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• W2117386260 abstract "SH2 domains bind to specific phosphotyrosine-containing sites in a fashion dictated by the amino acids flanking the phosphotyrosine. Attention has focused on the role of the three COOH-terminal positions (+1 to +3) in generating specificity. Autophosphorylation of Tyr1021 in the tail of the β-receptor for platelet-derived growth factor creates a specific binding site for the COOH-terminal SH2 domain of phospholipase C (PLC)-γ1. We show that the residues 4 and 5 amino acids COOH-terminal to Tyr1021 (+4 Leu and +5 Pro) are required for efficient PLC-γ1 binding, and that their replacement with the corresponding residues from a phosphatidylinositol 3′-kinase binding site abrogates stable association with PLC-γ1. In contrast, replacement of the +3 Pro with Met produces a Tyr1021 site with mixed specificity that binds both PLC-γ1 and phosphatidylinositol 3′-kinase. This motif is rendered specific for phosphatidylinositol 3′-kinase by further substitution of the +4 Leu. These results indicate that the +4 and +5 residues are important for the selective binding of specific SH2 domains. This study suggests that phosphotyrosine sites can be tailored to bind one or more SH2 domains with high affinity, depending on the combination of residues in the +1 to +5 positions. SH2 domains bind to specific phosphotyrosine-containing sites in a fashion dictated by the amino acids flanking the phosphotyrosine. Attention has focused on the role of the three COOH-terminal positions (+1 to +3) in generating specificity. Autophosphorylation of Tyr1021 in the tail of the β-receptor for platelet-derived growth factor creates a specific binding site for the COOH-terminal SH2 domain of phospholipase C (PLC)-γ1. We show that the residues 4 and 5 amino acids COOH-terminal to Tyr1021 (+4 Leu and +5 Pro) are required for efficient PLC-γ1 binding, and that their replacement with the corresponding residues from a phosphatidylinositol 3′-kinase binding site abrogates stable association with PLC-γ1. In contrast, replacement of the +3 Pro with Met produces a Tyr1021 site with mixed specificity that binds both PLC-γ1 and phosphatidylinositol 3′-kinase. This motif is rendered specific for phosphatidylinositol 3′-kinase by further substitution of the +4 Leu. These results indicate that the +4 and +5 residues are important for the selective binding of specific SH2 domains. This study suggests that phosphotyrosine sites can be tailored to bind one or more SH2 domains with high affinity, depending on the combination of residues in the +1 to +5 positions. The Src homology 2 (SH2) domains of intracellular signaling proteins bind phosphotyrosine-containing sites on a variety of autophosphorylated growth factor receptors and cytoplasmic phosphoproteins(1Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1427) Google Scholar, 2Koch C.A. Moran M.F. Anderson D. Liu X. Mbamalu G. Pawson T. Mol. Cell. Biol. 1992; 12: 1366-1374Crossref PubMed Scopus (38) Google Scholar). Such complexes are important for interactions of receptor tyrosine kinases with their targets(3Pawson T. Schlessinger J. Curr. Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (574) Google Scholar), for the activation of lymphoid cells by antigen(4Weiss A. Cell. 1993; 73: 209-212Abstract Full Text PDF PubMed Scopus (472) Google Scholar), and for the regulation of gene expression by cytokine and interferon receptors(5Shuai K. Horvath C.M. Tsai Huang L.H. Qureshi S.A. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (677) Google Scholar). SH2 domains possess a common ability to bind phosphotyrosine, but distinguish different phosphorylated sites through the recognition of residues flanking the phosphotyrosine(6Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2173) Google Scholar). Hence, SH2 domains bind phosphotyrosine-containing peptides of optimal sequence with a Kd of ~10-100 nM, whereas their affinities for phosphopeptides of random sequence are up to 1000-fold lower(7Payne G. Stolz L.A. Pei D. Band H. Shoelson S.E. Walsh C.T. Chemistry & Biology. 1994; 1: 99-105Abstract Full Text PDF PubMed Scopus (32) Google Scholar). Experiments employing specific phosphopeptides and degenerate phosphopeptide libraries have focused attention on the 3 residues immediately COOH-terminal to the phosphotyrosine (the +1 to +3 residues), and have shown that each SH2 domain has a distinct selectivity for these amino acids(8Escobedo J.A. Kaplan D.R. Kavanaugh W.M. Turck C.W. Williams L.T. Mol. Cell. Biol. 1991; 11: 1125-1132Crossref PubMed Scopus (180) Google Scholar, 9Fantl W.J. Escobedo J.A. Martin G.A. Turck C.W. del Rosario M. McCormick F. Williams L.T. Cell. 1992; 69: 413-423Abstract Full Text PDF PubMed Scopus (471) Google Scholar, 10Songyang Z. Shoelson S.E. Chadhuri 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.F. Chou M.M. Harafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2370) Google Scholar, 11Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo R.X. 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). The Src and Lck SH2 domains, for example, bind preferentially to peptides with the sequence pTyr-Glu-Glu-Ile (pYEEI). Structural data have shown that these SH2 domains contain two pockets, one of which is lined by basic residues that bind phosphotyrosine, while the second is a hydrophobic pocket that accommodates the side chain of the Ile at the +3 position(12Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Crossref PubMed Scopus (570) Google Scholar, 13Eck M.I. Shoelson S.E. Harrison S.C. Nature. 1993; 362: 87-91Crossref PubMed Scopus (441) Google Scholar). The +1 Glu, in contrast, contacts the surface of the domain. Hence, although the Src and Lck SH2 domains touch the peptide backbone at the −1 and +4 positions, specific contacts are restricted to the phosphotyrosine and the side chains of the +1 and +3 residues(12Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Crossref PubMed Scopus (570) Google Scholar, 13Eck M.I. Shoelson S.E. Harrison S.C. Nature. 1993; 362: 87-91Crossref PubMed Scopus (441) Google Scholar). In contrast to the Src SH2 domain, that binds preferentially to hydrophilic residues at the +1 position, the SH2 domains of phospholipase C (PLC)-γ1, 1The abbreviations used are: PLC-γ1phospholipase C-γ1PI3Kphosphatidylinositol 3′-kinaseSH2Src homology domain 2β-PDGFRβ-platelet derived growth factor receptorGAPGTPase activating proteinGSTglutathione S-transferaseN-SH2NH2-terminal SH2 domainC-SH2COOH-terminal SH2 domainFmocN-(9-fluorenyl)methoxycarbonyl. the SH2-containing phosphotyrosine phosphatases, and phosphatidylinositol 3′-kinase (PI3K) select hydrophobic residues at both the +1 and +3 positions(10Songyang Z. Shoelson S.E. Chadhuri 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.F. Chou M.M. Harafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2370) Google Scholar). We have previously suggested that residues in addition to the 3 amino acids directly following the phosphotyrosine are required to stabilize the interaction between PLC-γ1 and the Tyr1021 autophosphorylation site of the β-receptor for platelet-derived growth factor (β-PDGFR)(14Larose L. Gish G. Shoelson S. Pawson T. Oncogene. 1993; 8: 2493-2499PubMed Google Scholar). This notion has received support from structural analysis of the COOH-terminal PLC-γ1 SH2 domain (C-SH2) and the NH2-terminal Syp phosphotyrosine phosphatase SH2 domain both of which possess an extended hydrophobic groove that accommodates the +1 to +3 hydrophobic peptide residues, as well as residues in the +4 and +5 positions(15Pascal 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, 16Lee 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). Contacts with the +6 residue are also detected for PLC-γ1 SH2-C domain(15Pascal 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). phospholipase C-γ1 phosphatidylinositol 3′-kinase Src homology domain 2 β-platelet derived growth factor receptor GTPase activating protein glutathione S-transferase NH2-terminal SH2 domain COOH-terminal SH2 domain N-(9-fluorenyl)methoxycarbonyl. The β-PDGFR possesses autophosphorylation sites in the juxtamembrane region, the kinase insert, and the COOH-terminal tail that bind to SH2-containing proteins such as Src, PI3K, Nck, GAP, Syp, and PLC-γ1 (17Kypta R.M. Goldberg Y. Ulug E.T. Courtneidge S.A. Cell. 1990; 62: 481-492Abstract Full Text PDF PubMed Scopus (478) Google Scholar, 18Coughlin S.R. Escobedo J.A. Williams L.T. Science. 1989; 243: 1191-1194Crossref PubMed Scopus (287) Google Scholar, 19Li W. Hu P. Skolnik E.Y. Ullrich A. Schlessinger J. Mol. Cell. Biol. 1992; 12: 5824-5833Crossref PubMed Scopus (155) Google Scholar, 20Kazlauskas A. Ellis C. McCormick F. Pawson T. Science. 1991; 247: 1578-1581Crossref Scopus (212) Google Scholar, 21Kaplan D.R. Morrison D.K. Wong G. McCormick F. Williams L.T. Cell. 1990; 61: 125-133Abstract Full Text PDF PubMed Scopus (322) Google Scholar, 22Feng G.-S. Hui C.-C. Pawson T. Science. 1993; 259: 1607-1611Crossref PubMed Scopus (582) Google Scholar, 23Kumjian D.A. Wahl M.I. Rhee S.G. Daniel T.O. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8232-8236Crossref PubMed Scopus (122) Google Scholar). In most cases these interactions are relatively specific. For example, PLC-γ1 binds the receptor upon phosphorylation of Tyr1021 in the C-terminal tail(14Larose L. Gish G. Shoelson S. Pawson T. Oncogene. 1993; 8: 2493-2499PubMed Google Scholar, 24Rönnstrand L. Mori S. Arridsson A.-K. Eriksson A. Wernstedt C. Hellman V. Claesson-Welsh L. Heldin C.-H. EMBO J. 1992; 11: 3911-3919Crossref PubMed Scopus (168) Google Scholar, 25Kashishian A. Cooper J.A. Mol. Biol. Cell. 1993; 4: 49-57Crossref PubMed Scopus (48) Google Scholar, 26Valius M. Bazenet C. Kazlauskas A. Mol. Cell. Biol. 1993; 13: 133-143Crossref PubMed Google Scholar). Substitution of this Tyr with Phe abrogates PLC-γ1-binding and PDGF-induced hydrolysis of phosphatidylinositol 4,5-biphosphate, but has no effect on binding of other SH2-containing signaling proteins(27Valius M. Kazlauskas A. Cell. 1993; 73: 321-334Abstract Full Text PDF PubMed Scopus (568) Google Scholar). However, mounting evidence suggests that not all SH2-binding sites are specific for a single signaling protein. For example, both Nck and PI3K reportedly bind Tyr10211021 in the β-PDGFR(28Nishimura R. Li W. Kashishian A. Mondino A. Zhon M. Cooper J. Schlessinger J. Mol. Cell. Biol. 1993; 13: 6889-6896Crossref PubMed Scopus (152) Google Scholar). In the Met receptor tyrosine kinase, two closely spaced tyrosine residues bind multiple SH2-containing proteins, suggesting that a single tyrosine phosphorylation site might under some circumstances couple to multiple signaling pathways(29Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Zonca P. Giordano S. Graziani A. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (880) Google Scholar). Using the PLC-γ1 binding site of the β-PDGFR, we have investigated the determinants that restrict a phosphotyrosine-containing site to a single binding partner, or can allow binding to multiple SH2 proteins. The kinase insert (residues 698-797) and the carboxyl-terminal tail (residues 953-1103) were generated by the polymerase chain reaction using the human β-PDGF receptor as a template(30Claesson-Welsh L. Eriksson A. Moren A Severinsson L. Ek B. Ostman A. Betsholtz C. Heldin C.-H. Mol. Cell. Biol. 1988; 8: 3476-3486Crossref PubMed Scopus (243) Google Scholar), and subcloned into the bacterial expression vector pGEX-2t. Point mutations were generated by overlapping extension using polymerase chain reaction(31Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar). In vivo tyrosine phosphorylation of GST fusion proteins was produced by bacterial infection with a λgt 11 phage containing the Elk tyrosine kinase domain(32Lhotak V. Greer P. Letwin K. Pawson T. Mol. Cell. Biol. 1991; 11: 2496-2502Crossref PubMed Scopus (71) Google Scholar). GST fusion proteins were isolated using glutathione beads and their tyrosine phosphorylation was analyzed following their separation by SDS-polyacrylamide gel electrophoresis and immunoblotting with antiphosphotyrosine antibodies(33Kamps M.P. Sefton B.M. Oncogene. 1988; 2: 305-315PubMed Google Scholar). The level of phosphorylation of the GST fusion proteins on tyrosine residues was then determined with 125125I-protein A and autoradiography. Confluent Rat-2 cells overexpressing PLC-γ1 (34Sultzman L. Ellis C. Lin L.-L. Pawson T. Knopf J. Mol. Cell. Biol. 1991; 11: 2018-2025Crossref PubMed Google Scholar) were lysed in PLC lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EGTA, 10 μg ml-1 leupeptin, 10 μg ml-1 aprotinin, 1 mM phenylmethylsulfonyl fluoride, 200 μM sodium vanadate, 10 mM sodium pyrophosphate, and 10 mM sodium fluoride). The clarified lysate was incubated with immobilized tyrosine-phosphorylated GST fusion proteins (1-3 μg/ml) for 60 min at 4°C, the beads were washed with HNTG buffer (20 mM HEPES, pH 7.5, 150 mM sodium chloride, 0.1% Triton X-100, 10% glycerol, 200 μM sodium vanadate, 10 μg ml-1 aprotinin, 10 μg ml-1 leupeptin, and 1 mM phenylmethylsulfonyl fluoride), and associated proteins were then resolved by SDS-polyacrylamide gel electrophoresis followed by transfer to a nitrocellulose membrane. Immunoblotting was performed with anti-PLC-γ1 antibodies to residues 1064-1291 of bovine PLC-γ1 (35Decker S.J. Ellis C. Pawson T. Velu T. J. Biol. Chem. 1990; 265: 7009-7015Abstract Full Text PDF PubMed Google Scholar) or anti-p85α antibodies to residues 2-83 of bovine p85α and enhanced chemiluminescence (Amersham) or 125125I-protein A was used to detect the signal. In vitro association of PLC-γ1 with the GST fusion protein containing the COOH-terminal portion of the PDGF receptor was competed by increasing concentrations of pY1021, a 12-amino acid phosphopeptide corresponding to residues 1018-1029 of the PDGF receptor (DNDpYIIPLPDPK)(30Claesson-Welsh L. Eriksson A. Moren A Severinsson L. Ek B. Ostman A. Betsholtz C. Heldin C.-H. Mol. Cell. Biol. 1988; 8: 3476-3486Crossref PubMed Scopus (243) Google Scholar). Competition experiments were also performed with variant pY1021 peptides containing alanine substitution at positions +4 or +5 relative to Tyr1021. A phosphopeptide with the sequence SSNpYMAPYDNY corresponding to the binding site for GAP on the PDGF receptor (peptide pY771) (36Kazlauskas A. Kashishian A. Cooper J.A. Valius M. Mol. Cell. Biol. 1992; 12: 2534-2544Crossref PubMed Google Scholar) was used as a control. Peptides were prepared on an ABI 431A peptide synthesizer using Fmoc chemistry with HBTU activation, deprotected by 1 M TMSBr/trifluoroacetic acid treatment for 4 h at 0°C, and isolated by ether precipitation as outlined by the manufacturer (Applied Biosystems Inc., Foster City). Phosphotyrosine was incorporated using the reagent Fmoc-Tyr(PO3Me2)-OH (Bachem Bioscience). The peptides were purified by high performance liquid chromatography and their composition confirmed by mass spectroscopic analysis. The amount of PLC-γ1 bound was detected by immunoblotting with specific anti-PLC-γ1 antibodies and 125125I-protein A. The radioactive signal was quantified using a PhosphoImager scanner and the data were normalized according to the amount of tyrosine-phosphorylated GST-PDGF receptor tail used in each assay. cDNA fragments corresponding to the SH2 domains of bovine PLC-γ1 N-SH2 (residues 545-659), C-SH2 (residues 663-759), and N+C-SH2 (residues 545-759) were isolated by polymerase chain reaction and subcloned into pGEX-kt, a bacterial expression vector. GST fusion proteins were produced, purified, and eluted from glutathione-agarose beads as described earlier(37Gish G. Shen R. Larose L. Pawson T. Methods Enzymol. 1995; (in press)PubMed Google Scholar). Biosensor-based analysis of wild type and variant pY1021 phosphopeptides interacting with PLC-γ1 N-SH2, C-SH2, and N+C-SH2 domains were conducted as described previously(37Gish G. Shen R. Larose L. Pawson T. Methods Enzymol. 1995; (in press)PubMed Google Scholar, 38Marengere L.E.M. Songyang Z. Gish G.D. Schaller M.D. Parsons J.T. Stern M.J. Cantley L.C. Pawson T. Nature. 1994; 369: 502-504Crossref PubMed Scopus (161) Google Scholar). The PLC-γ1-binding site at Tyr1021 in the tail of the β-PDGFR is contained within the sequence pYIIPLPD(30Claesson-Welsh L. Eriksson A. Moren A Severinsson L. Ek B. Ostman A. Betsholtz C. Heldin C.-H. Mol. Cell. Biol. 1988; 8: 3476-3486Crossref PubMed Scopus (243) Google Scholar). In contrast, the SH2 domains of the PI3K p85 subunit bind Tyr740740 and Tyr10211021 in the kinase insert, which conform to the consensus for PI3K-binding, pYXXM(10Songyang Z. Shoelson S.E. Chadhuri 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.F. Chou M.M. Harafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2370) Google Scholar, 27Valius M. Kazlauskas A. Cell. 1993; 73: 321-334Abstract Full Text PDF PubMed Scopus (568) Google Scholar). GST fusion proteins containing the kinase insert or COOH-terminal tail of the β-PDGFR were phosphorylated on tyrosine, and incubated with lysates of Rat-2 fibroblasts. Under these conditions, the kinase insert binds the p85 subunit of PI3K, but not PLC-γ1 (Fig. 1, A and B, lanes 1). In contrast, the tail binds PLC-γ1 but fails to associate with PI3K (Fig. 1, A and B, lanes 2). To investigate which residues COOH-terminal to Tyr1021 in the tail were required for specific PLC-γ1 binding, the residues at the +3, +4, and +5 positions were substituted with the amino acids found at the corresponding positions of the Tyr740740 PI3K-binding site (which has the sequence pYMDMSKD). Hence, the +3 Pro of the Tyr1021 PLC-γ1-binding site was changed to Met, the +4 Leu to Ser, and the +5 Pro to Lys, within the context of the GST tail fusion protein. Since both PLC-γ1 and PI3K binding sites have Asp at +6, this residue was changed to Ala. Wild type and mutant fusion proteins were expressed and phosphorylated on tyrosine to similar levels as described under “Materials and Methods,” and assayed for binding to PLC-γ1 and PI3K. Substitution of the +3 Pro by Met, to yield a β-PDGFR tail with the sequence pY1021IIMLPD, had little discernible effect on PLC-γ1-binding (Fig. 1C). In contrast, substitutions of the +4 Leu to Ser (to give pYIIPSPD) or the +5 Pro to Lys (to pYIIPLKD) both greatly reduced PLC-γ1 binding activity (Fig. 1C). Substitution of the +6 Asp by Ala had no detectable effect in these assays. These results indicate that the +4 and +5 residues of the β-PDGFR Tyr1021 site are both important for high affinity PLC-γ1 binding. In each case their replacement with amino acids from the cognate positions of the PI3K-binding site effectively abolished high affinity binding to PLC-γ1, but did not induce PI3K binding (data not shown). However, replacing the +3 Pro with Met had little effect on PLC-γ1 binding, but induced a novel binding activity for the p85 subunit of PI3K (Fig. 1, A and B, lanes 3). Substitution of the +3 Pro with Met therefore appears to broaden the binding specificity of the Tyr1021 site by increasing its affinity for PI3K, without markedly affecting binding to PLC-γ1. Although the SH2 domains of PLC-γ1 and p85 select phosphopeptides with different amino acids at the +1 to +3 positions from a degenerate peptide library screen, they both bind preferentially to hydrophobic residues at +1 and +3(10Songyang Z. Shoelson S.E. Chadhuri 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.F. Chou M.M. Harafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2370) Google Scholar). Consistent with this finding, they both possess a Cys or Ile at SH2 residue βD5, which appears to be diagnostic of SH2 domains that bind residues COOH-terminal to the phosphotyrosine in a hydrophobic groove(10Songyang Z. Shoelson S.E. Chadhuri 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.F. Chou M.M. Harafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2370) Google Scholar). This being the case, it seems likely that the PLC-γ1 SH2 domains can accommodate a +3 Met in the phosphopeptide ligand without substantial loss in binding affinity, provided that the appropriate residues are present at the +4 and +5 positions. Although it does not inhibit PLC-γ1 binding, this substitution does create a PI3K-binding site, consistent with the observation that the binding of p85 SH2 domains is particularly dependent on a +3 Met. The residues normally present at the +4 and +5 positions of the PLC-γ1-binding site are apparently compatible with PI3K, once a +3 Met has been introduced. Consistent with this observation, a β-PDGFR tail mutant in which the +1 to +3 residues of the PLC-γ1-binding site are converted from Ile-Ile-Pro to Met-Asp-Met (i.e.pYMDMLPD) binds both PLC-γ1 and p85 (Fig. 2). These results predict that further substitution of the pYMDMLPD sequence at the +4 position, by replacement of the +4 Pro with a Ser, should discriminate against PLC-γ1 but not against PI3K, and thereby create a p85-specific binding site. Fig. 2 shows that the phosphorylated β-PDGFR tail containing the sequence pYMDMSPD binds p85, but shows no detectable PLC-γ1 binding activity. These data indicate that PLC-γ1 does not bind to the Tyr740740 PI3K-binding site in the kinase insert because the +4 and +5 residues in the natural PI3K site antagonize association with PLC-γ1. To investigate in more detail the notion that the residues at the +4 and +5 positions of the Tyr1021β-PDGFR autophosphorylation site are important for binding to PLC-γ1, we synthesized phosphopeptides corresponding to the Tyr1021 site (residues 1018-1029 of the β-PDGFR). These peptides contained either the wild type residues, or variant sequences in which the +4 Leu or +5 Pro were replaced with Ala. The wild type and variant phosphopeptides were analyzed for their ability to compete for the binding of the phosphorylated β-PDGFR tail to PLC-γ1 (Fig. 3A). The wild type phosphopeptide inhibited binding of PLC-γ1 to the phosphorylated tail of the β-PDGFR with an IC50 of approximately 10 nM, reflecting a high affinity interaction. In contrast, a variant peptide with Ala at +4 was about 16-fold less efficient in competing with the β-PDGFR tail for binding to PLC-γ1 (IC50, 160 nM). Substitution of the +5 Pro by Ala resulted in a 3-fold decrease in peptide inhibition of PLC-γ1 binding to the β-PDGFR tail (IC50, 30 nM). When the data were expressed as the relative percent of PLC-γ1 bound in the absence of phosphopeptide, all three competition curves showed a Hill coefficient of 1.0, suggesting only one site of interaction for PLC-γ1 on the β-PDGFR tail (Fig. 3B). This is consistent with the fact that in a previous study(14Larose L. Gish G. Shoelson S. Pawson T. Oncogene. 1993; 8: 2493-2499PubMed Google Scholar), we have demonstrated that Tyr1021 in the GST-PDGF receptor tail was the major phosphorylation site by the Elk kinase domain in the bacterial cells. Finally, these results indicate that the residues at the +4 and +5 positions of the Tyr1021 site are indeed required for optimal binding, and cannot be replaced even with another hydrophobic residue without affecting SH2-binding efficiency. The experiments discussed above employed full-length PLC-γ1 isolated from fibroblast cell lysates. Since the binding of PLC-γ1 to the phosphorylated β-PDGFR tail is apparently mediated by one or the other of the two PLC-γ1 SH2 domains, we have directly measured the association of these SH2 domains with a phosphopeptide containing the Tyr1021 site. For this purpose, wild type Tyr1021 phosphopeptide was immobilized on the sensor chip of a Pharmacia BIAcore instrument(37Gish G. Shen R. Larose L. Pawson T. Methods Enzymol. 1995; (in press)PubMed Google Scholar, 38Marengere L.E.M. Songyang Z. Gish G.D. Schaller M.D. Parsons J.T. Stern M.J. Cantley L.C. Pawson T. Nature. 1994; 369: 502-504Crossref PubMed Scopus (161) Google Scholar). Surface plasmon resonance was used to measure the binding of these immobilized phosphopeptides to bacterial GST fusion proteins containing the PLC-γ1 NH2-terminal SH2 domain (N-SH2), the COOH-terminal SH2 domain (C-SH2), or both SH2 domains together (N+C-SH2). The combined N+C-SH2 domains of PLC-γ1 bound the wild type Tyr1021 peptide with the highest affinity (Table 1). When the two SH2 domains were examined individually, substantial differences in their affinities for the wild type β-PDGFR phosphopeptide were observed. The C-SH2 domain bound Tyr1021 with a higher affinity than the N-SH2 domain (Table 1). These results suggest that the COOH-terminal SH2 domain is primarily responsible for specific PLC-γ1-binding to the Tyr1021 autophosphorylation site in the tail of the β-PDGFR, and are consistent with predictions based on screening of a degenerate phosphopeptide library(10Songyang Z. Shoelson S.E. Chadhuri 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.F. Chou M.M. Harafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2370) Google Scholar). Furthermore, this finding strengthens the value of models based on the NMR structure of the PLC-γ1 C-SH2 domain complexed to a phosphopeptide representing the β-PDGFR Tyr1021 autophosphorylation site(15Pascal 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). However, these results suggest that the two SH2 domains of PLC-γ1 might be acting cooperatively to support optimal affinity binding of PLC-γ1 to the β-PDGF receptor in vivo.Table I Open table in a new tab To assess the importance of residues at positions +4 and +5 in the Tyr1021 phosphopeptide for the binding of the combined N+C-SH2 domains of PLC-γ1 using the BIAcore system, Tyr1021 phosphopeptides with alanine substitutions at the +4 or +5 positions were used. Substitution of Leu for Ala at position +4 decreased the affinity of this peptide for N+C-SH2 domains by about 10-fold (Table 1). Substitution of Pro by Ala at position +5 resulted in a 2-fold decrease in binding affinity for GST-SH2 fusion protein containing both SH2 domains (Table 1). The principal kinetic effect of the +4 and +5 substitutions was on the off-rate (data not shown). These results indicate that the isolated SH2 domains of PLC-γ1 bind with relatively high affinity to a phosphopeptide spanning the Tyr1021 site of the β-PDGFR. In addition, substituting residues +4 and +5 with Ala had similar effects on binding to isolated PLC-γ1 SH2 domains, measured by surface plasmon resonance, and association with full-length PLC-γ1, assessed by competition with the phosphorylated β-PDGFR tail. Similar results were obtained when residue +5 Pro was substituted by Lys. However, a 100-fold decrease in affinity was observed when the +4 Leu residue was substituted for Ser instead of Ala (data not shown). The results presented here suggest that the control of SH2 domain binding specificity is more complex than previously supposed. By manipulating the sequence of the residues COOH-terminal to the PLC-γ1-binding site in the tail of the β-PDGFR, we have been able to change a site that is initially specific for PLC-γ1 and does not recognize PI3K, to a site with mixed binding specificity that complexes with both PLC-γ1 and PI3K. Further alteration creates a site that is specific for PI3K, and does not bind stably to PLC-γ1 (Table 2). Binding of PI3K appears especially sensitive to the presence of a Met in the +3 position, while PLC-γ1-binding is also modulated by the +4 and +5 residues. The data presented here suggest that the natural PI3K-binding site at Tyr740740 of the β-PDGF receptor has two elements involved in specificity. The residues at +1 and +3, especially the +3 Met, induce high affinity binding to PI3K. In contrast, the residues at +4 and +5 do not appear of particular importance for PI3K binding, but contribute to specificity by repelling other SH2-containing proteins that might otherwise bind this site, such as PLC-γ1. It is of interest in this regard that IRS-1, which upon phosphorylation binds PI3K but not PLC-γ1(39Lavan B.E. Kuhne M.R. Garner C.W. Anderson D. Reedijk M. Pawson T. Lienhard G.E. J. Biol. Chem. 1992; 267: 11631-11636Abstract Full Text PDF PubMed Google Scholar), has nine YXXM motifs of which four have Ser at the +4 position(40Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1265) Google Scholar). None of these sites has a +4 Leu. The two preferential PI3K-binding sites on IRS-1, at Tyr608 and Tyr939(41Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar), have Ser and Asp at the +4 position, respectively. These results suggest a means by which SH2-binding sites can be tailored to be either specific, as seen in the β-PDGFR, or promiscuous, as in the Met receptor.Table II Open table in a new tab We thank Dr. M. F. White for helpful discussions." @default.
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