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 studies have shown that the SH3 domains from several proteins recognize short, contiguous amino acid sequences that are rich in proline residues. While all SH3 recognition sequences identified to date share a conserved P-X-X-P motif, the sequence recognition specificity of individual SH3 domains is poorly understood. We have employed a novel modification of phage display involving biased libraries to identify peptide ligands of the Src, Fyn, Lyn, PI3K and Abl SH3 domains. With biased libraries, 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 RXXRPLPPLPP while the Abl SH3 domain selects phage containing the sequence PPPYPPPP(I/V)PXX. We have also analysed the binding properties of Abl and Src SH3 ligands. We find that although the phage-displayed Abl and Src SH3 ligands are proline rich, they are distinct. In surface plasmon resonance binding assays, these SH3 domains displayed highly selective binding to their cognate ligands when the sequences were displayed on the surface of the phage or as synthetic peptides. The selection of these high affinity SH3 peptide ligands provides valuable information on the recognition motifs of SH3 domains, serve as new tools to interfere with the cellular functions of SH3 domain-mediated processes and form the basis for the design of SH3-specific inhibitors of disease pathways.
Rickles
R J
RJ
ARIAD Pharmaceuticals, Cambridge, MA 02139.
Botfield
M C
MC
Weng
Z
Z
Taylor
J A
JA
Green
O M
OM
Brugge
J S
JS
Zoller
M J
MJ
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D015698:Genomic Library; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases
D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl; D051076:Proto-Oncogene Proteins c-fyn; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases