Systematic Mapping of Potential Binding Sites for Shc and Grb2 SH2 Domains on Insulin Receptor Substrate-1 and the Receptors for Insulin, Epidermal Growth Factor, Platelet-derived Growth Factor, and Fibroblast Growth Factor
1996; Elsevier BV; Volume: 271; Issue: 10 Linguagem: Inglês
10.1074/jbc.271.10.5603
ISSN1083-351X
AutoresColin W. Ward, K. H. Gough, Melisa Rashke, Soo San Wan, Gordon Tribbick, Jianxin Wang,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoMultipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and −1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways. Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and −1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways. INTRODUCTIONSH2 domains are one of the conserved protein modules that regulate signal transduction pathways involving phospholipid metabolism, protein phosphorylation and dephosphorylation, activation of Ras-like GTPases, gene expression, protein trafficking, and cytoskeletal architecture (1.Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2217) Google Scholar). SH2 domains promote protein-protein interactions by binding short, specific, phosphotyrosine peptide sequences on activated receptors and cytoplasmic proteins(1.Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2217) Google Scholar, 2.Pawson T. Schlessinger J. Curr. Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (574) Google Scholar). The three-dimensional structure of several SH2 domains have now been determined(3.Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Crossref PubMed Scopus (573) Google Scholar, 4.Eck M.J. Shoelson S.E. Harrison S.C. Nature. 1993; 362: 87-91Crossref PubMed Scopus (441) Google Scholar, 5.Pascal S.M. Singer A.U. Gish G. Yamazaki T. Shoelson S.E. Pawson T. Kay L.E. Forman-Kay J.D. Cell. 1994; 77: 461-472Abstract Full Text PDF PubMed Scopus (229) Google Scholar, 6.Lee C-H. Kominos D. Jacques S. Margolis B. Schlessinger J. Shoelson S.E. Kuriyan J. Structure. 1994; 2: 423-438Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 7.Xu R.X. Word J.M. Davis D.G. Rink M.J. Willard D.H. Gampe R.T. Biochemistry. 1995; 34: 2107-2121Crossref PubMed Scopus (86) Google Scholar), and the nature of the phosphopeptide binding site has been described.Individual SH2 domain binding sites have been identified and their specificity requirements determined by (i) isolation of specific phosphopeptides after receptor activation, (ii) site-specific mutagenesis, (iii) synthetic peptide binding studies (see Refs. 2 and 8), or (iv) analysis of degenerate phosphopeptide libraries(9.Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar, 10.Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky S. Feldman R.A. Cantley L.C. Mol. Cell Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar). It is generally accepted that the SH2 binding sites of receptor tyrosine kinases are located outside the kinase domain proper in the juxtamembrane region, the kinase insert region, or the C-terminal tail (1.Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2217) Google Scholar). However, there are some data (11.Obermeier A. Lammers R. Wiesmuller K.H. Jung G. Schlessinger J. Ullrich A. J. Biol. Chem. 1993; 268: 22963-22966Abstract Full Text PDF PubMed Google Scholar, 12.Roussel M.F. Shurtleff S.A. Downing J.R. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6738-6742Crossref PubMed Scopus (95) Google Scholar, 13.Roussel M.F. Cleveland J.L. Shurtleff S.A. Sherr C.J. Nature. 1991; 353: 361-363Crossref PubMed Scopus (138) Google Scholar) that suggest that Tyr(P) residues within the catalytic domain proper can act as binding sites for SH2 domain or other signaling proteins. The phosphatidylinositol 3′-kinase binding site in the tyrosine kinase receptor Trk-A is Tyr-751 (11.Obermeier A. Lammers R. Wiesmuller K.H. Jung G. Schlessinger J. Ullrich A. J. Biol. Chem. 1993; 268: 22963-22966Abstract Full Text PDF PubMed Google Scholar). This Tyr residue is located in the H-helix (see (14.Hubbard S.R. Wei L. Elis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar)), 24 residues upstream of the C-terminal residue of the catalytic domain as defined by the alignments of Hanks(15.Hanks S.K. Curr. Opin. Struct. Biol. 1991; 1: 369-383Crossref Scopus (130) Google Scholar). Similarly, the highly conserved activation loop residue Tyr-809 of the CSF-1 receptor (see (14.Hubbard S.R. Wei L. Elis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar)) has been shown to be required for induction and sustained transcription of c-myc and the control of ligand-dependent cell growth without significantly affecting receptor tyrosine kinase activity or immediate early gene expression(12.Roussel M.F. Shurtleff S.A. Downing J.R. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6738-6742Crossref PubMed Scopus (95) Google Scholar, 13.Roussel M.F. Cleveland J.L. Shurtleff S.A. Sherr C.J. Nature. 1991; 353: 361-363Crossref PubMed Scopus (138) Google Scholar).In this paper we describe a systematic approach to identify potential binding sites for SH2 domains on cytoplasmic proteins or transmembrane receptors that includes an analysis of every tyrosine-containing peptide. This approach is rapid and sensitive and complements the studies on direct identification of binding sites by mutagenesis and competition binding or specificity determination with degenerate peptide libraries(9.Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar, 10.Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky S. Feldman R.A. Cantley L.C. Mol. Cell Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar). It confirms many of the sites established by alternative methods and reveals new potential binding sites for Grb2 at Tyr-1110 in IR, 1The abbreviations used are: IRinsulin receptorIRSinsulin receptor substrateEGFRepidermal growth factor receptorPDGFRplatelet-derived growth factor receptorGSTglutathione S-transferaseFGFRfibroblast growth factor receptorbFGFRbasic FGFRPIDphosphotyrosine interaction domain. Tyr-1114 in EGFR, Tyr-743 in PDGFR, and Tyr-47, Tyr-727, and Tyr-939 in IRS-1 and for Shc at Tyr-960 in IR, Tyr-703 and Tyr-789 in EGFR, and Tyr-557 in PDGFR.MATERIALS AND METHODSGST-SH2 Fusion ProteinscDNAs encoding the whole of Grb2 (residues 1-215) or the SH2 domain of Shc (residues 366-473) cloned into pGEX vectors (16.Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi D. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1331) Google Scholar, 17.Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Grignani F. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1131) Google Scholar) were kind gifts from Dr. David Bowtell (Howard Flory Institute of Experimental Physiology and Medicine, Parkville, Victoria, Australia) and Dr. Tony Pawson (Samuel Lunenfeld Research Institute, Mount Sinai Hospital and University of Toronto, Ontario, Canada), respectively. The GST fusion proteins were produced in Escherichia coli and purified with glutathione-agarose beads(18.Smith D.B. Johnstone K.S. Gene (Amst.). 1991; 67: 31-40Crossref Scopus (5035) Google Scholar). Fusion protein concentrations were estimated by Coomassie dye binding (19.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213347) Google Scholar) using the kit obtained from Bio-Rad.Synthesis of PeptidesThe 256 peptides synthesized corresponded to the sequences centered around all of the tyrosine residues in rat IRS-1 (20.Sun X.J. Crimmins D.L. Myers M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar) and the cytoplasmic domains of the human insulin receptor(21.Ullrich A. Bell J.R. Chen E.Y. Herrara R. Petruzelli L.M. Dull T.J. Gray A. Coussens L. Liao Y-C. Tsubokawa M. Mason A. Seeburg P.H. Grunfeld C. Rosen O.M. Ramachandran J. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1505) Google Scholar), human EGFR(22.Ullrich A. Coussens L. Hayflick J.S. Dull T.J. Gray A. Tam A.W. Lee J. Yarden Y. Libermann T.A. Schlessinger J. Downward J. Mayes E.L. Whittle N. Waterfield M.D. Seeburg P.H. Nature. 1984; 309: 418-425Crossref PubMed Scopus (1979) Google Scholar), mouse PDGFR(23.Yarden Y. Escobedo J.A. Kuang W-J. Yang-Feng T.L. Daniel T.O. Tremble P.M. Chen E.Y. Ando M.E. Harkins R.N. Francke U. Fried V.A. Ullrich A. Williams L.T. Nature. 1986; 323: 226-232Crossref PubMed Scopus (763) Google Scholar), and chicken basic fibroblast growth factor receptors(24.Lee P.L. Johnson D.E. Cousens L.S. Fried V.A. Williams L.T. Science. 1989; 245: 57-59Crossref PubMed Scopus (476) Google Scholar). The peptides were synthesized as 11-mers joined to a biotin cap by the tetrapeptide linker SGSG in the format biotin-SGSGXXXXXYXXXXX where Y is either Tyr or Tyr(P). The biotin-SGSG- cap enabled attachment to streptavidin-coated plates in the enzyme-linked immunosorbent assay. Peptides containing Cys residues were also synthesized, with Ala replacing the Cys residue to check for nonspecific binding due to the free thiol side chain. In no case did this substitution have a significant effect on binding. Throughout the text the peptides are referred to by the protein source and Tyr residue number; e.g. EGFR-1068. Peptides were synthesized using the multipin peptide synthesis approach on polyethylene supports derivitized with an acid-labile handle(25.Valerio R.M. Bray A.M. Maeji N.J. Int. J. Pept. Protein Res. 1994; 44: 158-165Crossref PubMed Scopus (52) Google Scholar). The synthesis was carried out on detachable crown-shaped pin heads grafted with hydroxyethylmethacrylate and functionalized with the trifluoroacetic acid-labile 4-hydroxymethylphenoxyacetic acid handle, which yields peptide carboxylates on acid cleavage(25.Valerio R.M. Bray A.M. Maeji N.J. Int. J. Pept. Protein Res. 1994; 44: 158-165Crossref PubMed Scopus (52) Google Scholar).Peptide Binding AssayThe GST-SH2 fusion proteins for Shc and Grb2 (10 μg/ml final concentration) were incubated in solution phase with 0 μM, 0.2 μM, 1 μM, 5 μM, and 25 μM (final concentrations) of each Tyr and Tyr(P) peptide. Aliquots (125 μl) of each fusion protein (20 μg/ml) were mixed with 125 μl of biotinylated peptide (0 μM, 0.4 μM, 2 μM, 10 μM, and 50 μM) in individual wells in TitertekTM plates preblocked with 1% bovine serum albumin, 10 mM phosphate-buffered saline. The buffer used for the dilution of SH2 fusion proteins and biotinylated peptides was 10 mM phosphate-buffered saline, 0.1% (v/v) Tween 20. The mixtures were incubated for 1 h at room temperature (22°C) with shaking. After 1 h, duplicate 100-μl aliquots were transferred to Streptavidin-coated NUNC MaxisorpTM plates. The NUNC plates were incubated for 1 h at room temperature with shaking and washed with distilled water. The amount of GST fusion protein bound to the trapped biotinylated peptides was determined by enzyme-linked immunosorbent assay. Aliquots (100 μl) of rabbit anti-glutathione S-transferase antibody (AMRAD Corp. Ltd.) at 1:5000 dilution in 1% bovine serum albumin, 10 mM phosphate-buffered saline were added to each well, and the plates were incubated for 1 h at room temperature with shaking. The plates were washed with distilled water, and 100 μl of goat anti-rabbit IgG (H + L) horseradish peroxidase conjugate (Kirkegaard & Perry Laboratories, Inc.) at 1:2000 dilution in 0.1% bovine serum albumin, 10 mM phosphate-buffered saline was added to each well. The plates were incubated for 1 h at room temperature with shaking. After a final wash with distilled water, 100 μl of substrate, 2,2′-azino-di-[3-ethyl-benzthiazolinesulfonate] was added to each well. The optical density of the plates was read after 45 min at 405/492 nm with a Titertek MultiscanTM plate reader. The values are expressed as net optical densities and represent the difference between the value obtained for the phosphopeptide minus the value obtained for the corresponding nonphosphorylated control peptide. Net optical densities between 0.2 and 0.6 will be referred to as weak binding, those between 0.6 and 1.0 as medium binding, and those between 1.0 and 1.4 as strong binding.RESULTSPhosphopeptide Design and Binding AssayPhosphopeptides accounting for all the Tyr-containing sequences in IR, EGFR, PDGFR, and bFGFR (Fig. 1) and IRS-1 were synthesized as 11-mers with the Tyr or Tyr(P) residue in the sixth position. Preliminary studies using a solid phase, direct binding protocol on peptides that remained attached to the acrylic acid-coated polyethylene rods after synthesis (26.Geyson H.M. Rodda S.J. Mason T.J. Tribbick G. Schoofs P.G. J. Immunol. Methods. 1987; 102: 259-274Crossref PubMed Scopus (719) Google Scholar) gave strong binding for Tyr(P) peptides compared with the nonphosphorylated or Phe-substituted controls. However the solid phase assay did not give the required discrimination between specific Tyr(P) peptides, presumably because of the high concentration of peptide on the pin surface. Consequently, the final system chosen was a solution phase, direct binding assay with biotinylated peptides at concentrations of 0, 0.2, 1, 5, and 25 μM. Complex formation between GST-SH2 fusion proteins and biotinylated peptides was measured by enzyme-linked immunosorbent assay using anti-GST antibodies after transfer to streptavidin-coated plates. This assay was quick, sensitive, and discriminating when used with a set of test peptides including known positive controls (data not shown).Saturation binding curves for the Grb2-fusion protein with 100 μM of phosphopeptide EGFR-1068 (27.Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (924) Google Scholar) and the Shc-SH2-fusion protein with 100 μM PDGFR-708 (28.Yokote K. Mori S. Hansen K. Mcglade J. Pawson T. Heldin C.H. Claesson-Welsh L. J. Biol. Chem. 1994; 269: 15337-15343Abstract Full Text PDF PubMed Google Scholar) were carried out and showed linear binding within the range 1-20 μg/ml (data not shown). A concentration of 10 μg/ml of fusion protein was chosen for all screening assays. Ca2+ ions (2 mM), which have been shown to stabilize the conformation of the p85N-SH2 domain and enhance its binding to activated α-PDGFR (29.Mahadevan D. Thanki N. McPhie P. Beeler J.F. Yu J.C. Wlodawer A. Heidaran M.A. Biochemistry. 1994; 33: 746-754Crossref PubMed Scopus (18) Google Scholar), were found to be inhibitory to Grb2-SH2 binding to EGFR-1068 and Shc-SH2 binding to PDGFR-708 and were not included in the binding screens with Grb2- and Shc-SH2-fusion proteins.Grb2-SH2 Binding SitesThe results obtained from the phosphopeptide screens with the Grb2-fusion protein are shown in Figure 2:, Figure 3:, Figure 4:, Figure 5:, Figure 6:. The major sites for Grb2 binding in human EGFR (at 1068, 1086, and 1114) were all located in the C-tail region of the receptor (Fig. 2), while the two major sites of Grb2 binding in the mouse PDGFR (at 684 and 743) were both in the kinase insert region (Fig. 3). Chicken bFGFR contained only one potential binding site for Grb2 at Tyr-556, and this showed only weak to medium interaction (Fig. 4). The dominant site in human IR was peptide IR-1110 from the kinase domain (Fig. 5). Phosphopeptides corresponding to sites in the juxtamembrane or C-tail regions were either weak (IR-953, IR-972) or negligible (IR-960, IR-1316, IR-1322; Fig. 5). The strong binding sites in rat IRS-1 were peptides IRS1-47, −895, −939, and to a lesser extent −727 (Fig. 6).Figure 2:Grb2- and Shc-SH2 domain binding to phosphopeptides from human EGFR. Binding was carried out at as described under "Materials and Methods." The net optical density values obtained at 1.0 μM are shown for each Tyr residue in human EGFR. Peptides EGFR-777, −803, −845, −867, −891, −920, −954, −974, and −1173 showed no net binding by the SH2 domain of Grb2. Peptides EGFR-777, −803, −845, −867, −891, −920, −1045, −1068, −1086, −1101, −1114, and −1148 showed negligible binding to the Shc-SH2 domain. A schematic representation of the EGFR is shown to indicate the approximate location of each of the positively binding peptides in the sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:Grb2- and Shc-SH2 domain binding to phosphopeptides from mouse PDGFR. Binding was carried out at as described under "Materials and Methods." The net optical density values obtained at 1.0 μM are shown for each Tyr residue in mouse PDGFR. Peptides PDGFR-660, −719, −731, −746, −755, −832, and −889 showed no net binding by the SH2 domain of Grb2. Peptides PDGFR-530, −549, −651, −660, −684, −731, −746, −755, −776, −825, −872, −882, −889, −934, −938, and −977 showed negligible binding to the Shc-SH2 domain. A schematic representation of the PDGFR is shown to indicate the approximate location of each of the positively binding peptides in the sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4:Grb2- and Shc-SH2 domain binding to phosphopeptides from chicken basic FGFR. Binding was carried out at as described under "Materials and Methods." The net optical density values obtained at 1.0 μM are shown for each Tyr residue in chicken bFGFR. Peptides PDGFR-461, −561, −570, −581, −583, −603, −611, −651, −652, −675, −699, −728, −764, and −774 showed negligible binding by the SH2 domain of Grb2. Peptides bFGFR-461, −561, −570, −581, −583, −603, −611, −651, −652, −675, −699, and −774 showed negligible binding to the Shc-SH2 domain. A schematic representation of the bFGFR is shown to indicate the approximate location of each of the positively binding peptides in the sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5:Grb2- and Shc-SH2 domain binding to phosphopeptides from human IR. Binding was carried out at as described under "Materials and Methods." The net optical density values obtained at 1.0 μM are shown for each Tyr residue in IR. Peptides IR-960, −999, −1075, −1150, −1151, 1198, −1215, −1316, and −1322 showed negligible binding by the SH2 domain of Grb2. Peptides IR-953, −972, −999, −1075, −1110, −1146, −1150, −1151, −1215, −1316, and −1322 showed negligible binding to the Shc-SH2 domain. A schematic representation of the IR is shown to indicate the approximate location of each of the positively binding peptides in the sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6:Grb2- and Shc-SH2 domain binding to phosphopeptides from rat IRS-1. Binding was carried out at as described under "Materials and Methods." The net optical density values obtained at 1.0 μM are shown for each Tyr residue in rat IRS-1. Peptides IRS1-18, −87, −107, −147, −178, −426, −478, −489, −546, −567, −578, −608, −628, −690, −745, −746, −815, −907, and −1222 showed negligible binding to the Grb2-SH2 domain. Peptides IRS1-18, −46, −47, −87, −147, −178, −426, −478, −489, −546, −555, −567, −578, −608, −628, −746, −759, −760, −795, −815, −907, −987, −999, −1010 and −1172 showed negligible binding to the Shc-SH2 domain. A schematic representation of the IRS-1 is shown to indicate the approximate location of each of the positively binding peptides in the sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Shc-SH2 Binding SitesThe results obtained from the phosphopeptide screens with the Shc-SH2 fusion protein are also shown in Figure 2:, Figure 3:, Figure 4:, Figure 5:, Figure 6:. One peptide, EGFR-703 at the start of the kinase domain, bound Shc-SH2 strongly, while peptide EGFR-789 from the kinase domain showed medium binding (Fig. 2). Of the PDGFR peptides, PDGFR-557 bound strongly, while PDGFR-654, −708, and −848 exhibited medium binding to the Shc-SH2 domain (Fig. 3). The Shc-SH2 domain showed medium binding to the chicken bFGFR C-tail phosphopeptide bFGFR-764 and weak binding to bFGFR-556 and −728 in the kinase domain (Fig. 4). Only two phophopeptides from human IR bound the Shc-SH2-fusion protein. The best binder (medium strength) was IR-960 from the juxtamembrane region, while IR-1198 from the kinase domain showed weak binding (Fig. 5). As shown in Fig. 6, none of the 34 phosphopeptides from IRS-1 bound the Shc-SH2-fusion protein strongly, and only one, IRS1-690, bound with medium strength.DISCUSSIONSongyang and co-workers (9.Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar, 10.Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky S. Feldman R.A. Cantley L.C. Mol. Cell Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar) have used degenerate phosphopeptide or peptide libraries to systematically search for optimal sequences that serve as binding sites for SH2 domains or substrates for protein-tyrosine kinases(30.Songyang Z. Carraway K.L. Eck M.J. Harrison S.C. Feldman R.A. Mohammadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantley L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (841) Google Scholar). In this paper we describe a complementary approach in which all tyrosine-containing sequences in signaling proteins are synthesized and screened for their capacity to be bound by specific SH2 domains. It is obvious that the method could be extended to include a systematic analysis of the potential for these sites to be phosphorylated by any specific tyrosine kinase.To date most known tyrosine autophosphorylation sites are reported to be located within noncatalytic regions of receptors(2.Pawson T. Schlessinger J. Curr. Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (574) Google Scholar). However, the demonstration that some phosphorylation/dephosphorylation sites occur within the catalytic domain of tyrosine kinase proteins and that at least one SH2-containing protein has been shown to bind to a Tyr(P) sequence within the catalytic domain of TrkA (11.Obermeier A. Lammers R. Wiesmuller K.H. Jung G. Schlessinger J. Ullrich A. J. Biol. Chem. 1993; 268: 22963-22966Abstract Full Text PDF PubMed Google Scholar) highlights the need to examine all Tyr-containing sequences for their potential to bind SH2 (1.Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2217) Google Scholar, 2.Pawson T. Schlessinger J. Curr. Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (574) Google Scholar) or PID domains (31.Kavanaugh W.M. Williams L.T. Science. 1994; 266: 1862-1865Crossref PubMed Scopus (448) Google Scholar, 32.Bork P. Margolis B. Cell. 1995; 80: 693-694Abstract Full Text PDF PubMed Scopus (171) Google Scholar) if phosphorylated. Such studies do not establish that these sites are used in vivo but merely indicate their potential to be phosphorylated and participate in SH2-mediated signaling if they were exposed and accessible. Additional data are required to establish their in vivo significance and must take account of the fact that the stoichiometry of phosphorylation at specific sites at any given time in living cells is not known(33.Batzer A.G. Rotin D. Urena J.M. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar). In addition the net signaling outcomes will be influenced by direct competition between different SH2- or PID-containing proteins for the same or closely overlapping phosphorylation sites and by the effects of secondary phosphorylation of Ser or Thr residues on these phosphotyrosinemediated interactions.In this paper we have synthesized and screened all the tyrosine-containing sequences in IRS-1 and the cytoplasmic domains of IR, EGFR, PDGFR, and bFGFR for their capacity to bind to the SH2 domains of the signaling proteins Grb2 and Shc.EGFRTwo of the major sites identified for Grb2 binding on EGFR (Tyr-1068, Tyr-1086) have been reported(27.Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 33.Batzer A.G. Rotin D. Urena J.M. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar), but the additional major potential binding site at Tyr-1114 in the C-tail has not been described previously. Tyr-1114 has not been identified as a major phosphorylation site in EGFR by biochemical analysis(34.Case
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