Point mutation in the fibroblast growth factor receptor eliminates phosphatidylinositol hydrolysis without affecting neuronal differentiation of PC12 cells.

Fibroblast growth factors (FGF) stimulate growth arrest and differentiation in rat pheochromocytoma PC12 cells. We examined the role of phosphatidylinositol (PI) hydrolysis in FGF-induced differentiation of PC12 cells by exploring the biological and biochemical activity of a mutant FGF receptor 1 (flg) defective in stimulation of PI hydrolysis. We show that point mutation at Tyr-766 (Y766F) of the FGF receptor prevents tyrosine phosphorylation of phospholipase C gamma and eliminates acidic FGF (aFGF)-induced stimulation of PI hydrolysis in PC12 cells. Treatment of PC12 cells expressing either wild-type or the Y766F mutant with aFGF led to tyrosine phosphorylation of Shc, the association of Shc with GRB2, a shift in the electrophoretic mobility of the Ras guanine nucleotide-releasing factor, Sos (son of sevenless), and enhancement in mitogen-activated protein kinase phosphorylation. Moreover, stimulation with aFGF led to a typical neurite outgrowth of PC12 cells expressing either wild-type or the Y766F FGF receptor mutant. These experiments indicate that PI hydrolysis is not essential for FGF-induced neuronal differentiation of PC12 cells. Moreover, the aFGF-induced Ras signaling pathway, which is essential for PC12 cell differentiation, is not affected by elimination of PI hydrolysis.

Acidic or basic fibroblast growth factors (FGF)' and nerve growth factors (NGF) act by binding to cell surface receptors with tyrosine kinase activity (reviewed in Schlessinger and Ullrich (1992)). Ligand binding results in receptor activation leading to a variety of biological responses. Exposure of the rat PC12 pheochromocytoma cell line t o acidic FGF (aFGF), basic FGF, or NGF induces growth arrest and a dramatic change in cell morphology. PC12 cells, which are round and have an adrenal medullary chromaffh-like phenotype, respond to FGFs and NGF by growing long neurite extensions (Greene and Tischler, 1976;Togari et al., 1983Togari et al., , 1985Rydel and Green, 1987). These cells serve as a useful model system for studying neuronal differentiation and signaling pathways of the FGF and NGF receptors.
Accumulating evidence suggests that Ras (reviewed in Satoh et al. (1992) and Polakis and McCormick (1993)), a 21-kDa GTP-binding protein, is activated by FGF and NGF and plays * The costs of publication of this article were defrayed in part by the "advertisement" in accordance with 18 U.S.C. Section 1734 solely to payment of page charges. This article must therefore be hereby marked indicate this fact.
The abbreviations used are: FGF, fibroblast growth factors; PI, phosphatidylinositol; FGFR, FGF receptor; aFGF, acidic FGF; NGF, nerve growth factors; anti-P-Tyr, anti-phosphotyrosine; Sos, son of sevenless; MAF'K, mitogen-activated protein kinase. a crucial role in mediating FGF and NGF-induced differentiation of PC12 cells. Ras acts as a molecular switch Wittinghofer and Pai, 19911, which is active in the GTPbound form and inactive in the GDP-bound form. The regulation of Ras activation involves guanine nucleotide releasing factors (Sos or GRF) and proteins that stimulate its intrinsic GTPase activity (Ras-GAP or NF-1). Treatment of PC12 cells with either FGF or NGF increases the amount of active, GTPbound, Ras (Qiu and Green, 1991;Li et al., 1992;. FGF-or NGF-induced Ras activation was shown to be obligatory for activation of a kinase cascade including Raf, mitogen-activated protein kinase kinase (MAF'KK), and mitogen-activated protein kinase WU"AP) (Wood et al., 1992;Thomas et al., 1992;Robbins et al., 1992), which culminates in the phosphorylation of nuclear transcription factors (Pulverer et al., 1991;Marais et al., 1993;Hunter and Karin, 1992). The central role of Ras in PC12 cell differentiation is demonstrated by the ability of oncogenic Ras mutants to stimulate neurite outgrowth (Noda et al., 1985;Bar-Sagi and Feramisco, 1985;Satoh et al., 1987). Moreover, a dominant negative mutant of Ras or anti-Ras antibodies prevents differentiation induced by either FGF or NGF (Hagag et al., 1986;Szeberenyi et al., 1990).
GRBB is a small SH2-(src homology) and SH3-containing adaptor protein that links both receptor and nonreceptor tyrosine kinases to the Ras-signaling pathway (Lowenstein et al., 1992). GRBS binds to tyrosine-phosphorylated epidermal growth factor receptor through its SH2 domain (Lowenstein et al., 1992) and to the guanine nucleotide-releasing factor Sos through its SH3 domains Egan et al., 1993;Rozakis-Adcock et al., 1993;Gale et al., 1993;Buday and Downward, 1993;Chardin et al., 1993;Simon et al., 1993;Olivier et al., 1993;Skolnik et al., 1993). GRBS is also associated with the tyrosine-phosphorylated SH2 domain-containing adaptor protein Shc . It has been shown that overexpression of Shc in PC12 cells leads t o Ras-dependent neurite outgrowth of PC12 cells (Rozakis-Adcock et al., 1992), suggesting that the interaction between Shc and GRBB may be important in regulating Ras activation.
Treatment of target cells with FGF or NGF also stimulates tyrosine phosphorylation of phospholipase Cy (Burgess et al., 1990;Vetter et al., 1991). Growth factor-induced phosphorylation of phospholipase Cy on tyrosine residues enhances its catalytic activity (Nishibe et al., 1990) and is required for phospholipase Cy activation in intact cells (Kim et al., 1991). Phospholipase Cy hydrolyzes phosphatidylinositol (4, 5) bisphosphate to diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which releases Ca2+ from intracellular stores. The demonstration that phospholipase Cy is a substrate for the FGF and NGF receptors, and the stimulation of PI hydrolysis by NGF in PC12 cells (Traynor et al., 1982;Contreras and Guroff, 1987;Altin and Bradshaw, 1990;Pessin  Activation of the Ras-signaling pathway and PI hydrolysis has also been implicated in differentiation and development of Xenopus laevis embryos. Moreover, FGF is essential for mesoderm induction during early development of the Xenopus embryo (Slack et al., 1987). Microinjection of messenger RNA encoding a dominant inhibitory mutant of Ras into Xenopus eggs blocked FGF-induced mesoderm induction (Whitman and Melton, 1992), suggesting that Ras is involved in Xenopus mesoderm induction. The normal embryonal development can also be distorted by treatment with Li' , an inhibitor of PI hydrolysis, which results in deformed dorsal embryos (reviewed in Berridge et al. (1989) and Berridge (1993)). It is thought that the teratogenic effect of Li' on developmental processes in Xenopus embryos is mediated by inhibition of inositol formation (Busa and Gimlich, 1989;Maslanski et al., 1992), which is required for the regeneration of phosphatidylinositol (4, 5 ) bisphosphate. Hence, PI hydrolysis may play a role in amphibian embryonic mesoderm induction.
We have previously shown that Tyr-766 in the carboxyl-terminal tail of FGFR 1 (flg) functions as a high affinity binding site for the SH2 domain of phospholipase Cy (Mohammadi et al., 1991). The elimination of "766 by site-directed mutagenesis prevented both the association of phospholipase Cy with FGFR and the tyrosine phosphorylation of phospholipase Cy, leading to the elimination of FGF-induced PI hydrolysis and Ca2+ release (Mohammadi et al., 1992;Peters et al., 1992). However, the Y766F FGFR mutant was able to stimulate DNA synthesis in transfected L-6 myoblasts, indicating that PI hydrolysis is not essential for FGF-induced mitogenesis. These results are in agreement with others (Valius et al., 1993; Seedo r f et Ronnstrand et al., 1992) in demonstrating the lack of requirement for PI hydrolysis in the mitogenic signaling pathways of other growth factor receptors.
To shed light on the role of PI hydrolysis in the control of PC12 cell differentiation, we have tested the ability of the Y766F FGFR mutant defective in stimulation of PI hydrolysis to elicit the differentiation of PC12 cells in response to aFGF.
Here, we show that PC12 cells expressing either wild-type or the Y766F FGF receptors undergo aFGF-dependent neuronal differentiation by displaying typical dense neurite-like processes within 16-20 h. Furthermore, we show that the Rassignaling pathway is maintained in the absence of PI hydrolysis and that PI hydrolysis is not essential for FGF-induced differentiation of PC12 cells.

EXPERIMENTAL PROCEDURES
Materials-PC12 cells were kindly provided by Dr. M. V. Chao a t Cornel1 Medical Center, New York. Escherichia coli expressing recombinant human aFGF, anti-flg lC, and anti-flg 3B antibodies was previously characterized (Mohammadi et al., 1991;Jaye et al., 1988). Heparin was from Elkins-Sinn, Inc. "he following rabbit polyclonal antibodies were generated in our laboratory: anti-phosphotyrosine (anti-P-Tyr) antibodies, anti-phospholipase Cy antibodies raised against a synthetic peptide (residues 1256-1274) from the COOH terminus of phospholipase Cy, anti-Grb2 antibodies raised against a glutathione S-transferase fusion protein containing the N-SH3 domain of Grb2, and anti-Sos directed against the catalytic domain of human Sosl. Rabbit polyclonal anti-Shc antibodies are directed against a glutathione Stransferase-SH2 fusion protein . ['%I]- (carrier-free) and [3Hlmyoinositol were purchased from DuPont NEN. The cDNAs and expression vectors encoding wild-type and Y766F FGF receptor mutant (flg) receptors have been described elsewhere (Mohammadi et al., 1991(Mohammadi et al., , 1992.
Generation of Cell Lines-Parental rat PC12 cells were cotransfected by calcium-phosphate precipitation (Wigler et al., 1979) with 30 pg of cDNA encoding either wild-type human FGF receptor (flg) or the Y766F mutant (Mohammadi et al., 1991(Mohammadi et al., , 1992 and 0.5 pg of a selective plasmid carrying a neomycin resistance marker. Clones generated in me-

A.
Parental wt Y766F P a r e n t a l w t Y 7 6 6 F B.
-n ---- 1. A mutant FGF receptor in which "766 is replaced b y phenylalanine is phosphorylated on tyrosine upon FGF stimulation of PC12 cells. Parental PC12 cells and transfected PC12 cells expressing either wild-type (wt) human FGFR (flg) or the Y766F mutant were treated in the absence (-) or presence (+) of aFGF for 5 min at 37 "C. Cells were then lysed, immunoprecipitated with anti-FGFR antibodies (anti-flg 3B), and separated by 8% SDS-polyacrylamide gel , electrophoresis. The samples were then transferred to a nitrocellulose membrane and immunoblotted with either anti-FGFR antibodies (antiflg 3B) (Fig. lA), revealing the level of receptor expression, or anti-P-Tyr antibodies (Fig. IB), indicating the level of receptor autophosphorylation in each of these cell lines. Immunoblots were exposed for 12 h at -70 "C. dium containing Geneticin (G418) were screened for expression of FGF receptor constructs by using two rabbit anti-peptide antibodies. Rabbit anti-flg 3B antibodies directed against the flg kinase insert recognize both ratflg and the transfected human wild-type and mutant receptors. Rabbit anti-flg 1C antibodies directed against the COOH-terminal tail of flg recognize the human but not the rat receptor. Following binding studies of '2sI-aFGF (iodinated with chloramine T (Greenwood et al., 1963)) to the cell lines and Scatchard analysis of the binding data, a clone of PC12 cells expressing 2 x lo6 wild-type FGF receptordcell and one expressing 1.2 x los Y766F mutant receptordcell was chosen for further analysis.
Immunoprecipitation and Western Blotting Analysis-!l'ransfected PC12 cells were treated with aFGF, lysed, and subjected to immunoprecipitation and immunoblotting analysis according to published procedures (Mohammadi et al., 1992).

RESULTS AM) DISCUSSION
Parental PC12 cells expressing approximately 8,000-10,000 FGFFUcell were transfected with mammalian expression vectors, which direct the synthesis of either wild-type FGFR or the Y766F FGFR mutant (Mohammadi et al. 1992). Binding studies with '251-labeled aFGF indicated that the transfected PC12 cells express approximately 2 x lo5 wild-type receptors or 1.2 x lo5 Y766F mutant receptors/cell, with a dissociation constant of approximately 0.1 I~M (data not shown). The transfected cells were treated with aFGF, solubilized, and subjected to immunoprecipitation with anti-FGFR antibodies followed by immunoblotting with either anti-FGFR or anti-P-?'yr antibodies. The results presented in Fig. 1 show that both wild-type and the Y766F mutant FGF receptors undergo typical ligand-dependent tyrosine autophosphorylation. Stronger autophosphorylation of wild-type FGFR was observed as compared with the mutant receptor due to elimination of Tyr-766, which is a major autophosphorylation site of the FGFR (Fig. 1B) (Mohammadi et al., 1991(Mohammadi et al., , 1992.
We next compared the ability of the wild-type receptor and the Y766F receptor mutant to stimulate tyrosine phosphorylation of phospholipase Cy. Following ligand stimulation PC12 cells were lysed, subjected to immunoprecipitation with antiphospholipase Cy antibodies, and immunoblotted with either

A. I P -A n t i P L C -Y
' '

I P -A n t , P L C -Y
' ' I P -Anti F l p 36

FIG. 2. Point mutation of -766
of the F G F receptor inhibits association with and tyrosine phosphorylation of phospholipase Cy. PC12 cells expressing wild-type ( w t ) or mutant FGF receptors were stimulated with aFGF, lysed, and immunoprecipitated as described under "Experimental Procedures." A, anti-phospholipase Cy (PLC-y) immunoblotting of anti-phospholipase Cy immunoprecipitates. B, anti-P-Tyr immunoblotting of anti-phospholipase Cy immunoprecipitates. C, anti-phospholipase Cy immunoblotting of anti-FGFR (flg 3B) immunoprecipitates. Immunoblotting was performed as described for Fig. 1. Immunoblots were exposed for 18 h at -70 "C. anti-FGFR or anti-P-Tyr antibodies. The results presented in Fig. 2 show that aFGF stimulation led to association between wild-type FGF receptor and phospholipase Cy and tyrosine phosphorylation of phospholipase Cy (Fig. 2, B and C). However, the Y766F FGFR mutant was unable to associate with or tyrosine-phosphorylate phospholipase Cy (Fig. 2, B and C ) . Measurements of PI hydrolysis in Y766F-expressing cells indicated that the Y766F mutation abrogated aFGF-induced PI turnover (Fig. 3).
To examine the events that lead to Ras activation upon binding of aFGF to the wild-type receptor or the Y766F mutant, PC12 cells expressing either wild-type or Y766F FGFR were stimulated with FGF, immunoprecipitated with anti-GRB2 antibodies and blotted with either anti-Sos or anti-Shc antibodies, or immunoprecipitated with anti-Shc antibodies and blotted parental wt Y766F

" -
A. Parental and PC12 cells transfected with either wild-type or Y766F FGF receptors were incubated overnight in serum-depleted medium and stimulated with aFGF. Treated (+) and nontreated (-) cells were lysed, immunoprecipitated with anti-GRB2 antibodies, and immunoblotted with anti-Sos antibodies (A), anti-Shc antibodies (B), or immunoprecipitated with anti-Shc antibodies (covalently cross-linked to protein A-Sepharose) and blotted with anti-P-Tyr antibodies. Immunoblot of panel A was exposed for 18 h a t -70 "C, and panels B and C were exposed for 15 h. either wild-type (wt) or Y766F FGFRs. Parental and PC12 cells transfected with either wild-type or Y766F FGFRs were grown overnight in serum-depleted medium. Control starved cells (-) or cells treated with aFGF (+) were lysed and immunoprecipitated with anti-P-Tyr antibodies. The samples were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with polyclonal anti-microtubule-associated protein kinase antibodies, which detected two enzyme isoforms (42-and 44-kDa proteins). Immunoblots were exposed for 16 h at -70 "C.

-+ -+ -+ FGF
with anti-P-Tyr antibodies. Following aFGF treatment, both wild-type and Y766F FGF receptors induced tyrosine phosphorylation of Shc (Fig. 4C) and its association with GRBB (Fig.  4B). However, the FGFR could not be detected in the GRB2-Shc immunoprecipitates (data not shown), suggesting that GRBB and Shc do not associate with the FGFR or that the association is very weak. In this regard, FGF, NGF, and EGF receptors exhibit differential interaction with the adaptor proteins GRBB and Shc. The FGFR does not bind either GRB2 or Shc; the NGFR binds Shc (Obermeier et al., 1993) but not GRBB (Suen et al., 1993), while the epidermal growth factor receptor binds both GRBB (Lowenstein et al., 1992)  1992; Ruff-Jamison et al., 1993). GRB2 was associated with Sos in lysates of both stimulated and nonstimulated PC12 cells (Fig. 4A), and activation of either wild-type or the Y766F FGF receptors led to a shift in the mobility of Sos (Fig. 4A). We have shown that the shift in the mobility of Sos is due to phosphorylation on multiple threonine and serine residues.' Moreover, both wild-type and the Y766F FGF receptors were able to stimulate tyrosine phosphorylation of the MAP kinase isoforms (42 and 44 kDa) (Fig. 51, indicating activation of the Ras-signaling pathway. To investigate whether PI hydrolysis is required for FGFinduced differentiation of PC12 cells, control, wild-type, and Y766F-expressing PC12 cell lines were stimulated with aFGF, and neurite outgrowth was assessed 16-20 h later. Long and dense neurites were observed upon treatment with aFGF in PC12 clones expressing either wild-type or the Y766F mutant (Fig. 6, panels 4 and 61, whereas no neurites were observed in the parental PC12 cells (Fig. 6, panels 1 and 2). Thus, elimination of aFGF-induced PI hydrolysis does not affect PC12 cell differentiation.
It was proposed that PI hydrolysis plays a role in cell differentiation based on the observation that Li' disrupts both axis determination and mesoderm induction in the developing Xenopus embryo (Busa and Gimlich, 1989;Berridge et al., 1989), an effect that is reversed by addition of exogenous myoinositol (Maslanski et al., 1992). We assume that the difference between these studies and ours, regarding the requirement for PI hydrolysis, reflect the very different models examined. Our study employs PC12 cells, which are a cloned, uniform population of cells that respond to aFGF treatment by extension of neurites. In contrast, the Xenopus embryo contains a variety of cells with shape, and cell mobility. Therefore, the role of PI hydrolysis may not be directly comparable in these two very different systems. Alternatively, it is also possible that Li' has other effects in Xenopus that are not related to PI hydrolysis.
Our results indicate that the Y766F FGFR mutant activates the Ras-signaling pathway in a manner indistinguisable from activation by wild-type FGFR. Thus, the functional significance of activation of phospholipase Cy and stimulation of PI hydrolysis by wild-type FGFR in PC12 cells is unknown. Both the activated wild-type and Y766F mutant FGF receptors are able to stimulate phosphorylation of Shc on tyrosine residues, the association of Shc with GRB2, and to induce mobility shift of Sos (Fig. 4). Moreover, both receptors are able to activate microtubule-associated protein kinase (Fig. 5), indicating that the Ras-signaling pathway is also activated by the Y766F receptor mutant. In contrast to PI hydrolysis, Ras-dependent signals appear to be essential for FGF-(and NGF-) induced differentiation of PC12 cells (Hagag et al., 1986;Szeberenyi et al., 1990). However, other growth factors such as EGF, insulin, and IGF-1 also activate Ras in PC12 cells without inducing differentiation Qiu and Green, 19911, indi-cating that activation of Ras is essential for both mitogenesis and differentiation. Recent studies demonstrate that growth factors that promote neuronal differentiation of PC12 cells (e.g. FGF, NGF, and PDGF) lead to persistent elevation of mitogenactivated protein kinase activity and its translocation to the nucleus, while mitogenic growth factors (e.g. EGF and IGF-1) activate mitogen-activated protein kinase transiently (Chen et al., 1992;Qiu and Green, 1992;Heady and Johnson, 1992;Nguyen et al., 1993;Lenormand et al., 1993;Gonzalez et al., 1993). Moreover, treatment of PC12 cells overexpressing epidermal growth factor receptor or insulin receptor with either EGF or insulin leads to typical neuronal differentiation3 It is therefore thought that receptor number, duration, and amplitude of activation and the cellular localization of target proteins may also influence the cellular response of PC12 cells to a given growth factor. Full understanding of the pathways involved in FGF-induced differentiation of PC12 cells will require a thorough analysis and characterization of additional receptor mutants and their interaction with signaling proteins.