SITE-SPECIFICITY OF P72(SYK) PROTEIN-TYROSINE KINASE - EFFICIENT PHOSPHORYLATION OF MOTIFS RECOGNIZED BY SRC HOMOLOGY-2 DOMAINS OF THE SRC FAMILY

Protein tyrosine kinase p72(syk) purified from rat spleen has been assayed for its ability to phosphorylate a number of peptide substrates derived from naturally occurring phospho-acceptor sites, The phosphorylation efficiency is extremely variable, depending on the peptide sequence, with K-m values in the 3-1500 mu M range. The by far best peptide substrates, with K-m values of 3 and 4 mu M are those reproducing the phospho-acceptor sites of Vav and HS1 proteins, respectively, These sites include multiple acidic residues flanking tyrosine on both sides and they also display the consensus sequences (YEDL and YEEV) preferred by the SH2 domains of the Src family, Alteration of this consensus in the HS1. peptide, by replacing either the glutamic acid or valine, also reduces the phosphorylation efficiency by p72(syk), Also the replacement of acidic residues at position -1 and, to a lesser extent at positions -3 and -4 (but not at positions +3 and +5) are detrimental, These observations may suggest a role of p72(syk) in the recruitment of ligands/substrates for the Src family enzymes. We also show that the HS1 peptide can be used for the specific monitoring of p72(syk) since neither the two Src-related c-Fgr and Lyn kinases (needing a hydrophobic instead of acidic residue at position -1) nor CSK appreciably phosphorylate it.