Distinct ligand preferences of Src homology 3 domains from Src, Yes, Abl, Cortactin, p53bp2, PLCgamma, Crk, and Grb2.

Abstract

Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These (14) used a synthetic peptide library to identify Src and P13K SH3 ligands with the consensus sequence RXLPPZP (Z = L for Src and R for P13K) and demonstrated that each SH3 domain bound its respective ligand with higher affinity than did the other.In an effort to develop a more detailed understanding of the specificity of SH3-ligand interactions, we have constructed a phage-displayed library (termed the PXXP library) encoding peptides of the form X6PXXPX6, where X represents any of the 20 naturally occurring amino acids and P represents invariant proline residues. Using this library, we have identified peptide ligands for SH3 domains of Src, Yes, Abl, Cortactin, p53bp2, PLCy, Crk, and Grb2. Each SH3 domain selects a set of peptide ligands sharing a distinct consensus motif; these motifs reflect the unique ligand preferences of each SH3 domain. MATERIALS AND METHODSPreparation of Glutathione S-Transferase (GST)-SH3 Fusion Proteins. Constructs encoding GST fusions to the Grb2 N-terminal (Grb2 N, aa 1-58), Grb2 C-terminal (Grb2 C, aa 154-217), Nck N-terminal (Nck N, aa 1-68), Nck middle (Nck M, aa 101-166), Nck C-terminal (Nck C, aa 191-257), p53bp2 (aa 454-530), or Src (aa 87-143) SH3 domains were generated by PCR cloning of the appropriate cDNAs into pGEX-2T (Pharmacia). The integrity of the constructs was confirmed by DNA sequencing. pGEX-derived constructs expressing GST fusions to the SH3 domains of Yes, Cortactin, Crk, Abl, and PLC,y were kindly provided by M. Sudol (Rockefeller University), J. T. Parsons (University of Virginia), M. Matsuda

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G

“…However, all of the tested peptides bound very poorly or not at all to CrkSH3(1). Interestingly, some of these peptides bound with considerable a nity to the Grb2SH3(N) domain, which is in agreement with recent results from phage display screens that also demonstrated the binding of class-I peptides to GrbSH3(N) (Sparks et al, 1996). Furthermore, the corresponding D-amino acid peptides were tested for binding to CrkSH3(1).…”

“…SH2 domains usually bind to speci®c tyrosine-phosphorylated epitopes of proteins and many SH3 domains were shown to complex with amino acid motifs which are structurally similar to type II poly-proline-helices (Williamson, 1994;Feller et al, 1994b;Pawson, 1995). Additionally, basic amino acids can strongly contribute to the a nity and speci®city of binding to SH3 domains of proteins like Crk and Grb2 (Knudsen et al, 1994Sparks et al, 1996). In some of the SH3-binding sequences, the crucial basic residues are located at the C-terminus of the interacting sequence, resulting in the consensus motif PxxPxK/R (class II-peptides) (Feng et al, 1994;Sparks et al, 1996).…”

“…Additionally, basic amino acids can strongly contribute to the a nity and speci®city of binding to SH3 domains of proteins like Crk and Grb2 (Knudsen et al, 1994Sparks et al, 1996). In some of the SH3-binding sequences, the crucial basic residues are located at the C-terminus of the interacting sequence, resulting in the consensus motif PxxPxK/R (class II-peptides) (Feng et al, 1994;Sparks et al, 1996). On the other hand, class I-binding sites often contain basic residues in front of a proline-rich stretch (Feng et al, 1994;Sparks et al, 1996;Cheng et al, 1994;Weng et al, 1995).…”

“…In some of the SH3-binding sequences, the crucial basic residues are located at the C-terminus of the interacting sequence, resulting in the consensus motif PxxPxK/R (class II-peptides) (Feng et al, 1994;Sparks et al, 1996). On the other hand, class I-binding sites often contain basic residues in front of a proline-rich stretch (Feng et al, 1994;Sparks et al, 1996;Cheng et al, 1994;Weng et al, 1995). Some SH3 domains, including the SrcSH3 and Grb2SH3(N) domains, are able to bind peptides of both classes (Feng et al, 1994;Sparks et al, 1996).…”

“…On the other hand, class I-binding sites often contain basic residues in front of a proline-rich stretch (Feng et al, 1994;Sparks et al, 1996;Cheng et al, 1994;Weng et al, 1995). Some SH3 domains, including the SrcSH3 and Grb2SH3(N) domains, are able to bind peptides of both classes (Feng et al, 1994;Sparks et al, 1996). SH2 and SH3 domains are not only found as accessory protein interaction domains in various enzymes and structural proteins, they are also utilised as virtually exclusive building blocks of a class of signalling proteins known as the SH2/SH3 adapter family.…”