Catalytic (SH1) domains of protein tyrosine kinases (PTKs) demonstrate specificity for peptide substrates. Whether SH1 domains differentiate between tyrosines in a physiological substrate has not been confirmed. Using purified proteins, we studied the ability of Syk, Fyn, and Abl to differentiate between tyrosines in a common PTK substrate, c-Cbl. We found that each kinase produced a distinct pattern of c-Cbl phosphorylation, which altered the phosphotyrosine-dependent interactions between c-CbI and CrkL or phosphatidylinositol 3'-kinase (PI3K). Our data support the concept that SH1 domains determine the final sites of phosphorylation once PTKs reach their target proteins. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.