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Cell Growth & Differentiation Vol. 12, 255-264, May 2001
© 2001 American Association for Cancer Research

Selective Inhibition of Fibroblast Growth Factor (FGF)-stimulated Mitogenesis by a FGF Receptor-1-derived Phosphopeptide1

Dara J. Dunican2, Emma J. Williams, Fiona V. Howell and Patrick Doherty3

Molecular Neurobiology Group, MRC Centre for Developmental Neurobiology, Kings College London, London Bridge, London SE1 1UL, United Kingdom


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The activated fibroblast growth factor receptor (FGFR)-1 is phosphorylated on five tyrosine residues outside the catalytic site. Although one such residue, Tyr730, is flanked by potential binding sites for phosphotyrosine-interacting molecules, a physiological role for this region is still controversial. We report that a cell-permeant phosphopeptide mimic of this site, FGFR730(p)Y, inhibits FGF-mediated mitogenesis in cells with no effect on responses stimulated by other growth factors. A similar phosphopeptide corresponding to the phospholipase C{gamma} binding site on the receptor had no effect on the mitogenic response. The FGFR730(p)Y peptide did not inhibit phosphorylation of p90/FRS2 or Erk, suggesting that it does not act by inhibiting the Erk-kinase cascade. However, the FGFR730(p)Y peptide bound Shc in a manner requiring both phosphorylated tyrosine and a putative PTB domain binding determinant. These data suggest that the peptide might inhibit mitogenesis by competing with the corresponding site on the FGFR for the ability to bind Shc.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
FGFs4 play central roles in complex biological events such as morphogenesis, angiogenesis, and tumor formation (1 , 2) . They do so by interacting with one or more of the FGFRs (FGFR-1–4), which are members of the family of transmembrane receptors that contain a cytoplasmic tyrosine kinase domain (RTKs). RTKs can be activated by the interaction of their extracellular domains with specific ligands, which associate the receptors into active complexes (3 , 4) . In the active complex, the cytoplasmic domain of one receptor serves as a substrate for the catalytic activity of the second in a reciprocal fashion, and the resultant phosphorylation of specific tyrosine (Tyr) residues gives rise to high affinity binding sites for a range of downstream signaling molecules (5) . In general, binding of these molecules is via conserved pTyr-interacting domains, two forms of which have been characterized to date and are called SH2 or PTB domains (6) .

SH2 domains are evolutionarily conserved protein modules found in a wide range of signaling molecules. Different SH2 domains generally recognize specific short peptide motifs containing a phosphorylated Tyr in the context of specific residues found within 3–5 amino acids COOH-terminal to the pTyr (7) . The existence of PTB domains has been more recently established where they were originally described as pTyr-interacting modules in a manner analogous to SH2 domains (8 , 9) . Although it is now clear that this family of modules contains members that recognize unphosphorylated targets, phosphorylation-dependent PTB domains such as those in the adapters Shc or insulin receptor substrate-1 are involved in the recruitment of these molecules to phosphorylated RTKs (10) . PTB domains are structurally unrelated to SH2 domains and appear to complex the pTyr in a distinct manner. Their specificity of binding motif recognition differs from that of SH2 domains in that the critical residues for contact are primarily found on the NH2-terminal side of the pTyr (6 , 11 , 12) .

The FGFR-1 can be phosphorylated on seven cytoplasmic Tyr residues, five of which are not required for catalytic activity of the receptor (13) . This suggests that the receptor may use these noncatalytic pTyr residues to transduce signals in the manner outlined above, with these sites acting as docking sites for downstream signaling molecules. In this context, it has been shown previously that activation of the FGFR-1 stimulates Tyr phosphorylation and/or activation of a number of pTyr-binding signaling molecules such as PLC{gamma} (14 , 15) , Shc (16) , PI3-K (17) , Src (18) , SH2 domain-containing phosphatase-2 (19) , and FRS2 (20) . More importantly, the pTyr766 has been shown to mediate association of PLC{gamma} with FGFR-1 in a manner that is essential for FGF-stimulated phosphoinositide hydrolysis (15) and the ability of the receptor to mediate downstream events such as FGF-mediated axonal outgrowth in primary neuronal cells (21) . However, despite the established role of pTyr766 in PLC{gamma}-mediated cell signaling, the implication of the other pTyr sites in any downstream signaling events has proven difficult. Although isolated reports have described association of molecules such as Src or Shc/Grb2 with FGFR-1 in a pTyr-dependent manner (22 , 23) , these reports have failed to map the sites of interaction on the receptor and have not been widely confirmed. However, by precedent the most likely function of regions of the receptor containing the noncatalytic pTyr residues, such as Tyr730, is the recruitment of second messengers, albeit possibly by weaker or more transient interactions that make detection difficult.

On the basis of sequence analysis, phosphorylation of Tyr730 in the FGFR would be expected to create a binding site for Shc and/or PI-3 kinase (see "Discussion" and Table 1Citation for details). If phosphorylation of this site is important for recruitment of a downstream signaling molecule that participates in a mitogenic response, then the introduction into cells of phosphorylated peptide mimetics of the site should inhibit the biological response by competing with effector/receptor interactions. "Proof-of-principle" experiments using cell-permeant phosphopeptide mimetics of both the established Grb2 binding site on the EGF receptor and the PLC{gamma} binding site on the FGFR demonstrate the validity of this approach (21 , 24) . In both instances, nine amino acid phosphopeptides with specific SH2 domain binding activity were rendered cell-permeant by synthesizing them in tandem with a 16-amino acid sequence from the Antennapedia protein (25) . In the present study, we used the same strategy to made a cell-permeant phosphopeptide mimetic of the candidate recruitment site that would be created by phosphorylation of Tyr730 [termed the FGFR730(p)Y peptide]. The FGFR730(p)Y peptide inhibited the mitogenic response stimulated by FGF with significant effects detected at 1 µg/ml. The specificity of the inhibition was demonstrated by showing that the peptide had no effect on the mitogenic responses stimulated by serum or PDGF. The equivalent cell-permeant phosphopeptide mimetics of the PLC{gamma} binding site on the FGFR and the Grb2 binding site on the EGFR do not inhibit the mitogenic response stimulated by FGF (24) , demonstrating that the inhibition by the FGFR730(p)Y peptide is highly specific. The FGFR730(p)Y peptide did not inhibit events leading to Erk activation, suggesting that for entry into the cell cycle, signaling pathways in addition to those mediated by Erk are initiated by the FGFR-1. The inhibitory peptide was shown to bind to both Shc and the p85 of PI3-K, apparently via their PTB and SH2 domains, respectively. Mutational analysis of the peptide indicated that the inhibitory effect correlated with association of Shc and not p85. These data raise the interesting possibility that the peptide inhibits the mitogenic response by competing with a Shc-FGFR interaction; however, other targets for the peptide cannot be excluded. Nonetheless, the present data unequivocally demonstrate that an FGFR-derived phosphopeptide can differentially inhibit mitogenic responses stimulated by a range of growth factors and implicates, for the first time, Tyr730 as a functional mediator of the FGFR mitogenic signaling cascade.


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  		Table 1 A comparison of the FGFR730(p)Y sequence with those of known Shc PTB domain and p85 domain binding motifsa

 

   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Phosphorylation of Tyr730 in the FGFR-1 creates consensus binding motifs for both Shc and the p85 subunit of PI3-K as defined in a range of studies (12 , 26) . To test the potential for this sequence to act as a functional binding site in the activated FGFR, we synthesized a cell-permeant mimetic of this motif in tandem with a biotinylated Antennapedia internalization sequence [the FGFR730(p)Y peptide] and tested it for its ability to inhibit mitogenic responses stimulated by FGF.

A FGFR-1-derived Phosphopeptide Inhibits Mitogenesis in Response to FGF but not Other Growth Factors in Two Cell Types.
When added to two cell types, the FGFR730(p)Y peptide was capable of inhibiting FGF- mediated BrdUrd incorporation, an index of entry into S-phase. This peptide was able to abolish FGF-mediated mitogenesis at a concentration of 50 µg/ml in rat C2C12 muscle cells (Fig. 1A)Citation and 10 µg/ml in newt A1 myoblast cells (Fig. 1B)Citation . In additional studies using A1 cells, the FGFR730(p)Y peptide was seen to inhibit mitogenesis at concentrations as low as 1 µg/ml (Fig. 1C)Citation . In contrast, the peptide had no effect on the basal mitogenic activity in both cell types, suggesting that it acts in a specific way on the growth factor-mediated response. A similar cell-permeant mimetic of the well-characterized PLC{gamma} binding site on the FGFR-1 [FGFR766(p)Y peptide] had no effect on the FGF response at 100 µg/ml (Fig. 1, B and C)Citation , despite the fact that it can inhibit FGF-stimulated axonal growth at 1–10 µg/ml (21) . Likewise, a similar cell-permeant mimetic of the Grb2 binding site on the EGFR [EGFR1068(p)Y] that was demonstrated previously to inhibit mitogenic responses stimulated by EGF and PDGF does not inhibit an FGF mitogenic response (24) . Thus, we can conclude that it is the FGFR-derived portion of the FGFR730(p)Y peptide that is responsible for the inhibition of the FGF response, and that Antennapedia-based phosphopeptides do not have general, nonspecific effects on mitogenic responses.



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  		Fig. 1. The FGFR730(p)Y cell-permeable phosphopeptide inhibits mitogenic responses induced by FGF in two cell types. In a, C2C12 cells were serum starved and then cultured in control medium ({square}) or medium containing 5 ng/ml of bFGF ({blacksquare}) in the presence or absence of the FGFR730(p)Y peptide at 50 µg/ml, as described in "Materials and Methods." In b, the experiment was repeated with A1 cells using 10 µg/ml of either FGFR730(p)Y or FGFR766(p)Y peptide. In c, A1 cells were treated with control medium ({circ}, {square}) or medium containing 5 ng/ml of bFGF (•, {blacksquare}) in the presence of a range of concentrations of either the FGFR730(p)Y peptide ({square}, {blacksquare}) or the FGFR766(p)Y peptide ({circ}, •) as indicated. The results show the percentage of cells in S-phase as determined by BrdUrd incorporation. The percentage of cells in S-phase was calculated by dividing the number of nuclei incorporating BrdUrd by the total number of nuclei (see "Materials and Methods"); 150–300 cells were scored in three replicate wells for each experimental condition. The results in a and b show data pooled from three independent experiments (means; bars, SE). The results in c are taken from a single representative experiment.

 
The FGFR730(p)Y peptide was also examined for its effect on mitogenesis stimulated by other growth factors. Neither FCS nor PDGF-stimulated mitogenesis was appreciably inhibited by FGFR730(p)Y in A1 cells under conditions where the FGF response was again abolished (Fig. 2)Citation . Similar results were observed for C2C12 cells (data not shown). Together, these data indicate that the FGFR730(p)Y peptide specifically inhibits the FGF-mediated mitogenic response in contrast to all other agonists tested in this study.



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  		Fig. 2. Mitogenic responses induced by FCS or PDGF are unaffected by optimal concentrations of the FGFR730(p)Y inhibitory peptide. After serum starvation, A1 cells were cultured in control medium or medium supplemented with FGF (5 ng/ml), FCS (10% v/v), or PDGF (10 ng/ml) as indicated. In a set of test cultures, the medium was further supplemented with the FGFR730(p)Y peptide at 10 µg/ml as described in "Materials and Methods." After analysis of BrdUrd incorporation, results were expressed as the percentage of cells in S-phase. Data points represent the means of two experiments; bars, SE.

 
Effect of FGFR-1-derived Peptides on the Erk Pathway.
Recruitment of Grb2/SOS to the plasma membrane and the consequential activation of the Erk cascade are required for the mitogenic response stimulated by FGF as well as other growth factors (27) . In agreement, we have shown previously that a MAPK kinase inhibitor (PD098059) can fully block activation of the MAPK cascade and mitogenic responses stimulated by FGF, EGF, and PDGF in A1 muscle cells (24) . We examined the effect of the FGFR-derived peptides on Erk phosphorylation (an index of Erk activation) in response to FGF. The FGFR730(p)Y and FGFR766(p)Y peptides had no detectable effect on Erk phosphorylation in response to FGF (Fig. 3A)Citation , and this is consistent with the proposal that neither of these sites are required for activation of the MAPK cascade.



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  		Fig. 3. Erk and p90/SNT phosphorylation are unaffected by phosphopeptide treatment. In a, C2C12 cells were serum starved and then treated with control medium (No peptide) or medium supplemented with either the FGFR730(p)Y or FGFR766(p)Y peptide at 50 µg/ml for 1 h as indicated. FGF (0.1 ng/ml) or vehicle control was added as indicated. After 10 min, the cells were lysed directly into Laemmli sample buffer and analyzed for phosphorylation of Erk proteins by SDS-PAGE and Western blotting. The blot is representative of at least three experiments. In b, NIH3T3 cells were starved overnight before treatment with 25 µg/ml of the FGFR730(p)Y peptide or vehicle control for 1 h as indicated. Subsequently, FGF (50 ng/ml) was added for 10 min as indicated. The cells were then lysed, and aliquots of the lysates were boiled in Laemmli sample buffer and analyzed for tyrosine phosphorylation by SDS-PAGE and Western blotting with an anti-phosphotyrosine antibody. The blot is representative of three separate experiments.

 
Recently, an FGFR-associated molecule has been cloned that has been proposed to mediate the FGFR-induced recruitment of Grb2/SOS to the membrane and the consequent activation of the Erk pathway (28) . This Mr 90,000 protein termed p90, SNT, or FRS2 is highly phosphorylated in response to FGF on at least five sites that have been proposed to bind Grb2, both directly and through an intermediate protein (16 , 28) . p90/FRS2 has been shown to associate with the FGFR-1 via the receptor juxtamembrane sequence in a manner not requiring phosphorylation of the receptor (29) . To substantiate the likelihood that the FGFR730(p)Y peptide does not directly interfere with activation of the MAPK cascade, we tested the peptide for its ability to inhibit phosphorylation of p90. As reported by others, FGF treatment strongly stimulated phosphorylation of a Mr 90,000 protein (presumably p90/FRS2; Fig. 3BCitation ), and this response was not inhibited by treatment of cells with the FGFR730(p)Y peptide.

The FGFR-derived Peptides Interact with a Number of SH2 Domain-containing Proteins.
A number of phosphotyrosine-binding proteins have been proposed to be involved in FGFR signaling. These include PLC{gamma} (15) , PI3-K (17) , and Shc and Grb2 (16 , 23) . In an attempt to understand the inhibitory action of FGFR730(p)Y, we examined the ability of these molecules to interact with FGFR730(p)Y as compared with FGFR766(p)Y and a number of other RTK-derived peptides. This was carried out by the incubation of cell lysates with peptides immobilized on agarose and the subsequent analysis for the association of the candidate molecules Shc, p85, PLC{gamma}, and Grb2 with the peptides by affinity precipitation and immunoblotting.

Distinct binding preferences were observed for FGFR730(p)Y and FGFR766(p)Y (Fig. 4)Citation . FGFR766(p)Y was seen to bind PLC{gamma} strongly, reflecting the well-established role of the corresponding site on FGFR as the site mediating the binding and subsequent activation of this signaling molecule. In addition, weaker and more variable interaction of p85 was occasionally observed. In contrast, the FGFR730(p)Y peptide precipitated Shc isoforms, and p85 at robust levels comparable with positive control peptides (a peptide corresponding to the Tyr1309 Shc PTB domain binding site on the ErBB3 receptor, and a peptide corresponding to the Tyr740 p85 binding site on the PDGF receptor). Weaker binding of PLC{gamma} was also occasionally seen. Finally, Grb2 did not associate with either FGFR730(p)Y or FGFR766(p)Y, in contrast with its strong interaction with a "positive control" peptide derived from the EGFR. Thus, it is seen that the FGFR730(p)Y peptide binds robustly and preferentially to both p85 and Shc in a manner that correlates with its inhibitory effect on FGF-mediated mitogenesis.



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  		Fig. 4. An examination of the ability of FGFR-derived phosphopeptides to associate with a range of SH2/PTB-containing signaling molecules in cell lysates. A number of biotinylated Antennapedia-based phosphopeptides were coupled to agarose-streptavidin as described in "Materials and Methods." These included two FGFR-1-derived phosphopeptides [FGFR730(p)Y and FGFR766(p)Y] and a number of "positive control" peptides that have been established previously to interact with p85, Shc, or Grb2 (see text for details). FGFR766(p)Y was considered the positive control for a PLC{gamma} binding site. Equal amounts of control beads (No peptide) or peptide-coated beads (as indicated) were added to aliquots of NIH3T3 cell lysates and incubated at 4oC for 1–3 h. Beads were washed with lysis buffer supplemented with 0.1% SDS and boiled in Laemmli sample buffer. Samples were run by SDS-PAGE and Western blotting for PLC{gamma}, p85, Shc, and Grb2 as indicated. Each panel is representative of two to four separate experiments.

 
Treatment of the FGFR730(p)Y peptide with a tyrosine phosphatase inhibited the interaction with both p85 and Shc (Fig. 5)Citation . These data demonstrate that the FGFR730(p)Y peptide can interact, in a predictable and specific manner, with known effector molecules that have been implicated in mitogenic signaling cascades.



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  		Fig. 5. Shc and p85 bind FGFR730(p)Y in a phosphotyrosine-dependent manner. FGFR730(p)Y peptide was coupled to agarose-streptavidin beads as described in "Materials and Methods." The beads were divided into two aliquots and incubated at 37oC for 30 min in the presence or absence of 500 units of YOP protein tyrosine phosphatase in phosphatase buffer. The peptide treated with the phosphatase was termed FGFR730Y. Aliquots of the conjugated phosphorylated and dephosphorylated peptides were analyzed for their ability to interact with p85 and Shc from NIH3T3 cell lysates by affinity precipitation and Western blotting for these proteins.

 
The Inhibitory Ability of FGFR730(p)Y Correlates with Its Ability to Associate with Shc but not p85.
A comparison of the FGFR730(p)Y sequence with other physiological binding sites for Shc or p85 gives a clear insight into the critical amino acids required for the specific interaction of the phosphorylated sequence with these targets (Table 1)Citation . In this context, the FGFR730(p)Y peptide shares an Asn at position Tyr-3, which is in common with a range of Shc PTB domain binding sites (11) . In addition, the sequence of three Met residues immediately NH2-terminal to Tyr730 is highly indicative of a potential p85 SH2 domain binding site (26) . A number of observations demonstrate that the binding of the FGFR730(p)Y peptide to p85 cannot account for its inhibitory activity: for example, FGF-stimulated mitogenesis is not inhibited by wortmannin (30) , and whereas the FGFR766(p)Y peptide can also weakly bind p85 (Fig. 4)Citation , it does not inhibit mitogenesis (Fig. 1)Citation . To test whether Shc binding might correlate with inhibitory activity, a peptide was synthesized that was identical to FGFR730(p)Y, except that the Asn at position Tyr-3 was altered to a Pro (see Table 1Citation ). The effects of this "mutation" on the interaction of the FGFR730(p)Y peptide with Shc and p85 in living cells was tested by pretreating cells with the peptides and then stringently washing the cells to remove all extracellular peptide. The cells were then lysed, and the internalized peptide precipitated using streptavidin-agarose. The precipitates were then blotted for Shc and p85. The results demonstrated clearly that the FGFR730(p)Y peptide can associate with both p85 and Shc in living cells, and that substitution of the critical Asn with the Pro dramatically reduces the association with Shc in the absence of any detectable effect on the association with p85 (Fig. 6)Citation .



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  		Fig. 6. The effect of alteration of a potential Shc-binding determinant of FGFR730(p)Y on the ability of the peptide to interact with Shc and p85. NIH3T3 cells were grown to subconfluency and treated for 1 h with vehicle control, 25 µg/ml of FGFR730(p)Y, or a peptide identical except for a single Asn/Pro substitution NH2-terminal to the phosphotyrosine [N727P-FGFR730(p)Y]. The cells were washed extensively to remove extracellular peptides as detailed in "Materials and Methods" before lysis. Internalized peptides were captured from lysates by addition of 20 µl of agarose-streptavidin beads, and after several washings, the beads were boiled in Laemmli sample buffer. Associated proteins were examined by SDS-PAGE and Western blotting for both p85 and Shc.

 
Unexpectedly, when the "mutated" peptide was examined for its ability to affect mitogenic signaling in response to FGF, it was seen to have an inhibitory effect on basal levels of BrdUrd incorporation (data not shown). This may reflect a "gain of function" attributable to the amino acid alteration because none of the other peptides containing identical internalization sequences had such an affect. However, when the peptide incubation procedure was modified where cells were pretreated with both peptides for 1 h and then washed before the addition of growth factor, the FGFR730(p)Y peptide inhibited the FGF mitogenic response by 50%, whereas the "mutated" peptide was completely ineffective (Fig. 7A)Citation . Control experiments failed to detect any difference in the internalization of the two peptides (Fig. 7B)Citation , and this is supported by the fact that they can associate to a similar extent with p85 in living cells (Fig. 6)Citation . Thus, a single amino acid substitution that prevents the FGFR730(p)Y peptide from appreciably interacting with Shc, but not p85, results in a loss of the inhibitory activity of the peptide.



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  		Fig. 7. A comparison of native and Asn/Pro-substituted FGFR730(p)Y for the ability to inhibit FGF-mediated mitogenesis. After serum starvation, C2C12 cells were treated with either the FGFR730(p)Y or N727P-FGFR730(p)Y peptide at a concentration of 10 µg/ml for 2 h as indicated. The medium was removed, and the cells were washed before addition of FGF (5 ng/ml) or vehicle control as indicated. Cultures were maintained and analyzed for BrdUrd incorporation as described in "Materials and Methods." The results show the percentage of cells in S-phase in a single representative experiment (top panel). In addition, subconfluent C2C12 cells were treated with a biotinylated control peptide, the FGFR730(p)Y peptide, or the N727P-FGFR730(p)Y peptide (all at 10 µg/ml) as indicated for 30 min before cells were extensively washed and analyzed for relative levels of peptide incorporation by streptavidin/TRITC staining and fluorescence microscopy (bottom panel).

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Unlike other RTKs, such as the PDGFRs or EGFRs, it has proven difficult to link pTyr motifs of the FGFR to the recruitment and activation of downstream signaling molecules. This is despite observations that FGFR activation results in the phosphorylation and activation of a number of pTyr binding molecules that, in principle, could bind directly to the receptor. It is possible that such interactions may occur between the receptor and its downstream effector molecules but that they are too transient, or are of too low affinity, to allow detection by standard methodology such as coimmunoprecipitation.

In the present study, we evaluated the possibility that phosphorylation of Tyr730 in the FGFR-1 cytoplasmic domain might create a binding site for an effector molecule involved in mitogenic signaling by using an alternative strategy to immunoprecipitation studies. This was carried out by introducing a cell-permeant peptide mimetic of the phosphorylated Tyr730 site into cells and seeing if it had any effect on FGF-mediated cellular responses. This peptide was found to inhibit mitogenic responses stimulated by FGF but not other agonists in a manner that was specific to the 9-amino acid moiety derived from the sequence flanking Tyr730 and not other peptide sequences. This indicates that the introduction of general phosphorylated peptides into cells does not nonspecifically interfere with phosphorylation-dependent signaling mechanisms and thus highlights the pTyr in the context of its flanking residues as the mediator of the inhibitory signal. Additionally, the peptide does not interfere with processes that are universally used in mitogenic signaling but must inhibit a molecular interaction necessary for FGFR-1 but not general signal transduction in response to growth factor receptors.

Examination of the flanking residues of Tyr730 reveals consensus binding motifs for both the Shc PTB domain and the SH2 domain of p85, suggesting these molecules as potential candidates for the interacting protein. Indeed, the FGFR730(p)Y peptide interacted with both of these molecules in cell extracts and in whole cells in a specific and selective manner. This raises the possibility that the corresponding site on the receptor might act as a binding site for both of these molecules and that either, or both, may be involved in the mitogenic pathway inhibited by the peptide. Such a role of phosphorylated Tyr730 in mitogenic signaling is controversial because of the observation that in cells overexpressing FGFR-1 containing a Tyr730/Phe substitution, Shc phosphorylation and mitogenesis are not abolished (13) . One possibility is that Shc is activated in a fashion that does not require Tyr730 or the other five Tyr residues that were analyzed. A counter argument in favor of a direct interaction of Shc with the FGFR-1 is supported by the observation that in v-src-transformed fibroblasts, the FGFR-1 is phosphorylated, and phosphorylated Shc is constitutively associated with the receptor (23) . Although this is a very unphysiological system, it demonstrates that Shc and the receptor are capable of interacting and suggests that this may also occur to a lesser degree in untransformed cells. The site of interaction of Shc was not identified in this study.

A number of lines of evidence point to the site on the FGFR-1 containing Tyr730 acting as a potential mediator of SH2/PTB domain signaling pathways. Alignment comparison of the FGFR-1 with a range of other RTKs demonstrates that a Tyr residue is highly conserved at the equivalent position to FGFR730(p)Y in RTKs including PDGF, EGF, and Trk receptors (31) . More significantly, the equivalent Tyr on both EGFR and Trk (Tyr920 and Tyr751), respectively, have been implicated in acting as a phosphorylated binding site for the SH2 domains of p85 (32 , 33) . This indicates precedence for a phosphorylated tyrosine at this position on RTK catalytic domains, which can act as a binding site for signaling molecules. Additionally, Tyr730 has recently been implicated in the FGFR-mediated up-regulation of urokinase-type plasminogen activator, as seen by mutational analysis of the Tyr residues on the receptor, further indicating a signaling role for this residue (32 , 33) .

On the basis of a comparison of functional and biochemical properties of the FGFR730(p)Y peptide with other cell-permeant phosphopeptides, we can conclude that the FGFR730(p)Y peptide is not inhibiting mitogenesis by specifically interfering with p85/PI3-K function or other SH2 containing molecules such as Grb2 or PLC{gamma}. The lack of involvement of PI3-K is further confirmed by our previous demonstration that wortmannin has no effect on FGF-mediated mitogenesis (30) . Thus, the only candidate peptide target involved in mitogenic signaling that we have identified to date is Shc. In this context, we have shown that the FGFR730(p)Y peptide is as effective as an established Shc binding peptide in precipitating Shc from cell extracts, and that pretreatment of cells with the peptide results in internalization of the peptide and its association with Shc. At first glance, Shc might not appear to be a likely candidate given that a range of previous studies have suggested that Shc is involved in the mitogenic responses stimulated by other growth factors including PDGF, and the peptide does not inhibit these responses. However, Shc contains both an SH2 domain and a PTB domain, which have distinct phosphopeptide binding preferences (34) . On the basis of the comparison with other sequences (Table 1)Citation , the FGFR730(p)Y peptide would be expected to bind to the PTB domain of Shc and not necessarily the SH2 domain. As such, the peptide should not compete for interactions mediated by the SH2 domain of Shc. Studies of the mechanism of interaction of Shc with the PDGFR suggest that the SH2 domain is involved in the association of this molecule with receptors in addition to the PTB domain (35 , 36) . If the SH2 domain of Shc makes any significant contribution to binding with the PDGFR, then the FGFR730(p)Y peptide would be less likely to inhibit PDGFR responses.

Recent studies indicate that an Asn found at position pTyr-3 of particular phosphorylated sequences appears to be an absolute requirement for the interaction of the Shc PTB domain with these sequences (11 , 12) . The possibility that the FGFR730(p)Y peptide inhibits mitogenesis by binding to the Shc PTB domain is strengthened by the observation that "mutation" of the Asn at position Tyr-3 in the peptide inhibits the activity of the peptide. In this experiment, the requirement of this Asn for Shc binding and biological activity was tested by the production of a modified FGFR730(p)Y peptide, where the Asn was replaced by a Pro. Although the modified peptide entered into cells and bound to p85 to the same extent as the original FGFR730(p)Y peptide, it failed to interact with Shc, and it did not inhibit mitogenesis. This critical test strengthens the argument, but does not prove beyond doubt, that the ability of the peptides to both interact with Shc and inhibit the FGF response are directly related.

The most commonly ascribed role for Shc in intracellular signaling is the activation of the Erk pathway via recruitment of Grb2/SOS. However, in a number receptors that use Shc for signaling, Grb2 has also been proposed to interact with the receptor by additional Shc-independent means (31 , 35) . This is also the case for the FGFR-1, where Grb2 recruitment and thus Erk phosphorylation can be carried out by the SNT/FRS2 adapter protein, in addition to associating with Shc (16 , 28) . The FGFR730(p)Y peptide did not inhibit the phosphorylation of the prominent Mr 90,000 protein recently established to be SNT/FRS2, nor did it inhibit phosphorylation of Erk. Thus, if the FGFR730(p)Y peptide inhibits mitogenesis by inhibiting Shc function, it appears to do so in a manner that does not compromise the activation of the Erk signaling pathway in a detectable manner. To our knowledge, a direct comparison of the contribution of FRS2 and Shc to Erk activation in response to FGF has not yet been carried out. However, the initial study on FRS2 proposed up to four Grb2 binding sites on this docking molecule (28) , which presumably amplify downstream responses. This suggests the possibility that FRS2 may have a dominant role in mediation of the Ras/Erk pathway in response to FGF.

An increasing number of studies propose roles for Shc that go beyond the recruitment of Grb2 and activation of the Erk pathway. These are based on the demonstration of additional molecules that interact with Shc (37 , 38) , or characterization of protein binding sites on Shc other than the Tyr317 binding site for Grb2 (39) . Some studies directly implicate a requirement for Shc in mitogenesis (40) or transformation (41) in a manner independent of Erk activation by Shc. One such study proposed that a dual phosphorylation site on Shc (Tyr239/240) can mediate a signal leading to stabilization of c-Myc in a manner that does not require Erk phosphorylation (40) . This Erk-independent signal was proposed to be necessary for EGF-mediated mitogenesis. Another study also proposed a role for Shc in mitogenic signaling in a manner dependent on phosphorylation at Tyr239/240 and the expression of c-Myc (42) . In this work, the authors put forward the interesting hypothesis that in the case of PDGF-mediated mitogenesis, Src is involved in the selective phosphorylation of Tyr239/240 on Shc, leading to c-Myc-dependent mitogenesis. These studies are of particular interest in light of the established cooperativity seen between c-Myc and Ras in controlling the entry into S-phase (43) . Thus, it is increasingly likely that Shc is involved in other signals in addition to Erk phosphorylation that are important for mitogenesis. However, further work will be required to identify the precise mechanisms by which this adaptor molecule functions in different signaling contexts.

In conclusion, by synthesizing a cell-permeant mimetic of the Tyr730 site on the FGFR-1 we have generated a novel and specific inhibitor of FGF-mediated signaling. This peptide can act as a binding partner for both p85 and Shc in a phosphorylation-dependent manner, suggesting that the corresponding site on the receptor is a potential mediator of second messenger recruitment. The inhibitory effect of the peptide is correlated with the presence of an Asn at position Tyr-3 and its ability to interact with Shc, indicating that the peptide is inhibiting the interaction of Shc or a related PTB containing molecule with a phosphorylated target molecule, potentially FGFR-1. An implication of Shc in FGF mitogenic signaling is a novel one, and the inhibitory peptide described here will prove valuable in clarifying its mechanism of activation and its role in FGFR-1 signal transduction.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Peptide Synthesis.
Peptides were synthesized on a 431A Applied Biosystems peptide synthesizer using p-hydroxymethylphenoxymethyl polystyrene resin and standard Fmoc chemistry as described previously (21 , 24) . All peptides were made as linear sequences of the following configuration.

Biotin-Amino Hexanoic Acid-Antennapedia Sequence Phosphopeptide.
Amino hexanoic acid functions as a spacer, and the biotin (Novabiochem) at the NH2 terminus allows for capture to agarose-streptavidin (24) . Phosphorylated Fmoc tyrosine residues (Novabiochem) were used in the incorporation of pTyr residues. In general, the Antennapedia internalization sequence was RQIKIWFQNRRMKWKK (25 , 44) with the exception of the EGFR1068(p)Y peptide, where a functional analogue of this sequence was used (24) . The following phosphopeptides, derived from natural sequences present in the FGF, PDGF, EGF, and ErBB3 receptors, were synthesized in tandem with the Antennapedia sequence (Table 2)Citation .


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  		Table 2 Phosphopeptides used in this study

 
After synthesis and deprotection, the peptides were desalted on a Sephadex G-10 column and lyophilized. The peptides were analyzed for purity by analytical high-pressure liquid chromatography and were further purified by preparative high-pressure liquid chromatography where necessary. Peptides were generally made as stock solutions at 2.5 mg/ml in H2O, or in the case of FGFR766(p)Y, supplemented with acetic acid [2% (v/v)] and stored at -20oC before use. Final concentrations of acetic acid added to cells with the FGFR766(p)Y peptide never exceeded 0.08% (v/v), and this level of acetic acid had no effect on the measured biological response.

Cell Culture and Mitogenic Assays.
Newt A1 myoblast cells were grown in gelatin-coated plastic flasks in medium composed of 60% MEM with Earle’s salts (ICN), 27% distilled water, and 10% FCS supplemented with insulin, glutamine, and antibiotics as described previously (48) . Cells were maintained at 25oC in a humidified atmosphere of 2% CO2. Mammalian C2 myoblast cells (49) were cultured in DMEM (Life Technologies, Inc.) supplemented with 15% FCS and glutamine at 37oC in 8% CO2. Mitogenic assays were set up by seeding either 2000 A1 cells/well of 48-well plates (Nunc), coated previously with 0.75% gelatin, or 4000 C2 cells/well of 48-well plates. Cells were initially serum starved by culture in serum-free medium for 3 days. Serum-free medium was then replaced with medium containing 0.5% FCS alone or further supplemented with either bFGF (5 ng/ml), EGF (10 ng/ml), or PDGF (10 ng/ml; all from Collaborative), in the presence or absence of phosphopeptides. In some experiments, cultures were treated with the N727P-FGFR730(p)Y or FGFR730(p)Y peptides for 2 h before being washed three times, and the medium was replaced with growth factors in the absence of additional peptide (see "Results"). After 24 h, cultures were labeled for an additional 24 h with 1 mg/ml BrdUrd using the Amersham proliferation kit according to instructions. Cells were fixed for 5 min with ice-cold methanol and stained for BrdUrd, as described previously (24) . Briefly, after fixation cells were treated for 10 min with 1 M HCl, followed by 5 min with 100 mM Tris (pH 7.8), and then blocked for 10 min with PBS containing 10% goat serum; the primary and secondary antibodies were mouse anti-BrdUrd and antimouse FITC, respectively (diluted in PBS/10% fetal calf serum). Cell nuclei were counterstained with Hoechst dye (1:10000 v/v) and visualized with UV epifluorescence optics.

The percentage of cells in S-phase was calculated by dividing the number of nuclei stained with the BrdUrd antibody by the total number of nuclei stained with the Hoechst dye. Cells (150–300) were routinely scored in three replicate wells for each condition.

Examination of Erk and p90 Phosphorylation.
C2C12 or NIH3T3 cells were grown, serum starved, and stimulated with FGF in the presence or absence phosphopeptides as described above. After 10 min, the cells were lysed directly into Laemmli sample buffer and analyzed by SDS-PAGE and Western blotting with either anti-phospho Erk or anti-phosphotyrosine antibodies.

Affinity Precipitation Using Phosphopeptides.
From cell extracts, biotinylated peptides were precoupled to agarose-streptavidin beads as follows. The beads were washed once in NP40 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP40, 1 mM Vanadate and "Complete" protease inhibitors from Boehringer Mannheim). A 2.5-fold molar excess of peptide was added in 1 ml of NP40 lysis buffer and incubated by rotation at 4oC for 30 min, and subsequently, the beads were washed four times in lysis buffer. Lysates were generated by incubating cells in NP40 lysis buffer for 1 h at 4oC before the lysates were clarified by centrifugation at 1.4 x 104 x g for 20 min. Aliquots of cell lysate (300–500 µg of protein) were added to 20 µl of packed beads and rotated for 1–3 h at 4oC. The beads were then washed three times in NP40 lysis buffer supplemented with 0.5% deoxycholate and 0.1% SDS and boiled in SDS sample buffer. Samples were resolved by SDS-PAGE on a 12% gel. Samples were transferred to nitrocellulose and immunoblotted with different antibodies. The following antibodies were used in this study: anti-p85 monoclonal (a gift from D. Cantrell, Imperial Cancer Research Fund, London, United Kingdom; 1:500 dilution); anti-Grb2 monoclonal (Transduction Laboratories; 1:1000 dilution); anti- Shc polyclonal (Upstate Biotechnology; 1:1000 dilution); anti-PLC{gamma} (Upstate Biotechnology; 1:1000); anti-phospho Erk polyclonal (Promega Corp.; 1:20,000); and anti-pTyr 4G10 monoclonal (Upstate Biotechnology; 1:1000).

From intact cells, NIH3T3 cells were grown to subconfluence on six-well plates, serum starved, and then treated with 25 µg/ml of either FGFR730(p)Y or N727P-FGFR730(p)Y for 1 h. The cell monolayers were washed extensively with ice-cold PBS (three times), 2 M NaCl at pH 7.4 (three times), and 2 M NaCl/20 mM sodium acetate at pH 4 (two times) before lysis. Cells were then lysed in Triton lysis buffer [30 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.5% deoxycholate, 10 mM EDTA, 1 mM vanadate, and "Complete" protease inhibitors from Boehringer Mannheim] for 1 h at 4oC. Lysates were clarified as above and incubated for 30 min with streptavidin-agarose beads. The beads were subsequently washed three times in lysis buffer and finally boiled in SDS sample buffer and resolved by SDS-PAGE on a 12% gel, transferred to nitrocellulose, and immunoblotted with anti-Shc and anti-p85 antibodies.

Treatment of Peptides with Tyrosine Phosphatase.
Peptides to be treated were precoupled to agarose-streptavidin beads as described above, and the beads were then suspended in phosphatase reaction buffer [50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 5 mM DTT, 2.5 mM Na2EDTA, and 100 µg/ml BSA] in the presence or absence of 500 units of YOP protein tyrosine phosphatase (Novabiochem) and incubated at 30oC for 30 min. Beads were washed again in NP40 buffer before being used in affinity precipitation experiments.

Analysis of Internalization of Biotinylated Peptides by Histological Staining of Treated Cells with Streptavidin TRITC.
C2C12 cells were grown to a subconfluent state in eight-well chamber slides (Lab-Tek) before being treated with FGFR730(p)Y, N727P-FGFR730(p)Y, or an unrelated, biotinylated control peptide (all at 10 µg/ml) in DMEM (10% FCS) for 30 min. The cell monolayers were washed extensively with ice-cold PBS (three times), 2 M NaCl at pH 7.4 (three times), and 2 M NaCl/20 mM sodium acetate at pH 4 (two times) before being fixed with paraformaldehyde (4% w/v) in PBS (0.1 M; pH 7.4) for 10 min at room temperature, followed by three 5-min washes in PBS containing Tween (0.1% v/v). Nonspecific binding sites were blocked by incubation of cells with PBS/Tween containing BSA (2% w/v) for 30 min. The slides were then incubated with streptavidin/TRITC (Sigma Chemical Co.; 1:400) and Hoechst nuclear stain (1:10000) in PBS/Tween/BSA overnight at 4oC before mounting in Mowiol and viewed by fluorescent microscopy.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from Wellcome Trust, Contract BMH 4CT950524 from the European Union BIOMED 2 program, and Contract CT98 0227 from the European Union BIOTECH program. Back

2 Present address: Research Institute of Molecular Pathology, Dr. Bohr Gasse 7, A-1030 Vienna, Austria. Back

3 To whom requests for reprints should be addressed, at Molecular Neurobiology Group, MRC Centre for Developmental Neurobiology, New Hunts House, 4th Floor (South Wing), Guy’s Campus, Kings College London, London Bridge, London SE1 1UL, United Kingdom. Phone: 020-7848-6813; Fax: 020-7848-6816; E-mail: patrick.doherty{at}kcl.ac.uk Back

4 The abbreviations used are: FGF, fibroblast growth factor; bFGF, basic FGF; FGFR, FGF receptor; RTK, receptor tyrosine kinase; (p)Tyr, (phospho)tyrosine; SH2, Src homology 2; PTB, phosphotyrosine binding domain; FRS2, FGFR substrate 2; MAPK, mitogen-activated protein kinase; BrdUrd, 5-bromo-2-deoxyuridine/5-fluoro-2-deoxyuridine; EGF, epidermal growth factor; EGFR, EGF receptor; Erk, extracellular signal-regulated kinase; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PI3-K, phosphatidyl-3-OH kinase; PLC{gamma}, phospholipase C{gamma}. Back

Received for publication 1/23/01. Revision received 3/16/01. Accepted for publication 3/16/01.


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 Discussion
 Materials and Methods
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