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

• DOI: 10.1074/mcp.m500108-mcp200
• Publication Date: 2005-06-01T02:48:23Z
• Authors: Christina Y.H. Jia; Jing Nie; Chenggang Wu; Chengjun Li; Shawn S.-C. Li
• Keywords: Homology; Sequence homology

The Src homology (SH) 3 domain has been shown recently to bind peptide sequences that lack the canonical PXXP motif. diverse specificity in ligand recognition for a group of 15 SH3 domains now investigated using arrays peptides derived from proline-rich region SH2 domain-containing leukocyte protein 76 kDa (SLP-76). A screen individual or mixed allowed identification number candidate SH3-binding peptides. Although some contain conventional motif, most are devoid such motif and instead enriched basic residues. Fluorescent polarization measurements soluble purified demonstrated several domains, including those growth factor receptor-bound 2 (Grb2), NCK, phospholipase C (PLC)-γ1, bound with moderate affinities (10–100 μm) non-conventional Of particular interest, PLC-γ1 was found associate SLP-76 through at least three distinct sites, two which bore novel KKPP other contained classic sequence. Intriguingly mutation critical residues sites not only affected binding but also Grb2 C-terminal domain, indicating overlapped. Our studies suggest is an inherently promiscuous interaction module capable may Furthermore numerous implies global pool mammalian proteome be significantly greater than previously acknowledged. 1The abbreviations used are: SH, homology; SLP-76, kDa; Grb2, 2; PLC, C; SH3-C, SH3; PRR, region; PI3K, phosphatidylinositol 3-kinase; HEK, human embryonic kidney; LAT, linker activated T cells; ECF, enhanced chemifluorescence. one abundant modules eukaryotes (1Castagnoli L. Costantini A. Dall'Armi C. Gonfloni S. Montecchi-Palazzi Panni Paoluzi Santonico E. Cesareni G. Selectivity promiscuity network mediated by 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 Skupski M.P. Misra Ashburner M. Birney Boguski M.S. Brody T. Brokstein P. Celniker S.E. Chervitz S.A. Coates D. Cravchik Gabrielian Galle R.F. Gelbart W.M. George R.A. Goldstein L.S. Gong F. Guan Harris N.L. Hay B.A. Hoskins J. Z. Hynes R.O. Jones S.J. Kuehl P.M. Lemaitre B. Littleton J.T. Morrison D.K. Mungall O'Farrell P.H. Pickeral O.K. Shue Vosshall L.B. Zhang Zhao Q. Zheng X.H. Lewis Comparative genomics eukaryotes.Science. 2000; 287: 2204-2215Google 3Tong A.H.Y. Drees Nardelli Bader G.D. Brannetti Castagnoli Evangelista Ferracuti Quondam Zucconi Hogue C.W.V. Fields Boone combined experimental computational strategy define networks modules.Science. 2002; 295: 321-324Google Scholar). estimated over 400 copies spread among array proteins These non-catalytic 50–70 amino acids have mediate protein-protein interactions short thereby play important roles cell, ranging signal transduction regulation cytoskeletal rearrangements (4Mayer domains: complexity moderation.J. Cell Sci. 2001; 114: 1253-1263Google majority characterized date recognize class I and/or II share core 5Sparks A.B. Rider J.E. Hoffman N.G. Fowlkes D.M. Quilliam L.A. Kay B.K. Distinct preferences Src, Yes, Abl, Cortactin, p53bp2, PLCγ, Crk, Grb2.Proc. Natl. Acad. U. 1996; 93: 1540-1544Google 6Sparks Mapping phage-displayed random-peptide libraries.Methods Mol Biol. 1998; 84: 87-103Google classes ligands differ each principally location flanking residue (Arg Lys) dictates orientation respect surface (7Feng Kasahara Rickles R.J. Schreiber S.L. Specific outside ligands.Proc. 1995; 92: 12408-12415Google 8Feng Chen J.K. Yu H. Simon J.A. Two orientations domain: development general model SH3-ligand interactions.Science. 1994; 266: 1241-1247Google Thus, consensus (R/K)XXPXXP, while conform degenerated sequence PXXPX(R/K) (where X represents any acid). When however, both adopt polyproline type helical structure (8Feng 9Zarrinpar Bhattacharyya R.P. Lim W.A. function proline domains.Sci. STKE. 2003; 2003: RE8Google Extensive biochemical biophysical analysis their complexes past decade provided tremendous insights into structural basis Nevertheless recent domain-mediated (10Berry Nash Liu S.K.-W. Pawson McGlade C.J. high-affinity Arg-X-X-Lys confers between Gads cell signaling.Curr. 12: 1336-1341Google 11Liu Berry S.S. Structural specific RxxK motif-containing peptide: mode recognition.Mol. Cell. 11: 471-481Google 12Douangamath Filipp F.V. Klein A.T.J. Barnett Zou Voorn-Brouwer Vega M.C. Mayans O.M. Sattler Distel Wilmanns Topography independent α-helical PPII-helical peroxisomal domain.Mol. 10: 1007-1017Google 13Groemping Y. Lapouge K. Smerdon Rittinger Molecular phosphorylation-induced activation NADPH oxidase.Cell. 113: 343-355Google Scholar), it become increasingly clear our understanding this far being complete. growing body literature suggests possess beyond ligands. For example, tyrosine kinase substrate Eps8 related selectively containing PXXDY (14Mongiovi A.M. Romano P.R. Mendoza Wong W.T. Musacchio Paolo Di Fiore peptide-SH3 interaction.EMBO 1999; 18: 5300-5309Google Fyn Fyn-binding protein, Fyb/SLAP130, engage site associated 55 (SKAP55) RKXXYXXY (15Kang Freund Duke-Cohan J.S. Wagner Rudd C.E. proline-independent tyrosine-based RKxxYxxY immune adaptor SKAP55.EMBO 19: 2889-2899Google transducing molecule 1 (STAM1) STAM2/EAST/Hbp, family involved cytokine signaling receptor-mediated endocytosis exocytosis, PX(V/I)(D/N)RXXKP conserved STAM (AMSH) deubiquitinating enzyme UBPY (16Kato Miyazawa Kitamura N. interacts Hrs-binding via motifPX(V/I)(D/N)RXXKP.J. Chem. 275: 37481-37487Google Similar harboring (R/K)XX(K/R) identified immunoreceptor signaling, Gab1/Gab2, BLNK (B linker), Fyb, hematopoietic progenitor (HPK1), 17Harkiolaki Lewitzky Gilbert E.Y. Bourette Mouchiroud Sondermann Moarefi I. Feller S.M. Mona/Gads SLP-76.EMBO 22: 2571-2582Google 18Lewitzky Harkiolaki Domart M.-C. SH3C (HPK1) combines atypical R/KXXK, classical embedded (PPII) helix.J. 279: 28724-28732Google 19Lewitzky Kardinal Gehring N.H. Schmidt E.K. Konkol Eulitz Birchmeier Schaeper adapter binds high affinity Gab1 SH3-typical P-x-x-P motif.Oncogene. 20: 1052-1062Google In vitro vivo data showed these responsible respective (SH3-C) Collectively observations rather broad specificity. It should noted that, although its cognate usually weak typical dissociation constant 1–100 μm range, RSTK-containing submicromolar Proline-rich regions (PRRs), like highly prevalent eukaryotic proteomes (2Rubin length PRR varies few hundreds acids. often nucleates as WW, GYF, enabled VAsp (EVH) (9Zarrinpar 20Li S.S.C. Specificity versatility domains—structural implications cellular transduction.Biochem. 2005; (in press)Google This epitomized multidomain plays essential role (21Clements J.L. Known potential functions regulating T-cell development.Immunol. Rev. 191: 211-219Google addition N-terminal phosphorylation contains spans more 250 (Fig. 1). displays features common many known sequences. instance, very long covers significant part polypeptide chain. Moreover apart content Pro (∼25%), positively charged (34 total, underlined Fig. Curiously despite possibility domain-binding sequence, extensively investigated. To date, proteins, namely PLC-γ1, direct physical 22Liu S.K. signalling.Oncogene. 6284-6290Google 23Yablonski Kadlecek Weiss Identification C-γ1 (PLC-γ1) required NFAT.Mol. 21: 4208-4218Google Pro-rich provide excellent platform on explore Traditional methods determination, libraries either synthesized chemically displayed bacterial phages (5Sparks 24Songyang Peptide library screening determination phosphotyrosine-binding sequences.Methods Enzymol. 332: 183-195Google 25Songyang Shoelson Olivier Bustelo X.R. Barbacid Sabe Hanafusa Yi Ren Baltimore Ratnofsky Feldman Cantley L.C. motifs recognized Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, Vav.Mol. 14: 2777-2785Google 26Songyang Recognition primary-sequence modular domains.Prog. Biophys. Mol. 71: 359-372Google 27Rodriguez S.S.-C. Harper J.W. Songyang An oriented (OPAL) study interactions.J. 8802-8807Google generally require inclusion key positions achieve selections positions. Such strategy, powerful defining optimal overlook do preselected (27Rodriguez We present here map unbiased arrayed domains. study, we applied PRR. Through representing were identified. Remarkably small bear rich Synthetic corresponding solution NCK domain. measured above 10 μm, they comparable conventional, PXXP-containing ligand. Ala-scanning permutation sequences, physiological relevance variants bearing mutations cDNA fragments encoding v-Src, c-Src (chicken), Fgr, c-Abl, Fyn, (C), spectrin, (N, M, C), p85 subunit PI3K amplified PCR subcloned pGEX4T2 vector. letters N, parentheses denote N-terminal, middle, respectively. Proteins expressed Escherichia coli GST fusions affinity-purified glutathione-Sepharose beads (Amersham Biosciences) according manufacturer's protocols. Bound eluted 20 mm glutathione, 50 Tris, pH 8.0, 100 NaCl concentrated phosphate buffer, 7.0, prior use studies. (residues 162–412) represented series undecamer peptides, 120 generated "walking," N terminus, window 11 frameshift consecutive assembled format functionalized cellulose membrane multiple synthesis (28Frank SPOT-synthesis technique: synthetic supports—principles applications.J. Immunol. Methods. 267: 13-26Google Scholar) Auto-Spot Robot ASP 222 (Abimed). moistened sequentially ethanol water before probed GST-SH3 proteins. Specifically washed times TBS-T buffer Tris-HCl, 140 NaCl, 0.1% (v/v) Triton X-100, 7.6, blocked 5% BSA h room temperature. Approximately 1.0 total fusion added directly blocking incubated then once TBS anti-GST monoclonal antibody added. After 30-min incubation antibody, developed chemifluorescence (ECF) ECL Western blot kit Biosciences). signals captured analyzed Fluor-S Multi-Imager (Bio-Rad). Individual 0.1-mmol scale 431A Synthesizer (Applied Biosystems) standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. fluorescein labeling, appropriate amount 5-(and-6)-carboxyfluorescein succinimidyl ester (Molecular Probes) resin, coupling reaction proceed Upon cleavage resin trifluoroacetic acid, fluorescein-labeled separated unlabeled HPLC Luna C18 column (Phenomonex). Identities confirmed mass spectrometry. performed described varied titrated fluorescent PBS, NaCl. domain-peptide mixtures incubate dark 30 min anisotropy °C. Binding curves fitting isothermal hyperbola nonlinear regression Prism 3.0 (GraphPad Software, Inc., San Diego, CA), produced constants (Kd). Human kidney (HEK) 293 cells (from ATCC) cultured Dulbecco's modified Eagle's medium supplemented 10% fetal bovine serum, units/ml penicillin, μg/ml streptomycin. Cells transiently transfected expressing plasmids (pFLAG) wild-type mutant slp-76 means Lipofectamine (Invitrogen). All mutants QuikChange site-directed mutagenesis (Stratagene) except R237A, kind gift Jane McGlade. same creation SH3-C E171A E174A harvested 60 post-transfection Hepes, 7.5, 150 glycerol, 1% 1.5 MgCl2, EDTA, Na2P2O7, NaF protease inhibitors (1 pepstatin A, E64, bestatin, leupeptin, aprotinin, PMSF; Sigma). pull-down studies, aliquots cleared HEK lysate mg GST, immobilized glutathione-agarose 4 collected centrifugation resolved 12% SDS-PAGE. Protein bands blotted onto PVDF membranes (Roche Diagnostics) anti-FLAG (Santa Cruz Biotechnology, Inc.). co-immunoprecipitation FLAG-tagged Myc-tagged singularly combination cells. immunoprecipitated anti-Myc antibodies. interact bacteria assessed ability pulling down lysate. current taken variety different kinases (i.e. c-Src, Fyn), lipid PI3K), lipase (PLC-γ1), docking Gads, Crk), spectrin). examined, eight SLP-76. exhibited strong expected. Interestingly (PI3K), robust indicate can accommodate awaits further investigation, worth noting components multiprotein complex referred signalosome, nucleated (LAT) (29Huang Wange R.L. receptor signaling: complexes.J. 28827-28830Google identify overlapping SPOT technology nitrocellulose walking entire 161–412) sliding 3A). spots cover numbered 97 216 because originally larger array. Amino acid listed Supplemental Table S1. peptide-walking equal molar ratio. Because interested identifying all "information content" set proteomic did exhibit assay 2) included prevent nonspecific binding. As seen 3B, implying accommodating multitude interactions. words, information content. Inspection positive revealed interesting patterns. I, 14 (group I) contrast, another 39 II) second (Table I). one-third RXXK, KXXK, HXXK. There no apparent remai