Proline-rich sequences that bind to Src homology 3 domains with individual specificities.

Abstract

To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in Protein-protein interactions are central events in cellular signal transduction. These interactions are often mediated by noncatalytic conserved domains, three of which are the Src homology regions 2 and 3 (SH2 and SH3), first identified as part of the Rous sarcoma viral oncogene product (1, 2), and the plekstrin homology (PH) domain, more recently identified by protein sequence alignments (3, 4). To date, the best understood protein interactions are those mediated by the SH2 domains, which bind to phosphotyrosine residues on proteins that have been primed for signaling through phosphorylation.The function of SH3 domains is less clear. In relation to the oncogenic potential of protein-tyrosine kinases, these domains play an inhibitory role because mutation or deletion of them activates the transforming potential of c-Src and c-Abl protooncoproteins (5-7). The first clue to the function of SH3 domains was obtained by the cloning of fragments of two protein ligands for the Abl SH3: 3BP1 and 3BP2 (8). In these proteins, proline-rich peptides only 10 aa long serve as targets of SH3 binding (9). Since then, a number of interactions between SH3 domains and proline-rich ligands have been described, showing that SH3 domains are involved in epidermal growth factor receptor signaling (10), cellular localization of cytoplasmic proteins (11), upregulation of the GTPase activity of dynamin (12), and activation of phosphatidylinositol 3-kinase in response to IgM crosslinking (13).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.From these and other reports it is now clear that interactions between SH3s and their ligands occur through proline-rich sites. It is less clear, however, how the specificity of interaction is achieved. In this study we report the isolation of peptide fragments from proteins that exhibit differential binding specificity to various SH3s. Specificity of interaction is mediated by unique sequences that interact with binding pockets found on the surface of SH3 domains. Our data correlate well with recent reports that identify specific artificial ligands for different SH3 domains from chemically synthesized and phage display libraries (14-16) and suggest that these ligands do occur in vivo. MATERIALS AND METHODSLibrary Screening. A 16-day mouse embryo cDNA expression library (Novagen) was screened for SH3-binding proteins according to the manufacturer's protocols and as previously described (8). Biotinylated glutathione S-transfe...

Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity

“…Because fragment analysis has demonstrated that the COOH-terminal portion of synapsin I is involved in binding the SH3 domains of c-Src (this Relative to the homogenate, Src was enriched in highly purified vesicle fractions, and its association with the vesicle membrane was not affected by the salt treatment used to deplete synapsin I. H, homogenate; S1, postnuclear supernatant; S2, supernatant of P2; P2, crude synaptosomes; LP1, crude synaptic plasma membranes; LS1, supernatant of LP1; LP2, crude synaptic vesicles; LS2, synaptosol; SG2, synaptic vesicles; SG4, small synaptic membranes; USV, untreated synaptic vesicles; STSV, salt-treated synaptic vesicles. Analysis by screening libraries of phage-displayed or synthetic peptides has shown that the c-Src-SH3 domain exhibits a preference for the class I binding motif, RPLPPLP (26)(27)(28)(29), although it can also bind to class II ligands (28,30). In this respect, class II ligands have been reported to be more promiscuous in their binding to SH3 domains than class I ligands, and they exhibit more flexibility in the distance between the PXXP motif and the neighboring arginine residue, a property that can contribute to a broader specificity of this interaction (28,31).…”

Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor

“…SH2 domains bind phosphotyrosine moieties (Pawson, 1995). SH3 domains, which often accompany SH2 domains, recognize proline-rich sequences in target proteins (Alexandropoulos et al, 1995;Mayer and Baltimore, 1993;Pawson, 1995). Adapter proteins, including Crk, Nck and Grb2, are composed almost exclusively of SH2 and SH3 domains and lack any enzymatic or other functional activities (Mayer and Baltimore, 1993;Pawson, 1995).…”

Insights into Human Lck SH3 Domain Binding Specificity: Different Binding Modes of Artificial and Native Ligands

“…At least one peptide without a basic residue, actually with a tyrosine, at position P -3 was described that binds to the SH3 domain of Fyn. The 3BP2-derived peptide PPAYPPPPVP, which is a physiological Abl ligand, is able to bind Fyn SH3 (49), although this may not be physiologically relevant (50).…”