Novel Src Homology 3 Domain-binding Motifs Identified from Proteomic Screen of a Pro-rich Region

The Src homology (SH) 3 domain has been shown recently to bind peptide sequences that lack the canonical PXXP motif. The diverse specificity in ligand recognition for a group of 15 SH3 domains has now been investigated using arrays of peptides derived from the proline-rich region of the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76). A screen of the peptide arrays using individual or mixed SH3 domains has allowed the identification of a number of candidate SH3-binding peptides. Although some peptides contain the conventional PXXP motif, most are devoid of such a motif and are instead enriched in basic residues. Fluorescent polarization measurements using soluble peptides and purified SH3 domains demonstrated that several SH3 domains, including those from growth factor receptor-bound protein 2 (Grb2), NCK, and phospholipase C (PLC)-γ1, bound with moderate affinities (10–100 μm) to a group of non-conventional peptides. Of particular interest, the PLC-γ1 SH3 domain was found to associate with SLP-76 through at least three distinct sites, two of which bore a novel KKPP motif and the other contained the classic PXXP sequence. Intriguingly mutation of critical residues for the three sites not only affected binding of SLP-76 to the PLC-γ1 SH3 domain but also to the Grb2 C-terminal SH3 domain, indicating that the binding sites in SLP-76 for the two SH3 domains are overlapped. Our studies suggest that the SH3 domain is an inherently promiscuous interaction module capable of binding to peptides that may or may not contain a PXXP motif. Furthermore the identification of numerous non-conventional SH3-binding peptides in SLP-76 implies that the global ligand pool for SH3 domains in a mammalian proteome may be significantly greater than previously acknowledged. The Src homology (SH) 3 domain has been shown recently to bind peptide sequences that lack the canonical PXXP motif. The diverse specificity in ligand recognition for a group of 15 SH3 domains has now been investigated using arrays of peptides derived from the proline-rich region of the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76). A screen of the peptide arrays using individual or mixed SH3 domains has allowed the identification of a number of candidate SH3-binding peptides. Although some peptides contain the conventional PXXP motif, most are devoid of such a motif and are instead enriched in basic residues. Fluorescent polarization measurements using soluble peptides and purified SH3 domains demonstrated that several SH3 domains, including those from growth factor receptor-bound protein 2 (Grb2), NCK, and phospholipase C (PLC)-γ1, bound with moderate affinities (10–100 μm) to a group of non-conventional peptides. Of particular interest, the PLC-γ1 SH3 domain was found to associate with SLP-76 through at least three distinct sites, two of which bore a novel KKPP motif and the other contained the classic PXXP sequence. Intriguingly mutation of critical residues for the three sites not only affected binding of SLP-76 to the PLC-γ1 SH3 domain but also to the Grb2 C-terminal SH3 domain, indicating that the binding sites in SLP-76 for the two SH3 domains are overlapped. Our studies suggest that the SH3 domain is an inherently promiscuous interaction module capable of binding to peptides that may or may not contain a PXXP motif. Furthermore the identification of numerous non-conventional SH3-binding peptides in SLP-76 implies that the global ligand pool for SH3 domains in a mammalian proteome may be significantly greater than previously acknowledged. The SH3 1The abbreviations used are: SH, Src homology; SLP-76, SH2 domain-containing leukocyte protein of 76 kDa; Grb2, growth factor receptor-bound protein 2; PLC, phospholipase C; SH3-C, C-terminal SH3; PRR, proline-rich region; PI3K, phosphatidylinositol 3-kinase; HEK, human embryonic kidney; LAT, linker of activated T cells; ECF, enhanced chemifluorescence. domain is one of the most abundant protein modules found in eukaryotes (1Castagnoli L. Costantini A. Dall’Armi C. Gonfloni S. Montecchi-Palazzi L. Panni S. Paoluzi S. Santonico E. Cesareni G. Selectivity and promiscuity in the interaction network mediated by protein recognition modules.FEBS Lett. 2004; 567: 74-79Google Scholar, 2Rubin G.M. Yandell M.D. Wortman J.R. Gabor Miklos G.L. Nelson C.R. Hariharan I.K. Fortini M.E. Li P.W. Apweiler R. Fleischmann W. Cherry J.M. Henikoff S. Skupski M.P. Misra S. Ashburner M. Birney E. Boguski M.S. Brody T. Brokstein P. Celniker S.E. Chervitz S.A. Coates D. Cravchik A. Gabrielian A. Galle R.F. Gelbart W.M. George R.A. Goldstein L.S. Gong F. Guan P. Harris N.L. Hay B.A. Hoskins R.A. Li J. Li Z. Hynes R.O. Jones S.J. Kuehl P.M. Lemaitre B. Littleton J.T. Morrison D.K. Mungall C. O’Farrell P.H. Pickeral O.K. Shue C. Vosshall L.B. Zhang J. Zhao Q. Zheng X.H. Lewis S. Comparative genomics of the eukaryotes.Science. 2000; 287: 2204-2215Google Scholar, 3Tong A.H.Y. Drees B. Nardelli G. Bader G.D. Brannetti B. Castagnoli L. Evangelista M. Ferracuti S. Nelson B. Paoluzi S. Quondam M. Zucconi A. Hogue C.W.V. Fields S. Boone C. Cesareni G. A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules.Science. 2002; 295: 321-324Google Scholar). The human proteome is estimated to contain over 400 copies of SH3 domains spread among a diverse of (1Castagnoli L. Costantini A. Dall’Armi C. Gonfloni S. Montecchi-Palazzi L. Panni S. Paoluzi S. Santonico E. Cesareni G. Selectivity and promiscuity in the interaction network mediated by protein recognition modules.FEBS Lett. 2004; 567: 74-79Google Scholar). domains of been shown to by binding to proline-rich sequences and in the from to of B. SH3 in Scholar). The of SH3 domains to the peptides that a PXXP motif B. SH3 in Scholar, ligand of Src homology 3 domains from and S. A. Scholar, the specificity of SH3 domains with Scholar). The two of SH3 from other in the of a basic or that the of the peptide with to the SH3 binding S. C. the proline-rich of two of Src homology 3 S. A. Scholar, S. binding for peptides to the Src SH3 of a for Scholar). the the motif the peptides to the bound to SH3 domains, of the S. binding for peptides to the Src SH3 of a for Scholar, A. The and of recognition Scholar). and of numerous SH3 domains and in the specificity and of ligand recognition B. SH3 in Scholar, A. The and of recognition Scholar). with the identification of a number of non-conventional SH3 P. T. A SH3 binding motif specificity for the interaction and SLP-76 in T 2002; Scholar, Q. D. P. T. Li for binding of the SH3 domain to an SLP-76 a novel of peptide Scholar, A. P. P. T. M. B. M. for binding of and to a SH3 2002; Scholar, S.J. of of the has that of of interaction module is from A of that SH3 domains specificity the recognition of the conventional and the SH3 domains from the and bind to sequences a motif Panni S. M. A. Cesareni G. P. A novel J. Scholar). The SH3 domains of and a in the Src protein of kDa with a that is devoid of C. A. G. SH3 domain recognition of a motif in J. 2000; Scholar). The SH3 domains of and a of in and and bind to a motif in with the SH3 domain of and the M. A with the Src homology 3 domain of protein a novel binding 2000; Scholar). sequences an motif also been in a group of that in including and SLP-76 P. T. A SH3 binding motif specificity for the interaction and SLP-76 in T 2002; Scholar, Q. D. P. T. Li for binding of the SH3 domain to an SLP-76 a novel of peptide Scholar, M. M. Jones G. for SH3 binding and J. Scholar, M. M. Jones binding to an SH3 binding motif, with a PXXP motif in a 2004; Scholar, M. C. B. M. W. The C-terminal SH3 domain of the protein Grb2 with to sequences in and SLP-76 which lack the Scholar). and in experimental that the motif in was for with the C-terminal SH3 domains of Grb2 P. T. A SH3 binding motif specificity for the interaction and SLP-76 in T 2002; Scholar, Q. D. P. T. Li for binding of the SH3 domain to an SLP-76 a novel of peptide Scholar, M. C. B. M. W. The C-terminal SH3 domain of the protein Grb2 with to sequences in and SLP-76 which lack the Scholar). suggest that SH3 domains a be the of an SH3 domain for ligand is with a in the the C-terminal SH3 domain was shown to bind an peptide derived from SLP-76 with P. T. A SH3 binding motif specificity for the interaction and SLP-76 in T 2002; Scholar, Q. D. P. T. Li for binding of the SH3 domain to an SLP-76 a novel of peptide Scholar). SH3 domains, are in G.M. Yandell M.D. Wortman J.R. Gabor Miklos G.L. Nelson C.R. Hariharan I.K. Fortini M.E. Li P.W. Apweiler R. Fleischmann W. Cherry J.M. Henikoff S. Skupski M.P. Misra S. Ashburner M. Birney E. Boguski M.S. Brody T. Brokstein P. Celniker S.E. Chervitz S.A. Coates D. Cravchik A. Gabrielian A. Galle R.F. Gelbart W.M. George R.A. Goldstein L.S. Gong F. Guan P. Harris N.L. Hay B.A. Hoskins R.A. Li J. Li Z. Hynes R.O. Jones S.J. Kuehl P.M. Lemaitre B. Littleton J.T. Morrison D.K. Mungall C. O’Farrell P.H. Pickeral O.K. Shue C. Vosshall L.B. Zhang J. Zhao Q. Zheng X.H. Lewis S. Comparative genomics of the eukaryotes.Science. 2000; 287: 2204-2215Google Scholar). The of a from a to of A with the SH3 domain or other recognition modules such the and homology domains A. The and of recognition Scholar, and of SH3 and other recognition and for J. Scholar). is by SLP-76, a protein that an in T and and for the SLP-76 protein in and Scholar). to three sites and a C-terminal SH2 domain, SLP-76 a proline-rich region that than residues The SLP-76 some to proline-rich is and a of the SLP-76 from a of residues numerous residues in in the that SH3 sites may be in the SLP-76 has not been only two and been shown to associate with SLP-76 through with P. T. A SH3 binding motif specificity for the interaction and SLP-76 in T 2002; Scholar, D. The of in Scholar, D. T. A. of a phospholipase SH3 in SLP-76 for of PLC-γ1 and Scholar). an which to SH3 domain of specificity using peptide or ligand of Src homology 3 domains from and S. A. Scholar, the specificity of SH3 domains with Scholar, Z. for of SH2 or domain Scholar, Z. S.E. J. P. T. M. T. R. D. S. R.A. by the SH2 domains of and Scholar, Z. and of by Scholar, M. Li Z. peptide strategy to 2004; the of residues at critical of the peptide to at other a in the for a interaction domain, may that not contain a motif M. Li Z. peptide strategy to 2004; Scholar). a strategy by which to SH3 sites of proline-rich sequences using a group of SH3 strategy to the SLP-76 peptide arrays the using a group of 15 SH3 domains, numerous SH3-binding sequences a number of peptides from the screen the conventional PXXP motif, the of peptides lack such a motif and are in residues. peptides to the novel binding sequences affinities in for the Grb2 and C-terminal SH3 domains the PLC-γ1 SH3 Although the affinities to that an SH3 domain and a Furthermore the PLC-γ1 SH3 domain was found to at least three distinct sites in SLP-76 of which two contained a novel KKPP motif. and peptide arrays used to define critical residues for binding in and the of binding sites was investigated using SLP-76 of one or critical residues. the SH3 domains of Grb2 and and the of by and the The and C in the the and C-terminal SH3 domain, in and to the in and in to in binding The SLP-76 proline-rich region was by a of peptides. in by from the to the C through the of the with a of and a of two peptides. peptides in an a by peptide R. The peptide arrays and 2002; using the The peptide was with and by purified the peptide was three in and in in for at SH3 domains to the and with the for at The was three in and in an was a with the the was in and using the enhanced or The binding the peptide by and a peptides at a using an of was to a peptide and the was allowed to for at of a peptide from the using the peptide was from the peptide by a of the peptides by Fluorescent polarization measurements previously R. The peptide arrays and 2002; Scholar). 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The peptide arrays and 2002; Scholar). a of peptides was in an a by through the with a of two A of peptide to the from to the was a of a peptide sequences to the peptides are in The was for SH3 binding using the 15 SH3 domains mixed in in SH3-binding sites or the of the SLP-76 PRR, used the of SH3 domains in a screen of the peptide SH3 domains that not binding to SLP-76 in the to in numerous in the binding to the of SH3 domains, that the is capable of a of SH3 domain other is in of peptide sequences to the some shown in one group of peptides that affinities for the SH3 domains contained a PXXP motif. group of binding peptides devoid of conventional motif. The group of contained at least one and one basic in Furthermore of the non-conventional peptides bore one or basic such was for the of peptides in group for the of basic residues in The lack of conventional SH3-binding sites in SLP-76 is only one peptide has the of a SH3 such those to may a SH3-binding the number of SH3-binding sites be significantly than the number of in the of SLP-76 peptides that binding to SH3 domains in the sites sites in a The binding sites for individual SH3 domains using arrays to from of a used not The binding of the C-terminal SH3 domains of Grb2, and are shown in of and used to binding peptides. be the two the domain to bind to a by with from a P. T. A SH3 binding motif specificity for the interaction and SLP-76 in T 2002; Scholar). to the the Grb2 domain bound to several other sites, including the one by the domain to sites in the SLP-76 from the peptide and to the binding affinities of individual SH3 domains for non-conventional peptides in and and of the binding arrays using individual and mixed SH3 domains The peptides to those the but by two residues the and C to peptide in 2 three the peptide peptide and A was to the of peptide to binding using peptides and binding to the domains of Grb2, NCK, the of the peptides with SH3 domains by The SH3 domain, which binding in the was used a from domain binding in are in and in with the bound the and Grb2 domains with affinities P. T. A SH3 binding motif specificity for the interaction and SLP-76 in T 2002; Scholar, Q. D. P. T. Li for binding of the SH3 domain to an SLP-76 a novel of peptide Scholar). 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T. A. of a phospholipase SH3 in SLP-76 for of PLC-γ1 and Scholar). of region significantly the of T in through PLC-γ1 D. T. A. of a phospholipase SH3 in SLP-76 for of PLC-γ1 and Scholar). the binding sites for the PLC-γ1 SH3 domain, a the and of in the two residues than those in the such that peptide now contained the by purified PLC-γ1 SH3 domain to the identification of three binding one was in the region and the other two in the region and The PLC-γ1 SH3 domain is to bind to ligand with the ligand of Src homology 3 domains from and S. A. Scholar). Although peptide has the of a peptide a sequence. peptide with peptide that was shown to bind the Grb2 and the domains critical residues in peptide or that binding to the PLC-γ1 SH3 domain, an was for peptide and a was for peptide arrays with purified PLC-γ1 SH3 shown in the C-terminal or those in the motif to or of binding for peptide of residues the motif by an for the or the that peptide a in binding to PLC-γ1 SH3 domain the peptide that the residues the most in binding to the PLC-γ1 SH3 the of the of the peptide only be by basic or an a in the peptide be to and of residues to be at of the peptide the C-terminal in the sequence. at a was not for binding of the in the by other or enhanced the of the peptide that residues a in the interaction peptide and the PLC-γ1 SH3 The residues in peptide found to a in binding the PLC-γ1 SH3 not a ligand in that a motif. an that is C-terminal to the the of the motif and the for binding the PLC-γ1 SH3 domain, a of of peptide by of residues from the or the C of the peptide of the C-terminal two residues of the motif not a in was the residues A of binding was the residues of the the of the instead of the motif, in binding the PLC-γ1 SH3 residues to the a suggest that the for binding to the PLC-γ1 SH3 domain is A peptide in a of KKPP for binding the in for the two a motif, that is for binding to the PLC-γ1 SH3 the affinities of conventional sequences for the PLC-γ1 SH3 domain, peptides and and purified for binding studies in peptides two residues than the to A of also to the of and residues in to the of the KKPP motif to SH3 a was a peptide a PLC-γ1 SH3 domain ligand in a screen ligand of Src homology 3 domains from and S. 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A. found that the PLC-γ1 SH3 domain for the motif A motif bound with μm) to the PLC-γ1 SH3 domain and The affinities by the SLP-76 peptides suggest that are not for binding to the PLC-γ1 SH3 The of residues in peptide to binding using peptide of the of residues in the peptide by a of in or a of in to an and in that the of residues was for the in peptide by an not a the to a the of peptide for peptide binding in with those from the peptide studies the of the PLC-γ1 SH3-binding sites from the peptide studies in the of a of SLP-76 at of residues found to be for binding the C-terminal was shown to be for binding of peptide to the PLC-γ1 SH3 The in SLP-76, was to a to or was that mutation the of the but contained of and in SLP-76 that to the in the KKPP motif of peptide The of residues of peptide was to two residues in to and also to sites in an or in affinities for the PLC-γ1 SH3 domain, in and to using purified SH3 shown in the SH3 domain was to the SLP-76, to and was or most of in the was by the indicating that the a in SH3 domain Intriguingly the was of binding to the SH3 domain the and to moderate affinities for the PLC-γ1 SH3 that the three binding sites in SLP-76 may not from one in binding to the PLC-γ1 SH3 the of the may be affected by the of the PLC-γ1 SH3 domain, the Grb2 domain to sites in peptide which was shown to bind to the Grb2 domain, in with peptide that binding sites for the two SH3 domains in SLP-76 may also the of the binding sites of the Grb2 domain, used a group of SLP-76 in a and the for the PLC-γ1 SH3 domain, two and or was in a to the of the motif in peptide to binding Q. D. P. T. Li for binding of the SH3 domain to an SLP-76 a novel of peptide Scholar). or the other was to the of the motif in peptide to the the Grb2 and SLP-76 in SLP-76 and Grb2 or in The was to using or shown in Grb2 was found in SLP-76 that the two with other in the used purified domain to SLP-76 or a from Although the SLP-76 was by the SH3 domain, was that the in SLP-76 also binding to bound to the Grb2 domain with significantly than the SLP-76, that the an but in also a for the Grb2 the and the in binding the Grb2 suggest that the Grb2 domain may sites in the SLP-76 PRR, a by peptide and several sites by is not sites in binding or affected binding of SLP-76 to the Grb2 domain to is that that affected SLP-76 binding to the PLC-γ1 SH3 domain also interaction with the Grb2 that the C-terminal SH3 domains of Grb2, and are to the PLC-γ1 SH3 domain of group of SH3 domains is the of least residues in the a of residues in the of the domain was previously shown to a in binding SLP-76 Q. D. P. T. Li for binding of the SH3 domain to an SLP-76 a novel of peptide that residues in Grb2 a three of the Grb2 domain that contained of one or residues and in the studies using the critical of the two residues in binding to SLP-76 with or residues to an to bind the specificity of protein interaction modules such the SH3 domain is an the of and the of mediated by domains of using Scholar, B.A. B. A computational for the and of Scholar). The number of SH3 domains contained in a mammalian proteome such a in the the specificity and of SH3 of domains to The identification of an motif by the C-terminal SH3 domain of the protein Q. D. P. T. Li for binding of the SH3 domain to an SLP-76 a novel of peptide Scholar, The protein in with the SLP-76 and the that PXXP is the motif by SH3 of peptide recognition by the SH3 domain also been a of other a of including and the of the through non-conventional and of SH3 and other recognition and for J. and and of the a J. Scholar). The SH3 domain is capable of distinct binding by a conventional motif S. J. interaction a in the in 2004; or through B. A. J. M. F. D. J. C. to Scholar). the and promiscuity of SH3 domains and for in of interaction used peptide in with peptide and protein binding to a group of novel SH3 from the region of from are the SLP-76 is capable of a number of SH3 Although is not of are are in the suggest that may be to and to a of the of The is to other proline-rich that binding sites for recognition the of SH3-binding sites in the SLP-76 not to the or sites are in basic residues. from conventional PXXP SH3 domains are capable of binding to peptides. peptides in SLP-76 SH3 one or such and Although several SH3 domains, including those from Grb2, and bound with moderate affinities to is that are by SH3 domains, by SH3 domains that contain residues in is to in that a basic peptide devoid of to an SH3 domain the SH3 domain to a basic motif, in the through an interaction and the binding of the SH3 domain to C. A. M. S. T. of SH3 domain binding by J. 2004; Scholar). a SH3 domain distinct sites, and a may be by than one SH3 the Grb2 and the PLC-γ1 SH3 domains bind to sites in SLP-76, and the two SH3 domains at least one by studies not only the binding sites for of SLP-76 such also Although the of binding of SLP-76 to the SH3 domains of Grb2, and may be of in a by T Grb2 and associate with through SH2 domains T 2004; Scholar). The SH2 domain also bind to residues at the region of SLP-76 to by The of SH3 in the may with the SH2 domain the of the T with the by the peptide for the domain, most novel peptides from the or moderate affinities for SH3 Although is to that to specificity T. in from domain to 2004; most SH3 are of moderate is a that the of a particular the is and the of the the is or that most in be Furthermore the of to moderate binding sites in a for an SH3 domain may be a used by the to and of SH3 and other recognition and for J. Scholar). Although the SH3 domain may from one be by a the of binding sites the of the SH3 domain and of is by the binding protein 2 domain that only to a of a motif in two copies of the motif are in a for the domain by C. R. S. G. interaction of with the and the SH3 domain of J. 2002; Scholar). is that a the interaction SLP-76 and PLC-γ1 SH3 an by which to the specificity and interaction of SH3 The be to a of and SH3 may also be to other recognition been for the identification of binding for the domains a proteome J. Zhang D. L. S. J. C. M. L. M. R. M. J. A of domain 2004; Scholar). in with and other and to be a for the identification of the global ligand pool for peptide interaction with

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