©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Shc Binding to Nerve Growth Factor Receptor Is Mediated by the Phosphotyrosine Interaction Domain (*)

Ivan Dikic , Andreas G. Batzer , Pamela Blaikie , Axel Obermeier (1), Axel Ullrich (1), Joseph Schlessinger , Ben Margolis (§)

From the (1)Department of Pharmacology and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016 and the Max Planck Institut fur Biochemie, 8033 Martinsreid bei München, 82152 Munich, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Shc is an adaptor protein that contains two phosphotyrosine-binding domains, a Src homology 2 (SH2) domain and the newly described phosphotyrosine interaction (PI) domain. Shc interacts with several tyrosine-phosphorylated proteins and is itself tyrosine-phosphorylated in cells stimulated with a variety of growth factors and cytokines. Upon phosphorylation, Shc binds to the Grb2Sos complex leading to the activation of the Ras signaling pathway. Mutational analysis of the nerve growth factor (NGF) receptor (TrkA) suggested that the binding of Shc to the activated receptor is required for NGF-induced neuronal differentiation of PC12 cells. Here we report that the PI domain of Shc directly binds to tyrosine 490 on the autophosphorylated NGF receptor. The PI domain specifically recognizes an I/LXNPXpY motif (where p indicates phosphorylation) as determined by phosphopeptide competition assay. In addition, the PI domain is able to efficiently compete for binding of full-length Shc proteins to the NGF receptor. In PC12 cells, the Shc SH2 domain interacts with an unidentified tyrosine-phosphorylated protein of 115 kDa but not with the activated NGF receptor. The ability of Shc to interact with different tyrosine-phosphorylated proteins via its PI and SH2 domains may allow Shc to play a unique role in tyrosine kinase signal transduction pathways.


INTRODUCTION

Growth factors play an important role in controlling cell growth and differentiation. Growth factors bind and activate transmembrane receptors with intrinsic tyrosine kinase activity leading to the increased tyrosine phosphorylation of many cellular proteins(1) . Phosphorylated proteins in turn become targets for a variety of signaling proteins containing SH2() domains or other domains able to interact with phosphotyrosine residues(2, 3, 4) . This creates a network of specific protein-protein interactions involved in the signal transduction from cell surface receptors to the nucleus. For example, Grb2 is an adaptor protein that binds to activated growth factor receptors, such as EGFR, via its SH2 domain and is bound to Sos, a Ras guanine nucleotide releasing factor, via its two SH3 domains. It is thought that the translocation of Grb2Sos complex to the membrane regulates the conversion of Ras from an inactive GDP-bound form to an active GTP-bound form (reviewed in Ref. 5). Activated Ras then initiates a mitogen-activated protein kinase (MAPK) cascade that relays signals from the cell membrane to the nucleus (Ref. 6, and references therein).

In the case of other growth factor receptors, such as NGF receptor (TrkA) or fibroblast growth factor receptor, the Grb2Sos complex does not associate directly with the activated receptors, but binds to other tyrosine-phosphorylated proteins, such as Shc. Shc is an SH2 domain-containing adaptor protein that can bind to several growth factor receptors, including NGF receptor and EGFR, and is also tyrosine-phosphorylated in cells stimulated with a variety of growth factors and cytokines(7, 8, 9, 10, 11, 12, 13, 14) . Shc binding to tyrosine 490 of the NGF receptor has been shown to be required for the activation of the Ras signaling pathway and neuronal differentiation in PC12 cells(15, 16) . Shc binds to an NPXpY motif on several tyrosine-phosphorylated proteins(10, 11, 13, 14, 15, 16, 17, 18) . This is an unusual motif for SH2 domain binding, since SH2 domains binding specificity is dictated by residues carboxyl-terminal to the phosphotyrosine moiety(2, 3, 4) . This suggests that another domain, not the SH2 domain, might be responsible for Shc binding to the NPXpY motif.

We have recently identified a novel domain in Shc, encompassing amino acids 46-209 of the p52 Shc protein, and shown that it binds to activated growth factor receptors(19) . A similar region of Shc has been described and demonstrated to bind an unknown 145-kDa tyrosine-phosphorylated protein(20) . We have called this region the phosphotyrosine interaction domain (PI domain). We have also identified PI domains in several other proteins(21) . The nature of the PI domain binding to phosphotyrosine is not clear at present. We sought to delineate the role of the Shc SH2 or PI domains in the NGF receptor signaling pathway, where binding of Shc to the NGF receptor has an important biological role. Here we report that the PI domain of Shc specifically binds to the activated NGF receptor, interacting with phosphotyrosine 490 and surrounding amino acids. We also show that the SH2 domain does not bind to the activated NGF receptor, but interacts with an as yet unidentified 115-kDa tyrosine-phosphorylated protein. The data presented here demonstrate that in PC12 cells Shc can interact with different tyrosine-phosphorylated proteins through its two phosphotyrosine binding domains.


MATERIALS AND METHODS

Cell Lines, Antibodies, Immunoprecipitation, and Immunoblotting

A G418-resistant PC12 cell line expressing approximately 1.3 10 TrkA receptors (PC12-Trk) was established using high titer retroviruses generated in GPE-86 producer cell lines, as described previously(15) . GPE-86 stable cell lines expressing various chimeric receptors containing the extracellular domain of the PDGF receptor and the intracellular part of the TrkA receptor (PT) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS)(15) . PC12-Trk cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 7% FCS and 7% horse serum. Prior to stimulation, cells were starved for 36 h in 0.1% horse serum, 0.1% FCS, DMEM. Cells were then stimulated with 100 ng/ml NGF for 5 min at 37 °C, washed with ice-cold PBS, and lysed in lysis buffer (50 mM HEPES (pH 7.5), 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl, 1 mM EGTA, 25 mM NaF, 50 µM ZnCl, 500 µM sodium orthovanadate, 1 mM phenylmethylsulfonylflouride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Lysate protein content was normalized using the Bio-Rad protein assay(22) . Cell lysis, immunoprecipitation, and immunoblotting with HRP-protein A/chemiluminescence method were performed as described previously(23) . The following antibodies were used for immunoprecipitation: polyclonal rabbit anti-Shc and anti-Grb2 antibodies that were covalently cross-linked to protein A-agarose(9) , and affinity-purified polyclonal anti-Trk antibodies against a COOH-terminal peptide of Trk receptor (Oncogene Science). Polyclonal anti-phosphotyrosine, anti-GST(9) , anti-Grb2, and anti-Shc antibodies in Tris-buffered saline, 5% bovine serum albumin were used for immunoblotting, as described previously(23) .

Peptide Synthesis

A Fmoc-based strategy for peptide synthesis was used in conjunction with standard side chain-protecting groups as described previously(24) . Fmoc-Tyr(POMe)-OH (Bachem Bioscience) was used for incorporation of phosphotyrosine. Peptides were purified by ether precipitation, and preparative reverse-phase high performance liquid chromatography. Analytical high performance liquid chromatography demonstrated that the products were purified to homogeneity; mass spectroscopy was used to confirm the accuracy of synthesis. Several phosphopeptides corresponding to the autophosphorylation sites of Trk receptor or EGFR were synthesized ().

Fusion Proteins and Binding Assay

GST fusion proteins of Shc used in this study have been described previously(19) . Proteins were either used for binding assays on glutathione-agarose beads or eluted with 15 mM glutathione in buffer A plus 10% glycerol (pH 8.0), concentrated and stored at -70 °C in the presence of 20% glycerol, and used for peptide competition assay(9) . For binding assays, 3 µg of Shc PI-GST proteins or 7 µg of Shc SH2-GST were bound to glutathione-agarose beads. The beads were mixed with 2 mg of lysates from unstimulated or NGF-stimulated PC12-Trk cells for 1 h at 4 °C, washed four times with lysis buffer, and boiled in 20 µl of 2 sample buffer(23) . Bound proteins were separated on 9% SDS-polyacrylamide gel electrophoresis gels, transferred to nitrocellulose, immunoblotted with appropriate antibodies, and detected using HRP-protein A/chemiluminescence method.

Phosphopeptide and Shc PI Domain Competition Assays

TrkA receptors were immunoprecipitated from lysates of starved PC12-Trk cells or GPE-86 cells expressing PDGFR-Trk (PT) chimeric receptors, washed four times with lysis buffer, and subjected to in vitro autophosphorylation in the presence of 0.2 mM ATP and 10 mM MnCl for 5 min at room temperature. After washing, the autophosphorylated receptors were divided into aliquots and incubated with either Shc PI-GST or Shc-SH2-GST (human, residues 366-473) in the absence or presence of 2.5 µM of the various phosphopeptides in a volume of 100 µl for 1 h at 4 °C. After three washes with lysis buffer the samples were run on SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose and then immunoblotted using anti-phosphotyrosine or anti-GST antibodies and detected using HRP-protein A/chemiluminescence as described above. To study the ability of Shc PI-GST to compete full-length Shc binding to activated TrkA receptor, we used 293T cells for transient expression of full-length Shc with an HA tag subcloned in pCGN mammalian expression vector (kindly provided by Mikhail Gishicky). Eight micrograms of pCGN-p52 Shc-HA tag were transfected into 293T cells using the calcium phosphate precipitation method(25) . Trk receptor was immunoprecipitated from starved PC12-Trk cells, and after in vitro autophosphorylation, 120 µg of 293T cell lysate or 293T cell lysate expressing p52 Shc-HA tag protein were added to the receptors in absence or presence of increasing concentration of Shc PI-GST. Incubation and analysis was as described above using anti-phosphotyrosine, anti-Shc, or anti GST antibodies.


RESULTS

To study the interaction of Shc with the activated NGF receptor (TrkA), we used parental PC12 cells and PC12 cells overexpressing approximately 1.3 10 Trk receptors/cell (PC12-Trk). Unstimulated or NGF-stimulated cells were lysed, immunoprecipitated with anti-Shc, anti-Grb2, and anti-Trk antibodies, and subsequently immunoblotted with the indicated antibodies. In PC12-Trk cells Shc was found in a complex with two tyrosine-phosphorylated proteins: p140, corresponding to the activated TrkA receptor, and an as yet unidentified protein of approximately 115 kDa, called p115 (Fig. 1a). In addition, tyrosine-phosphorylated Shc and p115 proteins were coimmunoprecipitated with Grb2 in a ligand-dependent manner (Fig. 1a). In the same cell line, anti-TrkA antibodies coimmunoprecipitated tyrosine-phosphorylated p46 and p52 Shc proteins, a previously described tyrosine-phosphorylated protein of 38 kDa, and an unknown tyrosine-phosphorylated protein of 110-115 kDa that migrates in SDS gel close to the lower molecular mass form of TrkA (Fig. 1a and Ref. 26). The presence of the lower molecular mass form of TrkA in anti-Trk immunoprecipitates makes it difficult to determine if the p115 protein found in anti-Shc and anti-Grb2 immunoprecipitates is also present in anti-Trk immunoprecipitates. In these coimmunoprecipitation studies, it was difficult to simultaneously demonstrate complex formation of all four proteins: Trk, Shc, Grb2, and tyrosine-phosphorylated p115. This may be due to the transient nature of protein-protein interactions involved in complex formation and because of the limited sensitivity of these assays. In parental PC12 cells, Shc is also found in a complex with Grb2 and tyrosine-phosphorylated p115 (Fig. 1a). The coimmunoprecipitation of TrkA receptors with Shc in parental PC12 cells was below the detection limit in our assay (Fig. 1a). Taken together, these results suggest that Shc can form a complex with the activated NGF receptor, tyrosine-phosphorylated p115, and Grb2 upon NGF stimulation in PC12-Trk cells.


Figure 1: Intact Shc PI domain binds to the activated NGF receptor but not to mutant receptor TrkA Y490F. Parental PC12 cells (PC12) or PC12 cells overexpressing NGF receptors (PC12-Trk) cells were treated with (+) or without (-) 100 ng/ml NGF for 5 min, lysed, and subjected to immunoprecipitation with anti-Shc, anti-Grb2, and anti-Trk antibodies (a) or and precipitated with indicated GST fusion proteins or GST alone (b and c). Western blot analysis using indicated antibodies was performed as described under ``Materials and Methods.'' d, TrkA receptor, PDGFR-TrkA receptor (PT-R), or PDGFR-TrkA Y490F mutant receptor (PT-Y490F) were immunoprecipitated with anti-Trk antibodies from starved PC12-Trk cells or GPE-86 cells expressing various receptors, autophosphorylated in vitro, and then incubated with purified Shc PI-GST (0.5 µM) or Shc SH2-GST (1 µM) proteins. In addition, 10 µM peptide corresponding to the Shc binding site on the TrkA receptor (TrkA 490-10Y7) was added to the autophosphorylated NGF receptor and Shc PI-GST.



To determine which domain of Shc interacts with the activated NGF receptor and tyrosine-phosphorylated p115, we used GST fusion proteins, encoding various constructs of the Shc PI domain or the Shc SH2 domain, to precipitate proteins from unstimulated or NGF-stimulated lysates of PC12-Trk cells. Anti-phosphotyrosine blotting revealed that the Shc PI domain specifically precipitated activated TrkA receptor and the Shc SH2 domain precipitated tyrosine-phosphorylated p115 (Fig. 1b). None of the proteins was precipitated with GST alone (Fig. 1b). In these studies, we were not able to use full-length GST-Shc protein since it was rapidly degraded by proteolysis during bacterial expression. The binding of the Shc PI domain to the tyrosine-phosphorylated TrkA receptor required the intact PI domain, since Shc PI-GSTs with amino-terminal deletion of 85 amino acids or an internal deletion of amino acids 107-116 (19) were unable to precipitate activated TrkA receptors in the same binding assay (Fig. 1c). Anti-Trk immunoblotting showed that this interaction is dependent on activation and tyrosine phosphorylation of TrkA receptors since no TrkA receptors were precipitated from non-stimulated cell lysates (data not shown).

To study the binding site of the Shc PI domain on the NGF receptors, TrkA, PDGFR-TrkA chimera (PT-R), or PDGFR-TrkA-Y490F mutant (PT-Y490F) receptors were immunoprecipitated, in vitro autophosphorylated, and incubated with Shc PI-GST protein. The amounts of Shc-GST fusion protein bound to autophosphorylated receptors were detected by immunoblotting with anti-GST antibodies. TrkA and PT receptors bound equal amount of Shc PI domain, while PT-Y490F did not bind any detectable amount, although the phosphorylation level of all the receptors was very similar (Fig. 1d). In the presence of a synthetic phosphopeptide corresponding to Tyr-490 of the TrkA receptor, the binding of the Shc PI domain to the PT receptor was completely abolished (Fig. 1d). These results suggested a direct interaction between the Shc PI domain and Tyr-490 on the TrkA receptor. In the same assay, Shc SH2 domain did not bind to the activated Trk receptor (Fig. 1d).

To further analyze the nature of this interaction, we studied the ability of synthetic phosphopeptides () to compete for the binding of the PI domain to the NGF receptor. A peptide containing the Shc binding site of TrkA receptor (TrkA 490-10Y7) was able to compete the binding of PI domain to the activated TrkA receptors (Figs. 1d and 2a). In addition, two peptides corresponding to the autophosphorylation sites on the EGFR were tested. A peptide corresponding to Tyr-1148 (E-pY1148-7Y4), but not peptide E-pY1068-4Y9, efficiently competed the binding of the PI domain to the NGF receptor. Phosphotyrosine alone (30 mM) was only able to partially inhibit the binding of the PI domain (Fig. 2a). Both peptides, TrkA 490-10Y7 and E-pY1148-7Y4, have the NPXpY motif, suggesting that various peptides with NPXpY motifs are able to compete binding of the Shc PI domain to the NGF receptor. To delineate the contribution of several conserved amino acids amino-terminal of phosphotyrosine, we used mutant pY1148 phosphopeptides and tested their ability to compete the binding of the PI domain to the NGF receptor (). Changing the asparagine to alanine (E-pY1148-7Y4 NA) and leucine to glycine (E-pY1148-7Y4 LG) severely impaired ability of the peptides to compete for binding, whereas alteration of the proline to alanine (E-pY1148-7Y4 PA) had only a minor effect (Fig. 2b). Unphosphorylated E-Y1148-7Y4 peptide was unable to compete the binding of the PI domain to the NGF receptor (Fig. 2b). In addition, a peptide encompassing only four amino acids amino-terminal to phosphotyrosine E-pY1148-4Y10 was not able to compete the binding of the PI domain, confirming that other residues amino-terminal to NPXpY can play an important role (data not shown).


Figure 2: Inhibition of Shc PI domain binding to the NGF receptors with synthetic phosphopeptides. NGF receptor was immunoprecipitated from starved PC12-Trk cells, autophosphorylated in vitro, and then incubated without (-) or with Shc PI-GST (0.2 µM) in the absence or presence of 2.5 µM of the indicated phosphopeptides or 30 mM phosphotyrosine, respectively (a), and with Shc PI-GST (0.2 µM) in the absence or presence of 2.5 µM of the indicated peptides, respectively (b).



We next compared the ability of Shc PI-GST to compete binding of full-length Shc to the TrkA receptor. Equal amounts of 293T cells lysate or 293T cells lysate expressing p52-Shc-HA tag were incubated with the autophosphorylated TrkA receptors in the absence or presence of increasing concentration of the PI domain as indicated. The PI domain competed the binding of full-length Shc to the NGF receptor in a concentration-dependent manner (Fig. 3). This indicates that the PI domain of Shc is sufficient to mediate interaction with the activated Trk receptors in the context of full-length Shc molecules.


Figure 3: Inhibition of Shc binding to the NGF receptor by the PI domain. NGF receptor was immunoprecipitated from starved PC12-Trk cells, autophosphorylated in vitro, and then incubated with 293 cell lysates (-) or 293 cell lysates expressing p52-Shc-HA tag protein in the absence or presence of increasing concentrations of Shc PI-GST, as indicated.




DISCUSSION

We have analyzed the role of the PI domain or the SH2 domain of Shc in binding to the activated NGF receptor (TrkA) in PC12 cells. Using the phosphopeptide competition assay, we have demonstrated that the Shc PI domain, not the SH2 domain, binds to an NPXpY motif on the NGF receptor. We used two different NPXpY-containing peptides, one corresponding to tyrosine 490 in TrkA receptor and a second one corresponding to tyrosine 1148 in EGFR. Both peptides were equally efficient in competing the PI domain binding to the NGF receptor. However, phosphopeptides that contained only four amino acids amino-terminal of tyrosine could not compete. This indicated that other amino acids amino-terminal of NPXpY may have an important role. The alignment of Shc binding sites have revealed the conserved hydrophobic residues at the -5 position, asparagine at -3, and proline at -2 to the phosphotyrosine (). In addition, this is supported by studies of Campbell et al.(18) , who identified the conserved hydrophobic residue in the -5 position of Shc binding sites on middle T antigen and some receptor tyrosine kinases. By changing specific residues amino-terminal of phosphotyrosine in the EGFR Y1148 peptide, we have delineated their contribution in mediating binding to the Shc PI domain. The mutation of asparagine to alanine or leucine to glycine had a very strong effect on the ability of peptides to compete for the PI domain binding, whereas mutation of proline to alanine had a weaker effect. This is consistent with our results showing that the the PI domain of Shc binds to LXNPXpY motif surrounding the tyrosine 1148 on EGFR.()It seems very likely that the interaction of Shc with other tyrosine-phosphorylated proteins containing the I/L/FNPXpY motif () will be mediated via the PI domain of Shc.

In contrast, the SH2 domain of Shc did not bind to the activated NGF receptor in any of our assays, suggesting that the primary interaction domain of Shc with the NGF receptor is the PI domain. Nonetheless, studies have indicated that the specificity of Shc binding to some tyrosine-phosphorylated proteins is determined by the SH2 domain(9, 10) . In the case of EGFR, Shc SH2 domain directly binds to pY1173 and the PI domain binds to pY1148. However, in living cells, Shc binding to the EGFR is probably mediated by the cooperative binding of both the PI and the SH2 domains to two different tyrosine autophosphorylation sites. In NGF-stimulated PC12 cells, it appears that the SH2 domain does not cooperate with the PI domain but rather binds to an unknown tyrosine-phosphorylated protein of 115 kDa. Similarly, two other tyrosine-phosphorylated proteins of 120 and 180 kDa were identified as the direct targets of the SH2 domain of Shc in fibroblast growth factor- and PDGF-stimulated cells(20) . Whether the 115-kDa protein we observe is similar to the 120-kDa protein seen in these cells remains to be determined.

It has been demonstrated that the association of Shc with the activated NGF receptor is required for neuronal differentiation of PC12 cells and contributes to the activation of the Ras-MAPK pathway(15, 16) . The data presented here suggest a model for Shc interactions with the NGF receptor (Fig. 4). Our data indicate that the PI domain mediates Shc interaction with the NGF receptor by binding to the IXNPXpY motif present at tyrosine 490. This is in agreement with data indicating that mutation of Tyr-490 to phenylalanine impairs the ability of the NGF receptor to bind and phosphorylate Shc(14, 15, 16) . This Y490F mutation also leads to defects in NGF-induced association of Grb2 with Shc and impairs neuronal differentiation of PC12 cells(15, 16) . Thus we propose that the binding of Shc to Tyr-490 via its PI domain leads to Shc phosphorylation and the binding of the Grb2Sos complex. This complex, localized at the plasma membrane, activates Ras, eventually leading to MAP kinase activation. Further complexity is added, however, by the binding of the Shc SH2 domain to the highly tyrosine-phosphorylated 115-kDa protein. We have observed Shc interactions with tyrosine-phosphorylated p115 as early as 3 min and continuing for at least 20 min after NGF stimulation (data not shown). We have detected p115 in Grb2 immunoprecipitates, suggesting a complex is formed between Shc, Grb2, and p115. However, we have not been able to find TrkA in this complex, perhaps due to the lack of sensitivity of the immunoprecipitation analysis and the transient nature of the complex. It is also possible that Shc cannot bind to Trk and p115 at the same time. Further work will be necessary to address this issue.


Figure 4: Schematic diagram of the Shc interacting proteins in NGF-stimulated PC12 cells. The model depicts the diversity of the Shc interactions with a variety of proteins. The PI domain of Shc binds to the activated NGF receptor recognizing phosphotyrosine 490 in the context of IXNPXpY motif. Once phosphorylated on tyrosine 317, Shc serves as a binding site for the SH2 domain of Grb2 leading to Ras activation. The Shc SH2 domain can interact with other tyrosine-phosphorylated proteins such as p115. This suggests a role for Shc in mediating signals responsible for neuronal differentiation of PC12 cells more complex than that previously described. Whether Shc can simultaneously interact with both NGF receptor and p115, as illustrated here, remains to be determined.



It is possible that SH2 domain binding of Shc to p115 may provide the mechanism for signal amplification and diversification during neuronal differentiation. Particularly, different ways of Shc binding to the NGF or EGF receptors may reflect their ability to activate the Ras-MAPK pathway with different kinetics in PC12 cells. It has been shown that the activation of NGF receptor leads to the prolonged activation of Ras-MAPK pathway and neuronal differentiation of PC12 cells(27) . In contrast, stimulation of EGFR in PC12 cells leads to a short and transient MAPK activation, which is associated with the cell proliferation(28) . Although, we still do not fully understand the role of Shc in mediating neuronal differentiation of PC12 cells, the ability of two phosphotyrosine-binding domains of Shc to select different targets suggests that it may be more complex and diverse than initially envisioned.

  
Table: Sequences of phosphopeptides used in a competition assay


  
Table: Alignment of the Shc binding sites with the NPXY motifs



FOOTNOTES

*
This work was supported in part by grants from Sugen, Inc. and the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Lucille P. Markey and Kaplan Cancer Center Scholar. To whom correspondence should be addressed. Present address: Dept. of Internal Medicine and Biochemistry, University of Michigan Medical School, MSRBII, R. 1560, Ann Arbor, MI 48109-0676. Tel.: 313-936-4812; Fax: 313-763-0982.

The abbreviations used are: SH2, Src homology 2; EGFR, epidermal growth factor receptor; Grb2, growth factor receptor-bound protein 2; GST, glutathione S-transferase; HA, hemagglutinin; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PI, phosphotyrosine interaction; Shc, Src homologous and collagen protein; Sos, Son of Sevenless; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; HRP, horseradish peroxidase; Fmoc, N-(9-fluorenyl)methoxycarbonyl.

A. G. Batzer, P. Blaikie, K. Nelson, J. Schlessinger, and B. Margolis, submitted for publication.


ACKNOWLEDGEMENTS

We thank Kiki Nelson for phosphopeptide synthesis, Ron Beavis for mass spectroscopy analysis of the peptide, Mikhail L. Gishicky for the expression plasmids containing Shc-HA-tagged construct, and Valsan Mandiyan for the Shc 1-209 construct in pGEX-2T.


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