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 phosphotyrosine 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 (PTK) p55(fyn) (Fyn). Peptide libraries were incubated for a short time in the presence of Fyn and phages displaying efficiently phosphorylated peptides were selected by panning over anti-phosphotyrosine antibodies. The characterization of the peptides enriched after three phosphorylation/selection rounds allowed us to define a canonical substrate sequence for the kinase Fyn, E-(phi/T)YGx phi, where phi represents any hydrophobic residue. A peptide conforming to this sequence is a better substrate than a second peptide designed to be in accord with the consensus sequence recognised by the Fyn SH2 domain. When the library phosphorylation reaction is carried out in saturation conditions, practically all the tyrosine containing peptides are phosphorylated, irrespective of their context. These "fully modified" peptide libraries are a valuable tool to study the specificity of phosphotyrosine mediated protein recognition. We have used this new tool to identify a family of peptides that bind the PTB domain of the adapter protein Shc. Comparison of the peptide sequences permits us to confirm the essential role 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 productive contacts, at least in linear peptides.
Dente
L
L
Dipartimento di Biologia Università di Roma Tor Vergata, Rome, Italy.
Vetriani
C
C
Zucconi
A
A
Pelicci
G
G
Lanfrancone
L
L
Pelicci
P G
PG
Cesareni
G
G
D016384:Consensus Sequence; D005822:Genetic Vectors; D017911:Inoviridae / Q000235:genetics; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism*; D010748:Phosphopeptides; D010766:Phosphorylation; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D012641:Selection, Genetic; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
D019151:Peptide Library; D010455:Peptides; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D051076:Proto-Oncogene Proteins c-fyn