CATALYTIC SPECIFICITY OF PHOSPHOTYROSINE KINASES BLK, LYN, C-SRC AND SYK AS ASSESSED BY PHAGE DISPLAY

Protein tyrosine kinases (PTKs) are implicated in cell proliferation, differentiation, and receptor-mediated signalling events. Recruitment of intracellular PTKs into the signalling complex, often localized at the inner surface of the cell membrane, involves SH2 and SH3 domains a ttached to the catalytic kinase domain. While the interaction of SH2 a nd SH3 domains with their target sequences is well documented in a num ber of cases, the contribution of the catalytic domain itself in confe rring specificity to a given signal cascade is not fully understood. W e addressed this question and employed the phage display technique to assess the substrate requirements for the highly related Src-like PTKs c-Src, Blk, Lyn and the distantly related Syk. A diverse peptide libr ary on phage was established, and after multiple rounds of phosphoryla tion and selection of phage displaying phosphotyrosine-containing pept ides, canonical substrate sequences for each of the PTKs were enriched . The PTKs Blk and Lyn implicated in B cell signalling were found to p refer peptide substrates of the structure I/L-Y-D/E-X-L which resemble critical features of the ITAM motifs found in, e.g. the intracellular components Ig-alpha and Ig-beta of the beta cell receptor. All Src-li ke PTKs had a requirement for isoleucine or leucine in the position -1 with respect to the phosphorylated tyrosine residue in position 0. Wh ile Blk and Lyn had a strong preference for a negatively charged amino acid in position +1, c-Src preferred tryptophan or glycine in this po sition. Syk, not belonging to the Src-like PTK family, revealed a dist inct substrate requirement for aspartic acid in position -1 and glutam ic acid in position +1. In general, all PTKs we have tested had a stro ng preference for a particular amino acid in the positions -1 and +1 a djacent to the tyrosine residue. (C) 1996 Academic Press Limited