DOCSLIB.ORG

Evidence for SH3 Domain Directed Binding and Phosphorylation of Sam68 by Src

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Evidence for SH3 Domain Directed Binding and Phosphorylation of Sam68 by Src Oncogene (1999) 18, 4647 ± 4653 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Evidence for SH3 domain directed binding and phosphorylation of Sam68 by Src Zhiwei Shen1,2, Andreas Batzer3,6, Jackie A Koehler1,2, Paul Polakis4, Joseph Schlessinger3, Nicholas B Lydon5 and Michael F Moran*,1,2 1Banting and Best Department of Medical Research, University of Toronto, Canada; 2Department of Molecular and Medical Genetics, University of Toronto, Canada; 3Department of Pharmacology, New York University, New York, USA; 4ONYX Pharmaceuticals, Richmond, California, USA; 5Kinetix Pharmaceuticals Inc, Medford, Massachusetts, USA Sam68 is a 68 kDa protein that associates with and is which aect Src subcellular localization and substrate phosphorylated by the c-Src kinase at mitosis. It interactions. Src becomes activated near mitosis contains a KH domain implicated in RNA binding and (Chackalaparampil and Shalloway, 1988; Morgan et several proline-rich motifs that resemble known SH3 al., 1989), and is required for transition from the G2 binding sites. The SH3 domains of c-Src, phosphatidy- phase to mitosis (Roche et al., 1995). linositol 3-OH kinase, phospholipase C-g and Grb2 Sam68 is a 68-kDa protein that is reported to protein (containing two SH3 domains), but not other associate with the c-Src protein-tyrosine kinase during SH3 domains tested, were capable of binding Sam68 in mitosis (Fumagalli et al., 1994; Taylor and Shalloway, vitro. Synthetic peptides corresponding to the proline 1994). Although it was ®rst cloned as a Ras-GAP motifs of Sam68 inhibited with dierent eciencies the associated phosphotyrosine-containing protein (Wong binding of SH3 domains to Sam68 suggesting that the et al., 1992), it is unrelated to the major phosphoty- proline motifs of Sam68 function as speci®c SH3 domain rosine-containing 62-kDa protein, Dok, which is binding sites. Mutation of Sam68 SH3 binding sites known to associate with Ras-GAP (Carpino et al., further indicated that the SRC SH3 domain mediates 1997; Yamonashi and Baltimore, 1997). Sam68 binding of Src to unphosphorylated Sam68. Phosphor- contains a KH domain with nucleic acid-binding ylation of Sam68 by Src kinase was inhibited when the properties, multiple potential tyrosine phosphorylation Src SH3 binding site of Sam68 was mutated or when sites and several proline-rich motifs which resemble corresponding peptides were added to in vitro kinase known src homology-3 (SH3) binding sites (Wong et reactions indicating that binding of the Src SH3 domain al., 1992). The SH2 and SH3 domains of Src can to a speci®c site near the amino-terminus of Sam68 independently interact with Sam68 and contribute to (including residues 38 ± 45: PPLPHRSR) facilitates the in vivo association of Src and Sam68 (Taylor and phosphorylation of Sam68 by the Src kinase domain. Shalloway, 1994). The KH domain of Sam68 was Sam68-based proline peptides had no eect on the found to ®nd poly(U) speci®cally (Taylor and Shallo- phosphorylation of another in vitro substrate of Src, way, 1994), but its interactions with RNA in vivo enolase. These results suggest that Src eectively mounts remains to be established. The RNA binding capability Sam68 through its SH3 domain, possibly as a mechan- of Sam68 is apparently regulated by its tyrosine ism to position the kinase domain close to substrate phosphorylation status and interaction with SH3 tyrosine residues in the carboxyl-half of the protein. domains (Wang et al., 1995; Taylor et al., 1995). Suggestions for the biological role of Sam68 include Keywords: p68; SH3; Src; kinase regulation of RNA splicing, nucleocytoplasmic trans- port, mRNA stability, translation and signal transduc- tion (Courtneidge and Fumagalli, 1994; Richard et al., 1995), and recent evidence implicates Sam68 in Introduction poliovirus RNA replication (McBride et al., 1996). In addition, Sam68 is a substrate for Cdc2 kinase which The c-Src protein tyrosine kinase (Src) and the related further suggests it may represent an important eector Src-family kinases are normally inactive in vivo as a of cell cycle progression (Fumagalli et al., 1994; consequence of intramolecular interactions involving Resnick et al., 1997; Taylor and Shalloway, 1994). their SH3 and SH2 domains (Sicheri and Kuriyan, Tyrosine phosphorylation of Sam68 in chicken 1997). Activation of these non-receptor kinases can embryo ®broblasts transfected with activated c-Src occur by a variety of mechanisms that perturb these requires an intact Src SH3 domain suggesting that intramolecular interactions. In addition to allowing the the Src SH3 domain may function to recruit substrates catalytic domain to function, activation of Src by this for the Src kinase domain (Weng et al., 1994). The Src- mechanism may also involve the interaction of its SH2 family kinase Fyn was also found to interact with and SH3 domains with substrates or other proteins Sam68 through its SH2 and SH3 domains (Richard et al., 1995). SH3 domains consisting of about 60 amino acids are present in a wide range of proteins involved *Correspondence: MF Moran, Charles H. Best Institute, 112 College in signal transduction and associated with the Street, Toronto, Ontario, M5G 1L6, Canada cytoskeleton (Pawson, 1994). They consist of two 6 Current address: Hoechst Marion Roussel, Deutschland GmbH, DG small b-sheets and three variable loops (Musacchio et Rheumatologie, H528, Postfach 35 40, D-65174 Wiesbaden, Germany al., 1994; Noble et al., 1993) and contain an extended Received 1 June 1999; revised 14 July 1999; accepted 14 July 1999 hydrophobic patch that interacts in a sequence-speci®c SH3-mediated Sam68-Src interactions ZShenet al 4648 manner with the proline-rich core of ligands. Their To better understand the role of the c-Src SH3 binding sites typically contain proline residues that domain in its interaction with substrates, we sought to adopt a left-handed polyproline type II (PPII) helix identify SH3 binding sites on Sam68, and to address conformation. Proline-rich sequence motifs having the whether phosphorylation of Sam68 by Src is facilitated consensus sequences RXXPPXP (class I motifs) or by interaction of the Src SH3 domain with proline PPXPXR (where X corresponds to no speci®c amino motifs on Sam68. In this communication we report acid, class II motifs) bind SH3 domains with opposite results from in vitro experiments which indicate that orientations (Feng et al., 1994; Lim et al., 1994). Ionic two proline motifs in Sam68 are involved in Sam68 interactions between acidic residues on the SH3 binding to the Src SH3 domain, and the interaction of domain and the arginine residue proximal to the the Src SH3 domain with these sites is an important PPXP motif also contribute to SH3-ligand binding. eector of Sam68 tyrosine phosphorylation by Src. Human Sam68 contains ®ve proline-rich candidate SH3 binding sites (Wong et al., 1992). Results To identify SH3 domains capable of binding Sam68, GST fusion proteins (1 mg, immobilized on glutathione agarose) containing SH3 domains from various proteins were incubated with 25 nM unphosphorylated puri®ed human Sam68-KT3. After extensive washing of the beads, bound Sam68-KT3 was detected following SDS ± PAGE and immunoblotting with the KT3 antibody. Sam68 bound to the SH3 domains of the Src tyrosine kinase, p85, PLCg, and to a fusion protein containing the entire Grb2 protein which is comprised of two SH3 domains separated by a single SH2 domain (Figure 1a). Similar experiments employ- ing various mutant versions of Grb2 indicated that the interaction with Sam68 did not involve the Grb2 SH2 domain, and was primarily a function of the amino terminal SH3 domain of Grb2 (Z Shen and A Batzer, unpublished observations). Sam68 did not bind to SH3 domains derived from the Abl tyrosine kinase, Crk, the individual SH3 domains of Grb2, or the neural isoform of Src (Src+) which contain a six residue insertion, Figure 1 Anity precipitation of human Sam68 with SH3 domain-containing GST fusion proteins, and mapping of SH3 domain binding sites. (a) Puri®ed KT3 epitope-tagged Sam68 at concentration of 25 nM was recovered following incubation with 1 mg of the indicated SH3 fusion proteins, or GST alone. Bound Figure 2 Direct interaction of Sam68 and Src SH3 domain. Sam68-KT3 was identi®ed by monoclonal anti-KT3 antibody Proteins from cell lysates bound to GST-SrcSH3 beads (lanes 1 staining and chemiluminescence imaging. (b) Schematic presenta- and 2) or puri®ed GST (0.7 mg) and GST-fusions containing tion of human Sam68. The relative positions of ®ve proline-rich proline-rich motifs of Sam68, GST-I (0.4 mg), GST-II (0.6 mg) or motifs are indicated and labeled I to V. (c) Cell lysates containing GST-III (0.7 mg), were separated by SDS ± PAGE and transferred Sam68-KT3 (0.1 mM) were combined with the indicated immobi- to nitrocellulose. Sam68-KT3 in lane 1 was detected by anti-KT3 lized GST fusion proteins in the presence of no added peptide Western blotting as described above. Other lanes were probed (lane 1) or 1.0 mM of the indicated proline-rich peptides. Sam68- with biotinylated GST-Src SH3 followed by horseradish KT3 recovered on the beads was detected by anti-KT3 Western peroxidase-streptavidin. Bound GST-Src SH3 proteins were blotting as described above identi®ed by chemiluminescence SH3-mediated Sam68-Src interactions ZShenet al 4649 relative to the c-Src SH3 domain, at one end of the other three proline-rich peptides. Peptides I and III ligand binding site (Yu et al., 1992). both inhibited the binding of the PLCg SH3 domain to To determine if the proline sites of Sam68 were Sam68, but peptide I was less eective at blocking the involved in the interaction of Sam68 with SH3 interaction than peptide III. Peptides I and IV blocked domains, synthetic peptides (numbered I ± V) corre- the association of the p85 SH3 domain with Sam68, sponding to the Sam68 proline-rich motifs shown in whereas peptide III had a minor inhibitory eect Figure 1b were tested for their ability to competitively (Figure 1c).
Load more

Recommended publications
  • Paxillin: a Focal Adhesion-Associated Adaptor Protein
    Oncogene (2001) 20, 6459 ± 6472 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Paxillin: a focal adhesion-associated adaptor protein Michael D Schaller*,1 1Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center and Comprehensive Center for In¯ammatory Disorders, University of North Carolina, Chapel Hill, North Carolina, NC 27599, USA Paxillin is a focal adhesion-associated, phosphotyrosine- The molecular cloning of paxillin revealed a number containing protein that may play a role in several of motifs that are now known to function in mediating signaling pathways. Paxillin contains a number of motifs protein ± protein interactions (see Figure 1) (Turner that mediate protein ± protein interactions, including LD and Miller, 1994; Salgia et al., 1995a). The N-terminal motifs, LIM domains, an SH3 domain-binding site and half of paxillin contains a proline-rich region that SH2 domain-binding sites. These motifs serve as docking could serve as an SH3 domain-binding site. Several sites for cytoskeletal proteins, tyrosine kinases, serine/ tyrosine residues conforming to SH2 domain binding threonine kinases, GTPase activating proteins and other sites were also noted. In addition, the N-terminal adaptor proteins that recruit additional enzymes into domain of paxillin contains ®ve copies of a peptide complex with paxillin. Thus paxillin itself serves as a sequence, called the LD motif, which are now known docking protein to recruit signaling molecules to a to function as binding sites for other proteins (see speci®c cellular compartment, the focal adhesions, and/ Table 1) (Brown et al., 1998a). The C-terminal half of or to recruit speci®c combinations of signaling molecules paxillin is comprised of four LIM domains, which are into a complex to coordinate downstream signaling.
    [Show full text]
  • The PX Domain Protein Interaction Network in Yeast
    The PX domain protein interaction network in yeast Zur Erlangung des akademischen Grades eines DOKTORS DER NATURWISSENSCHAFTEN (Dr. rer. nat.) der Fakultät für Chemie und Biowissenschaften der Universität Karlsruhe (TH) vorgelegte DISSERTATION von Dipl. Biol. Carolina S. Müller aus Buenos Aires Dekan: Prof. Dr. Manfred Kappes Referent: Dr. Nils Johnsson Korreferent: HD. Dr. Adam Bertl Tag der mündlichen Prüfung: 17.02.2005 I dedicate this work to my Parents and Alex TABLE OF CONTENTS Table of contents Introduction 1 Yeast as a model organism in proteome analysis 1 Protein-protein interactions 2 Protein Domains in Yeast 3 Classification of protein interaction domains 3 Phosphoinositides 5 Function 5 Structure 5 Biochemistry 6 Localization 7 Lipid Binding Domains 8 The PX domain 10 Function of PX domain containing proteins 10 PX domain structure and PI binding affinities 10 Yeast PX domain containing proteins 13 PX domain and protein-protein interactions 13 Lipid binding domains and protein-protein interactions 14 The PX-only proteins Grd19p and Ypt35p and their phenotypes 15 Aim of my PhD work 16 Project outline 16 Searching for interacting partners 16 Confirmation of obtained interactions via a 16 second independent method Mapping the interacting region 16 The Two-Hybrid System 17 Definition 17 Basic Principle of the classical Yeast-Two Hybrid System 17 Peptide Synthesis 18 SPOT synthesis technique 18 Analysis of protein- peptide contact sites based on SPOT synthesis 19 TABLE OF CONTENTS Experimental procedures 21 Yeast two-hybrid assay
    [Show full text]
  • Insulin Receptor Tyrosine Kinase Substrate Links the E. Coli O157:H7
    Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspFU during pedestal formation Didier Vingadassaloma, Arunas Kazlauskasb, Brian Skehana, Hui-Chun Chengc, Loranne Magouna, Douglas Robbinsa, Michael K. Rosenc, Kalle Sakselab, and John M. Leonga,1 aDepartment of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655; bDepartment of Virology, Haartman Institute, University of Helsinki and HUSLAB, Helsinki University Central Hospital, FIN-00014, Helsinki, Finland; and cDepartment of Biochemistry and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390 Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved March 2, 2009 (received for review September 12, 2008) Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effec- host cell plasma membrane with N- and C-terminal intracellular tors to trigger localized actin assembly in mammalian cells, result- domains and a central extracellular domain that binds to the ing in filamentous actin ‘‘pedestals.’’ One effector, the translocated bacterial outer membrane protein intimin. Clustering of Tir in the intimin receptor (Tir), is localized in the plasma membrane and host cell membrane upon intimin binding initiates a signaling clustered upon binding the bacterial outer membrane protein cascade, ultimately leading to actin pedestal formation. intimin. The second, the proline-rich effector EspFU (aka TccP) For the canonical EPEC strain, serotype O127:H6, Tir is the activates the actin nucleation-promoting factor WASP/N-WASP, only translocated effector required for pedestal formation, and and is recruited to sites of bacterial attachment by a mechanism after becoming phosphorylated on tyrosine residue 474 (Y474) dependent on an Asn-Pro-Tyr (NPY458) sequence in the Tir C- by mammalian kinases, recruits the SH2 domain-containing terminal cytoplasmic domain.
    [Show full text]
  • From Src Homology Domains to Other Signaling Modules: Proposal of the `Protein Recognition Code'
    Oncogene (1998) 17, 1469 ± 1474 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc From Src Homology domains to other signaling modules: proposal of the `protein recognition code' Marius Sudol The Department of Biochemistry, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, USA The study of oncogenes has illuminated many aspects of domains have identi®ed a set of common features cellular signaling. The delineation and characterization which aids in their description. The domains are of protein modules exempli®ed by Src Homology composed of 40 ± 150 amino acids which fold to domains has revolutionized our understanding of the generate one or more ligand-binding surfaces, known molecular events underlying signal transduction path- as `recognition pockets'. Generally, conserved residues ways. Several well characterized intracellular modules of the domain are directly involved in mediating which mediate protein-protein interactions, namely SH2, contacts with the amino acids of the ligand and in SH3, PH, PTB, EH, PDZ, EVH1 and WW domains, maintaining the structure of the domain. Ligands are directly involved in the multitude of membrane, interact with their complementary domains through cytoplasmic and nuclear processes in multicellular and/or short sequence motifs (also called core motifs), unicellular organisms. The modular character of these composed of only 3 ± 6 amino acids (Figure 1). The protein domains and their cognate motifs, the univers- sequence of the core determines the selection of the ality of their molecular function, their widespread domains (Songyang and Cantley, 1995). The most occurrence, and the speci®city as well as the degeneracy `important' amino acid within the core is traditionally of their interactions have prompted us to propose the designated `0' and the remaining amino acids are concept of the `protein recognition code'.
    [Show full text]
  • Meltrin a Cytoplasmic Domain Interacts with SH3 Domains of Src and Grb2 and Is Phosphorylated by V-Src
    Oncogene (2000) 19, 5842 ± 5850 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Meltrin a cytoplasmic domain interacts with SH3 domains of Src and Grb2 and is phosphorylated by v-Src Akiko Suzuki1, Nae Kadota1, Tomokazu Hara1, Yoshiko Nakagami1, Toshiaki Izumi1, Tadaomi Takenawa2, Hisataka Sabe3 and Takeshi Endo*,1 1Department of Biology, Faculty of Science, Chiba University, Yayoicho, Inageku, Chiba, Chiba 263-8522, Japan; 2Department of Biochemistry, Institute of Medical Science, University of Tokyo, Shirokanedai, Minatoku, Tokyo 108-8639, Japan; 3Department of Molecular Biology, Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan Meltrin a/ADAM12 is a member of the ADAM/MDC receptor tyrosine kinases, tyrosine kinase-coupled family proteins characterized by the presence of cytokine receptors, TGF-b family receptor serine/ metalloprotease and disintegrin domains. This protein threonine kinases, and seven-pass G protein-coupled also contains a single transmembrane domain and a receptors. Integrins and cadherins not only serve as relatively long cytoplasmic domain containing several transmembrane adhesion molecules but also participate proline-rich sequences. These sequences are compatible in intracellular and extracellular signaling (Schwartz et with the consensus sequences for binding the Src al., 1995; Ben-Ze'ev, 1997; Yamada and Geiger, 1997). homology 3 (SH3) domains. To determine whether the ADAM/MDC family proteins have emerged as proline-rich sequences interact with SH3 domains in candidate molecules for proteolytic processing, cell ± several proteins, binding of recombinant SH3 domains to cell attachment, and cell fusion (Wolfsberg et al., 1995; the meltrin a cytoplasmic domain was analysed by pull- Wolfsberg and White, 1996; Huovila et al., 1996; Black down assays.
    [Show full text]
  • C-Abl: Activation and Nuclear Targets
    Cell Death and Differentiation (2000) 7, 10 ± 16 ã 2000 Macmillan Publishers Ltd All rights reserved 1350-9047/00 $15.00 www.nature.com/cdd Review c-Abl: activation and nuclear targets Y Shaul*,1 fibroblasts it resides predominantly in the nucleus while in primary haematopoietic cells and neurons c-Abl is more 1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot cytoplasmic. In sharp contrast, all the transforming Abl 76100, Israel variants are exclusively cytoplasmic. The cellular subcellular * Corresponding author: Y Shaul, Department of Molecular Genetics, Weizmann localization of c-Abl is controlled by NLSs (nuclear localization Institute of Science, Rehovot 76100, Israel. Tel: 972-8-934-2320; signals) and an NES (nuclear export signal). This pattern of Fax: 972-8-934-4108; E-mail: [email protected] cellular distribution of c-Abl hints at its possible involvement in multiple molecular pathways, and indeed various nuclear and Received 30.9.99; accepted 26.10.99 cytoplasmic functions have been attributed to c-Abl (reviewed 1 Edited by R Knight in ). Cellular processes involving the nuclear c-Abl will be discussed below. Some of the functional domains of c-Abl have been Abstract characterized (Figure 1). Common features to this family are the myristoylation site (found at the N terminus of the The c-Abl tyrosine kinase and its transforming variants have alternatively spliced human Ib and mouse IV transcripts), been implicated in tumorigenesis and in many important the tyrosine kinase domain with substrate specificity,2 the cellular processes. c-Abl is localized in the nucleus and the Src homology 2 (SH2) and 3 (SH3), both regulating c-Abl cytoplasm, where it plays distinct roles.
    [Show full text]
  • Novel Roles of SH2 and SH3 Domains in Lipid Binding
    cells Review Novel Roles of SH2 and SH3 Domains in Lipid Binding Szabolcs Sipeki 1,†, Kitti Koprivanacz 2,†, Tamás Takács 2, Anita Kurilla 2, Loretta László 2, Virag Vas 2 and László Buday 1,2,* 1 Department of Molecular Biology, Institute of Biochemistry and Molecular Biology, Semmelweis University Medical School, 1094 Budapest, Hungary; [email protected] 2 Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary; [email protected] (K.K.); [email protected] (T.T.); [email protected] (A.K.); [email protected] (L.L.); [email protected] (V.V.) * Correspondence: [email protected] † Both authors contributed equally to this work. Abstract: Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on tyrosine kinase action fundamentally changed our concept of cellular regulation. The induced assembly of subcellular hubs via the recognition of local protein or lipid modifications by modular protein interactions is now a central paradigm in signaling. Such molecular interactions are mediated by specific protein interaction domains. The first such domain identified was the SH2 domain, which was postulated to be a reader capable of finding and binding protein partners displaying phosphorylated tyrosine side chains. The SH3 domain was found to be involved in the formation of stable protein sub-complexes by constitutively attaching to proline-rich surfaces on its binding partners. The SH2 and SH3 domains have thus served as the prototypes for a diverse collection of interaction domains that recognize not only proteins but also lipids, nucleic acids, and small molecules.
    [Show full text]
  • Fyn/Lck Src Homology 3 Domain-Binding Protein (T-Celil Actvation/Signal Transduction/Protein-Tyrws Kna/Src-Famfly Kinases) KRIS A
    Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4135-4139, May 1994 Immunology Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein (T-celil actvation/signal transduction/protein-tyrws kna/Src-famfly kinases) KRIS A. REEDQUIST*, TORU FUKAZAWA*, BRIAN DRUKERt, GOVINDASWAMY PANCHAMOORTHY*, STEVEN E. SHOELSONt, AND HAMID BAND*§ *Lymphocyte Biology Section, Department of Rheumatology and Immunology, and tResearch Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, SGMB 514, 250 Longwood Avenue, Boston, MA 02115; and tDivision of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, OR 97201. Communicated by K. Frank Austen, December 30, 1993 ABSTRACT Tyrosine phosphorylation ofcellular proteins phosphotyrosyl (pY) peptide motifs and mediate assembly is the earliest identifiable event following T-cell antigen recep- of signaling complexes (15, 16). The SH3 domains bind to tor (TCR) stimulation and is essential for activating down- small proline-rich peptide motifs to mediate protein-protein stream signaling machiner. Two Src-family protein-tyrosine interactions (17, 18). A clear physiological role for SH3 kinas, the TCR-associated pS9f (Fyn) and the CD4/8- domains of the Sem-5/Grb-2 protein in linking receptor associated p561c (Lck), have emerged as the likely mediators of tyrosine kinases to the Ras pathway has been demonstrated early tyrosine phosphorylation in T cells. Here, we show direct (19). Recently, the SH3 domains of Src-family kinases have binding of a 120-kDa TCR-induced phosphotyrosyl polypep- been shown to bind to the p85 subunit of phosphatidylino- tide, p120, to glutathione S-transferase fusion proteins of the sitol (PI) 3-kinase (3-K) (20, 21), paxillin (a focal adhesion Src homology 3 (SH3) domains of Fyn, Lck, and p60 (Src) protein) (22), actin-associated protein p110 (23), and micro- but not other proteins.
    [Show full text]
  • Systematic Identification of SH3 Domain-Mediated Human Protein–Protein Interactions by Peptide Array Target Screening
    Proteomics 2007, 7, 1775–1785 DOI 10.1002/pmic.200601006 1775 RESEARCH ARTICLE Systematic identification of SH3 domain-mediated human protein–protein interactions by peptide array target screening Chenggang Wu1*, Mike Haiting Ma1*, Kevin R. Brown2, Matt Geisler1, Lei Li1, Eve Tzeng1, Christina Y. H. Jia1, Igor Jurisica2 and Shawn S.-C. Li1 1 Department of Biochemistry and the Siebens-Drake Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada 2 Ontario Cancer Institute, Northeast Structural Genomics Consortium, Toronto, Ontario, Canada Systematic identification of direct protein–protein interactions is often hampered by difficulties Received: December 12, 2006 in expressing and purifying the corresponding full-length proteins. By taking advantage of the Revised: February 1, 2007 modular nature of many regulatory proteins, we attempted to simplify protein–protein interac- Accepted: February 23, 2007 tions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein–protein interactions, we exam- ined a set of interactions mediated by the PLCg1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCg1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCg1 SH3 domain negatively regulated HPK1 kinase activity.
    [Show full text]
  • Peptide Array X-Linking (PAX): a New Peptide-Protein Identification Approach
    Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Okada, Hirokazu et al. “Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach.” Ed. Pontus Aspenstrom. PLoS ONE 7.5 (2012): e37035. As Published http://dx.doi.org/10.1371/journal.pone.0037035 Publisher Public Library of Science Version Final published version Citable link http://hdl.handle.net/1721.1/71732 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/2.5/ Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach Hirokazu Okada1, Akiyoshi Uezu1, Erik J. Soderblom2, M. Arthur Moseley, III2, Frank B. Gertler3, Scott H. Soderling1* 1 Department of Cell Biology, Duke University Medical School, Durham, North Carolina, United States of America, 2 Institute for Genome Science & Policy Proteomics Core Facility, Duke University Medical School, Durham, North Carolina, United States of America, 3 Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America Abstract Many protein interaction domains bind short peptides based on canonical sequence consensus motifs. Here we report the development of a peptide array-based proteomics tool to identify proteins directly interacting with ligand peptides from cell lysates. Array-formatted bait peptides containing an amino acid-derived cross-linker are photo-induced to crosslink with interacting proteins from lysates of interest. Indirect associations are removed by high stringency washes under denaturing conditions. Covalently trapped proteins are subsequently identified by LC-MS/MS and screened by cluster analysis and domain scanning.
    [Show full text]
  • Sh3 Domain Mediated Virus- Host Cell Interactions by the Nonstructural Protein 1 (Ns1) of Influenza a Virus
    SH3 DOMAIN MEDIATED VIRUS- HOST CELL INTERACTIONS BY THE NONSTRUCTURAL PROTEIN 1 (NS1) OF INFLUENZA A VIRUS LEENA YLÖSMÄKI UNIVERSITY OF HELSINKI FACULTY OF MEDICINE SH3 domain mediated virus-host cell interactions by the nonstructural protein 1 (NS1) of influenza A virus Department of Virology Faculty of Medicine University of Helsinki Finland Leena Ylösmäki ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Medicine, University of Helsinki, for public examination in the Lecture Hall 2, Haartmaninkatu 3, on June 28th, 2016, at 12 noon. Helsinki 2016 SUPERVISED BY: Kalle Saksela, MD, PhD, Professor Department of Virology Faculty of Medicine University of Helsinki Finland REVIEWED BY: Tero Ahola, PhD, Docent Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki Finland Denis Kainov, PhD, Docent Institute for Molecular Medicine Finland (FIMM) University of Helsinki Finland OPPONENT: Varpu Marjomäki, PhD, Docent Department of Biological and Environmental Science Faculty of Mathematics and Science University of Jyväskylä Finland ISBN 978-951-51-2283-4 (paperback) ISBN 978-951-51-2284-1 (PDF) Picaset Oy http://ethesis.helsinki.fi Helsinki 2016 2 To my family 3 Table of Contents LIST OF ORIGINAL PUBLICATIONS .................................................................................. 7 ABSTRACT ..................................................................................................................... 8 ABBREVIATIONS ...........................................................................................................
    [Show full text]
  • Mutagenic Analysis of SH3 Binding Site in the Avian Chb6 Alloantigen
    DePaul University Via Sapientiae College of Science and Health Theses and Dissertations College of Science and Health Fall 11-22-2016 Mutagenic Analysis of SH3 Binding Site in the Avian ChB6 Alloantigen Rohini Bhattacharya DePaul University, [email protected] Follow this and additional works at: https://via.library.depaul.edu/csh_etd Part of the Biology Commons Recommended Citation Bhattacharya, Rohini, "Mutagenic Analysis of SH3 Binding Site in the Avian ChB6 Alloantigen" (2016). College of Science and Health Theses and Dissertations. 200. https://via.library.depaul.edu/csh_etd/200 This Thesis is brought to you for free and open access by the College of Science and Health at Via Sapientiae. It has been accepted for inclusion in College of Science and Health Theses and Dissertations by an authorized administrator of Via Sapientiae. For more information, please contact [email protected]. “Mutagenic Analysis of SH3 Binding Site in the Avian ChB6 Alloantigen” A Thesis Presented in Partial Fulfillment of the Requirement for the Degree of Master of Science Oct 2016 By Rohini Bhattacharya Department of Biological Science College of Science and Health DePaul University Chicago, Illinois 1 Table of contents: Introduction 5 Literature Review 7 B Lymphocytes 7 Problems of antibody 11 Cell signaling 13 SLAM 17 Src Family kinases 19 Bursa of Fabricius 21 chB6 23 Statement of purpose 26 Materials and method 28 Results 33 Discussion 37 References 48 Figure legends 54 Figures 59 2 List of figures Figure # Figure Page # 1 VDJ recombination 59 2 Structure of antibody 60 3 chB6 sequence 61 4 P282A and P279A P282A enzyme digest 62 Table 1 Percent immunofluorescences after anti chB6 63 and FITC staining.
    [Show full text]