserum. The Vec cell line was generated by transfecting PAE cells with empty pcDNA1/neo plasmid as described above. Scatchard analysis was performed as previously described (Wennström et al., 1992). In vitro kinase assay PAE cells were grown in 6-well plates until subconfluent. The medium was then changed to serum-free Ham’s F12 containing 0.25% (w/v) BSA and cells were incubated for a further 24 hours. Cells were then stimulated with either medium or FGF-2 (100 ng/ml) for 8 minutes at 37°C. Following stimulation, cells were lysed in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol (DTT), 100 µM Na3VO4, 1% aprotinin and 1 mM phenymethylsulfonyl fluoride (PMSF). Lysates were transferred to Eppendorf tubes and centifuged at 14000 g for 20 minutes at 4°C. The supernatants were then transferred

to fresh Eppendorf tubes and incubated with antisera for 1 hour at 4°C followed by incubation for 30 minutes with Immunosorb A (EC Diagnostics AB, Uppsala, Sweden). Immune complexes were collected by centifugation and washed twice with lysis buffer and twice with phosphate-buffered saline. Kinase assays were performed for 15 minutes at 37°C in 40 µl of kinase buffer (20 mM Hepes, pH 7.5, 10 mM MnCl2 and 0.05% Triton X-100, containing 5 µCi [γ-32P]ATP). Reactions were terminated by addition of 40 µl of SDS-sample buffer (8% SDS, 0.4 M Tris/HCl, pH 8.8, 1 M sucrose, 10 mM EDTA, 0.02% Bromophenol Blue and 4% β-mercaptoethanol) and samples boiled for 5 minutes. Proteins were resolved by SDS-PAGE on 8% polyacrylamide gels. After fixation in 10% glutaraldehyde the gel was treated with 1 M KOH at 55°C for 45 minutes to remove serine phosphorylation. The gel was then dried and analysed by autoradiography. Assay of phospholipase activity in whole cells Phospholipase C activity was determined by the measurement of [3H]inositol phosphate accumulation in the presence of 10 mM LiCl from [3H]myo-inositol-labelled cells (Plevin et al., 1990). PLD activity was measured by the accumulation of [3H]phosphatidylbutanol in the presence of 30 mM butan-1-ol from cells labelled with [3H]palmitate. This method utilises the transphosphatidylation reaction catalysed by PLD, in which the phosphatidyl moiety liberated by choline release is transferred to an accepting nucleophile, R-OH. In vivo this accepting nucleophile is water, giving rise to a hydrolytic activity. However, in the presence of short chain primary aliphatic alcohols, such as butanol, phosphatidylalcohols are formed at the expense of phosphatidic acid since the primary alcohols are stronger nucleophilic acceptors than water. The [3H]phosphatidylbutanol was extracted and separated by thin layer chromatography as described (Cook et al., 1991). Phospholipase A2 activity was measured by determining the release of [3H]arachidonate from cells labelled with [3H]arachidonate (Stewart et al., 1997). [3H]arachidonate was separated from other lipids by thin layer chromatography using silica gel PE SIL G plasticbacked plates in a solvent system of n-hexane/diethyl ether/acetic acid (70:30:2 v/v/v). The [3H]arachidonate band was identified by comigration with arachidonate carrier, which was visualised by iodine staining and quantified by scintillation counting. Measurement of PI 3-kinase activity in vivo In vivo activity was measured by labelling cells for 90 minutes in phosphate-free DMEM containing 0.25 mCi/ml [32P]orthophosphate. Lipids were extracted, deacylated with methylamine (33% in methanol at 53°C for 50 minutes) and the resulting glycerophosphates deglycerated using mild sodium meta-periodate treatment (Wong et al., 1992). Phosphate-containing head groups were separated by anion exchange HPLC on a 25 cm partisphere 5 SAX column eluted with a linear gradient of ammonium dihydrogen phosphate (0.5 M, pH 3.8 with phosphoric acid) in water at 1 ml/minute over 110 minutes. Fractions were collected every 0.5 minutes and 32P-radioactivity was quantified by liquid scintillation counting. Fluorescence staining of actin filaments Cells were seeded onto adherent microscope slides at a density of 3×105 cells per 100 mm Petri dish and grown for 24 hours to subconfluency. The cells were quiesced by replacing the medium with serum-free Ham’s F-12 for 24 hours. Cells were washed in Hank’s buffered saline solution (pH 7.4) containing 20 mM Hepes, 10 mM glucose and 0.1% (w/v) BSA and stimulated with FGF-2. Incubations were terminated by washing the slides in ice-cold PBS. Cells were fixed in 4% paraformaldehyde for 8 minutes, washed twice in PBS and permeabilised by immersing in acetone at −20°C for 10 minutes. Cellular actin was stained with 0.33 µM rhodamine-conjugated phalloidin for 30 minutes. Slides were then washed in PBS, mounted in glycerol/DABCO and actin filaments visualised by fluorescence microscopy.

Tyrosine 766 in the fibroblast growth factor receptor Immunoblotting Cells were seeded at 2×105 per well of 6-well plates and grown for 2 days. The medium was then changed to Ham’s F12 containing 0.5% FCS and cells incubated for a further 24 hours. Cells were then stimulated with either medium or FGF-2 (100 ng/ml) for 10 minutes at 37°C. Following stimulation, cells were lysed in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 500 µM Na3VO4, 1% aprotinin 10 µg/ml leupeptin and 1 mM phenymethylsulfonyl fluoride (PMSF). Lysates were transferred to Eppendorf tubes and centifuged at 14000 g for 20 minutes at 4°C. The supernatants were then transferred to fresh Eppendorf tubes and Laemmli sample buffer added. Samples were boiled for 5 minutes and proteins were resolved by SDS-PAGE on 10% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Hybond-C extra, Amersham Pharmacia Biotech). Membranes were blocked for 2 hours at 4°C in TBS containing 0.1% Tween 20 and 5% (w/v) BSA and incubated overnight at 4°C with anti-phospho-MAP kinase antisera. The membranes were washed in TBS/Tween and incubated for 1 hour at 4°C with a peroxidase-coupled anti-rabbit secondary antibody (Amersham Pharmacia Biotech). Bound antibodies were visualised by chemiluminescent detection (Amersham Pharmacia Biotech). The nitrocellulose membrane was stripped in 62.5 mM Tris-HCl, pH 6.7, 2% SDS and 100 mM β-mercaptoethanol at 55°C for 30 minutes. The membrane was then reprobed with an anti-p42 MAP kinase antisera. Measurement of cell proliferation Cells were seeded at 3×104 cells per well in 24-well plates in Ham’s F-12 containing 10% (v/v) FCS. After 6 hours the medium was changed to Ham’s F-12 containing 0.2% (v/v) FCS. 24 hours later the cells were stimulated with a range of FGF-2 concentrations in Ham’s F-12 containing 0.02% (v/v) FCS. Fresh growth factor was added on days 2 and 4. On day 5, cells were trypsinised and cell numbers determined by use of a Coulter counter.

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cells expressing FGFR-1wt and mutant FGFR-1 Y766F, of which the latter had been previously shown not to mediate FGF-2-stimulated PLC-γ phosphorylation (Landgren et al., 1995). FGF-2-stimulated PLC, PLD and PLA2 activity in the FGFR-1wt cells was dose-dependent, with maximal activity for all three phospholipases observed at a concentration of 100 ng/ml (data not shown). This concentration was used for all subsequent experiments. FGF-2-stimulation of cells expressing FGFR-1wt resulted in a twofold increase in [3H]inositol phosphate accumulation (Fig. 2A), indicative of PLC catalysed PtdIns(4,5)P2 hydrolysis. However, FGF-2 failed to stimulate [3H]inositol phosphate accumulation in cells expressing the FGFR-1 Y766F above the level observed in control transfected (Vec) cells. These results are consistent with previous proposals that the binding site for PLC-γ in FGFR-1 is tyrosine 766 (Mohammadi et al., 1991). FGF-2-stimulation of cells expressing FGFR-1 resulted in a fourfold increase in PLD activity, measured by [3H]phosphatidylbutanol generation (Fig. 2B) and a fourfold increase in PLA2 activity, measured by [3H]arachidonate release (Fig. 2C). In both cases, cells expressing the FGFR-1 Y766F failed to stimulate PLD and PLA2 activity above the level in Vec cells. This suggests that FGFR-1 mediated PLD and PLA2 activity are dependent upon tyrosine 766. As a control, thrombin stimulated PLD and PLA2 activities to the same degree in cells expressing the FGFR-1wt and FGFR-1 Y766F, confirming that activation of the phospholipases was still possible in cells expressing the FGFR1 Y766F. Since FGF-2-stimulated PLC activity was lost in the FGFR1 Y766F cells, we postulated that activation of this enzyme, and the subsequent increase in DAG and Ca2+, might be

RESULTS Characterisation of transfected PAE cell lines PAE cells were stably transfected with cDNAs encoding the wild-type FGFR-1 (FGFR-1wt) and the point mutation Y766F receptor (FGFR-1 Y766F). To ensure that both receptors were expressed at similar levels and able to undergo ligandstimulated autophosphorylation, an in vitro kinase assay was performed. PAE cells expressing either the FGFR-1wt or FGFR-1 Y766F were stimulated with FGF-2, lysed and immunoprecipitated with an FGFR-1 specific antiserum. As a control, cells transfected with the empty vector plasmid were included. The immunoprecipitates were incubated with [γ32P]ATP and the samples analysed by SDS-PAGE. Only cells expressing the FGFR-1wt and FGFR-1 Y766F showed increased incorporation of 32P-radioactivity in response to FGF-2, indicating that they were both functional tyrosine kinases (Fig. 1). Scatchard analysis revealed that the cells expressed the following number of receptors per cell: wild type, 4.4×105 and Y766F, 3.1×105. Both receptors bound FGF2 with affinities of 0.85 nM and 0.5 nM, respectively (data not shown). Vec cells expressed 1×105 molecules/cell of a receptor that bound FGF-2 with a Kd of 0.2 nM. This receptor was different from FGFR-1 since it was not detected by an FGFR1 specific antiserum (Wennström et al., 1992). Tyrosine 766 is required for FGF-stimulated phospholipase activity FGFR-1 mediated phospholipase activation was studied in PAE

Fig. 1. In vitro kinase assay on FGFR-1 immunoprecipitates from transfected PAE cells. Cells were stimulated with either FGF-2 (100 ng/ml) or buffer, lysed and immunoprecipitated with an FGFR-1 specific antiserum. The immobilised immunoprecipitates were incubated with [γ-32P]ATP in the presence of kinase buffer. Proteins were separated by SDS-PAGE and visualised by autoradiography. The migration rates of marker proteins are indicated on the left, whilst the position of the FGFR-1 is indicated on the right.

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Fig. 2. The effect of Y766F mutation on FGFR-1-mediated phospholipase activation. Cells were labelled with (A) [3H]inositol, (B) [3H]palmitate or (C) [3H]arachidonate. Cells were stimulated with either 100 ng/ml FGF-2 or 10 U/ml thrombin for 10 minutes. PLC activity was determined by the accumulation of [3H]inositol phosphates (A), PLD activity was determined by the accumulation of [3H]phosphatidylbutanol (B), and PLA2 activity was determined by the generation of [3H]arachidonate (C), as described in Materials and Methods. Results are expressed as % of basal response (mean ± s.d., n=3) from a single experiment representative of two. The relevant basal d.p.m. were: (A) Vec, 5473±300; FGFR-1wt, 4792±295; Y766F, 5062±188; (B) Vec, 2351±256; FGFR-1wt, 2444±342; Y766F, 2836±36; (C) Vec, 800±150; FGFR-1wt, 694±29; Y766F, 1238±93.

Fig. 3. Regulation of PLC, PLD and PLA2 activity. Cells expressing the FGFR-1wt were labelled with (A) [3H]inositol, (B) [3H]palmitate or (C) [3H]arachidonate. They were then preincubated with either 5 µM GF 109203X (GFX) or vehicle (DMSO, 0.1% v/v) for 30 minutes. Other cells were pre-incubated with 100 mM butan-1-ol for 5 minutes. Cells were then stimulated with either 100 ng/ml FGF-2, 100 nM PMA or 5 µM A23187 for 20 minutes. PLC activity was determined by the accumulation of [3H]inositol phosphates (A), PLD activity was determined by the accumulation of [3H]phosphatidylbutanol (B), and PLA2 activity was determined by the generation of [3H]arachidonate (C), as described in Materials and Methods. Results are expressed as % of basal response (mean ± s.d., n=3) from a single experiment representative of two. The relevant basal d.p.m. were: (A) 3231±123, (B) 1551±64, (C) 1020±54

responsible for the activation of PLD and PLA2. The phorbol ester PMA and the calcium ionophore, A23187, were both able to stimulate PLD activty (Fig. 3B) and PLA2 activity (Fig. 3C)

in the FGFR-1wt cells. PMA did not stimulate PLC activity, whilst A23187 showed only a small stimulation (Fig. 3A). This suggests that PKC and Ca2+ can regulate both PLD and PLA2

Tyrosine 766 in the fibroblast growth factor receptor activities in these cells. To address the role of PKC in regulating FGF-2-stimulated PLD and PLA2 activity we used the PKC inhibitor GF 109203X (Toullec et al., 1991). Pretreatment with this compound led to approximately 60% inhibition of the FGF-2-stimulated PLD activity (Fig. 3B) and 85% inhibition of the FGF-2-stimulated PLA2 activity (Fig. 3C). As a control, GF 109203X compound also inhibited PMA-stimulated PLD activity (Fig. 3B) and PLA2 activity (Fig. 3C), confirming that this compound was a PKC inhibitor. The GF 109203X compound had no effect on FGF-2stimulated PLC activity (Fig. 3A). This data suggests that FGFR-1 mediated activation of PLD and PLA2 is regulated via PKC and possibly Ca2+. This would place both PLD and PLA2 downstream of PLC-γ. To determine if FGF-2-stimulated PLA2 activity was downstream of PLD activity, cells were preincubated with the primary alcohol, butan-1-ol, to reduce the formation of phosphatidic acid (PA) derived from PLD activation. FGF-2-stimulated PLC activity (Fig. 3A) and PLA2 activity (Fig. 3C) were not affected by butan-1-ol, suggesting that both PLC and PLA2 are not downstream of PLD. Tyrosine 766 is required for FGF stimulated PI 3kinase activity PI 3-kinase has been shown to associate directly with a number of tyrosine kinase receptors via the SH2 domains present in the p85 subunit of the enzyme (Stephens et al., 1993). However, in the PAE FGFR-1wt cells, direct association of PI 3-kinase with the FGFR-1 has not been detected (Wennström et al., 1992).We analysed PI 3-kinase activity in vivo by measuring

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Cell line Fig. 4. Effect of the Y766F mutation on FGFR-1 stimulated, 32Plabelled lipid levels. PAE Vec, FGFR-1wt and Y766F cells were labelled with [32P]orthophosphate, stimulated with either Hank’s buffer or 100 ng/ml FGF-2 for 30 seconds. Lipids were extracted, deacylated and deglycerated. Lipid head groups were separated by anion exchange HPLC as described in Materials and Methods. The radioactivity in [32P]PtdIns(3,4)P2 and [32P]PtdIns(3,4,5)P3 was quantified and results are shown as % of basal response for each cell line (mean ± range, n=2) from a single experiment representative of two. The relevant mean basal c.p.m. were: Vec, 1900; FGFR-1wt, 931; Y766F, 1225.

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the levels of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in [32P]orthophosphate-labelled cells. FGF-2-stimulation of cells expressing the FGFR-1wt resulted in a 2.5-fold increase in [32P]PtdIns(3,4,5)P3 and a 1.5-fold increase in [32P]PtdIns(3,4)P2 (Fig. 4), indicating that the FGFR-1 was able to stimulate PI 3-kinase activity. In cells expressing the FGFR-1 Y766F, no apparent increase in PtdIns(3,4,5)P3 or PtdIns(3,4)P2 formation was observed, suggesting that PI 3kinase activity was dependent upon tyrosine 766 in the FGFR1. As a control, 10% FCS was able to stimulate PtdIns(3,4,5)P3 and PtdIns(3,4)P2 formation to the same degree in cells expressing the FGFR-1wt and FGFR-1 Y766F (data not shown). Tyrosine 766 is required for FGFR-1 mediated cytoskeletal reorganisation Activation of a number of receptor tyrosine kinases has been shown to induce actin reorganisation. In Swiss 3T3 fibroblasts PDGF, EGF and insulin have been shown to induce the rapid appearance of membrane ruffles or lamellipodia in a Racdependent manner and the later appearance of actin stress fibres in a Rho-dependent manner (Ridley et al., 1992). FGF2 stimulation of PAE FGFR-1wt cells did not result in the rapid appearance of lamellipodia (Fig. 5B′). However, after 30 minutes stimulation an increase in stress fibre formation was observed (Fig. 5C′). After 24 hours exposure to FGF, cells expressing the FGFR-1wt had undergone a shape change with the generation of a number of processes staining densely for actin (Fig. 5D′, arrows). FGF-2 stimulation of Vec cells (Fig. 5A-D) and cells expressing the mutant FGFR-1 Y766F (Fig. 5 A′′-D′′) failed to stimulate stress fibres or a shape change. This data suggests that tyrosine 766 is required for both short-term and longer-term FGFR-1 mediated cytoskeletal reorganisation. Tyrosine 766 is not required for FGFR-1 mediated MAP kinase activation and proliferation in PAE cells FGFR-1 mediated mitogenesis in L6 myoblasts has been previously shown to be independent of PLC-γ activation (Peters et al., 1992; Mohammadi et al., 1992). We determined the ability of FGF-2 to stimulate MAP kinase and cell proliferation in the PAE cells. FGF-2 stimulated MAP kinase phosphorylation in the FGFR-1wt as well as in the FGFR-1 Y766F cells, albeit to a reduced level (Fig. 6). FGF-2 also stimulated a low level of phosphorylation of MAP kinase in the Vec cells, conceivably due to activation of an endogenous FGF receptor, distinct from FGFR-1 (Wennström et al., 1992). Stimulation of cells expressing either the FGFR-1wt or the FGFR-1 Y766F resulted in a threefold proliferative response over basal levels (Fig. 7). FGF-2 stimulation of Vec control cells resulted in a smaller, 2-fold increase in cell number. Thus, in PAE cells, FGFR-1 mediated cell proliferation was independent of tyrosine 766 and the subsequent stimulation of PLC, PLD, PLA2, PI 3-kinase and stress fibre formation. DISCUSSION Tyrosine 766 is an autophosphorylation site in the FGFR-1 responsible for binding PLC-γ (Mohammadi et al., 1991) . FGFR-1 receptors with a mutation at tyrosine 766, have been shown to mediate mitogenesis in fibroblasts and neuronal

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differentiation of PC12 cells, suggesting that PLC-γ activation is not obligatory for cell proliferation or differentiation. However, FGF stimulates other lipid signalling enzymes and has effects on the actin cytoskeleton. Therefore, we have used PAE cells expressing the FGFR-1wt and FGFR-1 Y766F to determine the role of tyrosine 766 in regulating phospholipase activation, PI 3-kinase and cytoskeletal reorganisation. The FGFR-1 Y766F was unable to hydrolyse PtdIns(4,5)P2 (Fig. 2A), consistent with its previously reported inability to bind PLC-γ (Landgren et al., 1995). FGFR-1 mediated activation of PLD and PLA2 was also dependent upon tyrosine 766, since FGF-2stimulated activity of these enzymes was prevented in cells expressing the FGFR-1 Y766F (Fig. 2B,C). A requirement for the PLC-γ binding site in stimulating PLD activity has also been reported for the FGFR-1 in L6 myoblasts (van Dijk and van Blitterswijk, 1998) and for the PDGF receptor in TRMP canine kidney epithelial cells cells (Yeo et al., 1994). Furthermore, targeted disruption of the Plcg1 gene, encoding PLC-γ1, in mouse embryo fibroblasts resulted in a loss of both PDGF-stimulated PLC and PLD activity (Hess et al., 1998), indicating that in the case of the PDGF receptor, PLD activity is downstream of PLC-γ activity. A number of reports have shown that growth-factor stimulation of PLD is both PKC- and Ca2+-dependent (Cook et al., 1991). Cytosolic PLA2 is also known to be calcium-dependent (Leslie et al., 1988; Kramer et al., 1991) and PKC has also been implicated in its regulation (Nemenoff et al., 1993). In the PAE cells, FGFR-1 mediated activation of both PLD and PLA2 diplayed a PKC and Ca2+ dependence (Fig. 3B,C), suggesting that both enzymes are downstream of PLC-γ mediated DAG and InsP3 generation, which results in the activation of PKC and an increase in the intracellular calcium concentration respectively. Indeed, it has been previously shown that an FGFR-1 Y766F receptor failed to induce PKC translocation Fig. 5. Effect of the Y766F mutation on FGFR-1 mediated cytoskeletal reorganisation. Cells were stimulated with 100 ng/ml FGF-2 for 1 minute (B,B′,B′′), or 30 minutes (C,C′,C′′) or with 30 ng/ml FGF-2 for 24 hours (D,D′,D′′). For the basal level (A,A′,A′′), cells were stimulated with buffer for 1 minute, 30 minutes or with serum-free medium for 24 hours; identical staining was obtained for all these basal conditions. Cells were fixed, permeabilised and stained with rhodamine-conjugated phalloidin as described in Materials and Methods. Results are from a single experiment representative of three. Arrows in D′ indicate cellular processes staining densely for polymerised actin. Bar, 25 µm.

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Fig. 6. Effect of the Y766F mutation on FGFR-1 mediated MAP kinase activation. Cells were treated with (+) or without (−) 100 ng/ml FGF-2 for 10 minutes. Total cell lysates were analysed by western blotting using an antibody against phospho-MAP kinase. The blot was then reprobed with an antibody against p42 MAP kinase.

from cytosol to membrane fractions (Huang et al., 1995). To determine if FGF-2-stimulated PLA2 activity was downstream of PLD activity we attempted to inhibit PLD derived PA production by use of butan-1-ol. This primary alcohol is able to function as a nucleophilic acceptor in the PLD-catalysed transphosphatidylation reaction, resulting in the generation of phosphatidylbutanol instead of PA. FGF-2-stimulated PLA2 activity was not affected by butan-1-ol (Fig. 3C), suggesting that PLA2 was not downstream of PLD activity. Therefore, considering the data regarding a PKC/Ca2+ regulation discussed above, and the fact that the known isoforms of PLD and cPLA2 lack SH2 domains, it is most probable that both enzymes are downstream of PLC-γ activity, and do not interact directly with tyrosine 766. However, the possibility that other

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Conc. FGF-2 (ng/ml) Fig. 7. Effect of the Y766F mutation on FGFR-1 mediated proliferation. PAE Vec, FGFR-1wt and Y766F cells were stimulated with a range of FGF-2 concentrations for 5 days. Cell numbers were determined by use of a Coulter counter. Results are expressed as % of basal response for each cell line (mean ± s.d., n=3) from a single experiment representative of three. The relevant basal values were: Vec, 18564±1479 cells; FGFR-1wt, 16269±1428 cells; Y766F, 33864±3774 cells.

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signalling molecules bind to tyrosine 766 in the FGFR-1 and regulate PLD and PLA2 activity, cannot be discounted. FGFR-1-mediated activation of PI 3-kinase was also dependent upon tyrosine 766 (Fig. 4). It has been previously reported that PI 3-kinase does not directly associate with the FGFR-1 in PAE cells (Wennström et al., 1992), discounting the possibility that PI 3-kinase may somehow directly interact with tyrosine 766. Instead, the regulatory p85 subunit of PI3-kinase is known to interact with the consensus motif Y(p)-M-X-M present in receptor tyrosine kinases such as the PDGFR and EGFR (Songyang et al., 1993). Interestingly, this site is also present in the FGFR-1. However, in the FGFR-1, tyrosine phosphorylation of this site has not been demonstrated and the location of this site, in the second kinase domain, may preclude its availability for SH2 domain binding. This would suggest that the FGFR-1 is able to couple indirectly to PI 3-kinase, possibly through activation of Ras, which has been shown to stimulate the p110 subunit of PI 3-kinase (Rodriguez-Viciana et al., 1994; Kodaki et al., 1994). Since tyrosine 766 is required for activation of PI 3-kinase, it is possible that PLC-γ and the subsequent activation of a PKC/Ca2+ signalling are critical for regulating this activity. However, since tyrosine 766 affects all phospholipase activation, a number of candidate pathways exist for regulating PI 3-kinase activity. Activation of PI 3-kinase has been implicated in PDGF mediated membrane ruffling in PAE cells (Wennström et al., 1994). PtdIns(3,4,5)P3 activates the small molecular mass GTPase Rac (Hawkins et al., 1995), which has also been implicated in membrane ruffling (Ridley et al., 1992). However, although the FGFR-1 was able to stimulate PtdIns(3,4,5)P3 production, no membrane ruffling was detected (Fig. 5). We suggest that the level of PtdIns(3,4,5)P3 formation may be below the threshold for activation of Rac mediated membrane ruffling. In support of this, PDGFstimulation of PAE cells transfected with the PDGF-β receptor results in the appearance of membrane ruffles (Wennström et al., 1994), and induces a level of PtdIns(3,4,5)P3 that is fourfold higher than that seen with FGF-2 in our cells. Recently, it has been shown that PDGF stimulation of PI 3kinase activity, via both p85-mediated binding to the receptor and complex formation with Ras, results in a substantial activation of PI 3-kinase, which stimulates membrane ruffling and PKB/Akt activation; in contrast, bFGF stimulation of PI 3kinase, via only Ras, induces a relatively small activation of PI 3-kinase, resulting in only activation of PKB/Akt (van Weering et al., 1998). The FGFR-1 mediated cytoskeletal reorganisation was also dependent upon tyrosine 766, as stress fibre formation and cell shape-changes were not apparent in cell expressing the FGFR1 Y766F (Fig. 5). Since all phospholipase and PI 3-kinase activities are lost with this mutation, there exist a number of candidate pathways that may be responsible for mediating FGF-2-stimulated cytoskeletal reorganisation. We have previously reported that in PAE cells, exogenously added LPA was able to stimulate stress fibre formation in a manner that was dependent upon PLD-derived phosphatidic acid (Cross et al., 1996). It would therefore seem possible that phosphatidic acid, from FGF-2-stimulated PLD activity, may be responsible for the generation of stress fibres; such a mechanism would explain the loss of cytoskeletal reorganisation in cells expressing the Y766F mutant (Fig. 5C″,D″), in which no

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activation of PLC or PLD was observed in response to FGF-2. Another report has implicated PLA2 in EGF-stimulated stress fibre formation (Peppelenbosch et al., 1995). Furthermore, PI 3-kinase has also been implicated in stress fibre formation since Rac has been shown to induce the appearance of actin stress fibres in cells via the activation of Rho (Ridley et al., 1992). Since stress fibre formation was observed in the absence of membrane ruffling in PAE cells expressing the FGFR-1wt (Fig. 5C′), it is unlikely that PtdIns(3,4,5)P3-mediated Rac activation was involved in this response. However, a role for PI 3-kinase in cytoskeletal reorganisation cannot be discounted, since the lipid products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 have been implicated in binding the actin-capping proteins profilin and gelsolin (Lu et al., 1996; Chellaiah et al., 1998). It has been suggested that agonist-stimulated PtdIns(4,5)P2 generation may be responsible for sequestering actin-binding proteins, resulting in actin polymerisation (Janmey, 1994; Hartwig et al., 1995). However, no significant increase in PtdIns(4,5)P2 synthesis was observed upon stimulation of the FGFR-1wt cells with FGF-2 for up to 30 minutes (result not shown). FGFR-1-mediated mitogenesis has been previously shown to be independent of PLC-γ activity. Whilst the FGFR-1 Y766F receptor was unable to activate the phospholipases or PI 3kinase, it was able to stimulate MAP kinase (Fig. 6) and cellular proliferation (Fig. 7). MAP kinase activation in the cells expressing the FGFR-1 Y766F was lower than that in cells expressing the FGFR-1wt, in agreement with previously reported findings (Huang et al., 1995). To conclude, we have shown that in PAE cells, FGFR-1 mediated proliferation was not dependent upon activation of PLC-γ, PLD, PLA2 and PI 3-kinase. However, tyrosine 766 in the FGFR-1 was required for FGF-2-stimulated PLC, PLD, PLA2, PI 3-kinase and cytoskeletal reorganisation, showing that this site has the potential to regulate a number of intracellular signalling pathways. The only other report of a phenotype associated with the FGFR-1 Y766F mutation is the loss of FGFR internalisation (Sorokin et al., 1994). Recently, a cell-permeable peptide encompassing the Y766 PLC-γ binding site inhibited FGF-stimulated neurite outgrowth in cerebellar granule cells (Hall et al., 1996). Therefore, it is possible that loss of phospholipase activation and/or cytoskeletal reorganisation were responsible for these observations. In the latter study, direct addition of arachidonic acid resulted in neurite outgrowth, raising the possibility that FGF-mediated PLA2 activity is critical for this response. We thank Charlotte Wikner for technical assistance. This work was supported by grants from the Wellcome Trust, the Cancer Research Campaign, the Medical Research Council (UK) and the Swedish Medical Research Council (project no K98-03X-12552-01A). M.J.C acknowledges the receipt of a Royal Society Fellowship. M.N.H is supported by a Beit Memorial Fellowship.

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