IDENTIFICATION OF SRC, FYN, LYN, PI3K AND ABL SH3 DOMAIN LIGANDS USING PHAGE DISPLAY LIBRARIES

Many proteins involved in intracellular signal transduction contain a small, 50-60 amino acid domain, termed the Src homology 3 (SH3) domain . This domain appears to mediate critical protein-protein interactions that are involved in responses to extracellular signals. Previous stu dies have shown that the SH3 domains from several proteins recognize s hort, contiguous amino acid sequences that are rich in proline residue s. While all SH3 recognition sequences identified to date share a cons erved P-X-X-P motif, the sequence recognition specificity of individua l SH3 domains is poorly understood. We have employed a novel modificat ion of phage display involving biased libraries to identify peptide li gands of the Src, Fyn, Lyn, PI3K and Abl SH3 domains. With biased libr aries, we probed SH3 recognition over a 12 amino acid window. The Src SH3 domain prefers the sequence XXXRPLPPLPXP, Fyn prefers XXXRPLPP(I/L )PXX, Lyn prefers RXXRPLPPLPXP, PI3K prefers RXXRPLPPLPPP while the Ab l SH3 domain selects phage containing the sequence PPPYPPPP(I/V)PXX. W e have also analysed the binding properties of Abl and Src SH3 ligands . We find that although the phage-displayed Abl and Src SH3 ligands ar e proline rich, they are distinct. In surface plasmon resonance bindin g assays, these SH3 domains displayed highly selective binding to thei r cognate ligands when the sequences were displayed on the surface of the phage or as synthetic peptides. The selection of these high affini ty SH3 peptide ligands provides valuable information on the recognitio n motifs of SH3 domains, serve as new tools to interfere with the cell ular functions of SH3 domain-mediated processes and form the basis for the design of SH3-specific inhibitors of disease pathways.