MODIFIED PHAGE PEPTIDE LIBRARIES AS A TOOL TO STUDY SPECIFICITY OF PHOSPHORYLATION AND RECOGNITION OF TYROSINE-CONTAINING PEPTIDES

Tyrosine phosphorylation and protein recognition, mediated by phosphot yrosine containing peptides, play an important role in determining the specific response of a cell, when stimulated by external signals. We have used peptide repertoires displayed by filamentous phage as a tool to study the substrate specificity of the protein tyrosine kinase (PT K) p55(fyn) (Fyn). Peptide libraries were incubated for a short time i n the presence of Fyn and, phages displaying efficiently phosphorylate d peptides were selected by panning over anti-phosphotyrosine antibodi es. The characterization of the peptides enriched after three phosphor ylation/selection rounds allowed us to define a canonical substrate se quence for the kinase Fyn, E-(phi/T)YG chi phi, where phi represents a ny hydrophobic residue. A peptide conforming to this sequence is a bet ter substrate than a second peptide designed to be in accord with the consensus sequence recognised by the Fin SH2 domain. When the library phosphorylation reaction is carried out in saturation conditions, prac tically all the tyrosine containing peptides are phosphorylated, irres pective of their context. These ''fully modified'' peptide libraries a re a valuable tool to study the specificity of phosphotyrosine mediate d protein recognition. We have used this new tool to identify a family of peptides that bind the PTB domain of the adapter protein She. Comp arison of the peptide sequences permits us to confirm the essential re lic of N at position -3, while P often found at position -2 in natural targets is not absolutely required. Furthermore, our approach permits us to reveal an ''extended'' consensus indicating that residues that do not seem to influence binding in natural peptides can make producti ve contacts, at least in linear-peptides. (C) 1997 Academic Press Limi ted.