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)-gamma1, bound with moderate affinities (10-100 microm) to a group of non-conventional peptides. Of particular interest, the PLC-gamma1 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-gamma1 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.
Jia
Christina Y H
CY
Department of Biochemistry, Schulich School of Medicine, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Nie
Jing
J
Wu
Chenggang
C
Li
Chengjun
C
Li
Shawn S-C
SS
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D020816:Amino Acid Motifs; D001665:Binding Sites; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis*; D010750:Phosphoproteins / Q000378:metabolism*; D011392:Proline; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D018909:src Homology Domains / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D010446:Peptide Fragments; D010750:Phosphoproteins; D011506:Proteins; C092947:SLP-76 signal Transducing adaptor proteins; D011392:Proline; D005982:Glutathione Transferase