The substrate specificity of the catalytic domain of Abl plays an important role in directing phosphorylation of the adaptor protein Crk

c-Abl preferentially phosphorylates peptide substrates that contain proline at the P+3 site (relative to the phosphorylated tyrosine). We previously d escribed a mutant form of the Abl catalytic domain (Y569W) with altered sub strate specificity at the P+3 position, as measured using synthetic peptide s. In this study, we examine the phosphorylation of Crk, a protein substrat e of Abl that is phosphorylated in the sequence Tyr221-Ala-Gln-Pro. In vitr o, phosphorylation of Crk by Y569W Abl is greatly reduced relative to wild- type Abl. Overexpression of Y569W mutant Abl in 293T kidney cells produces a similar overall pattern of tyrosine phosphorylation as wild-type Abl, ind icating that not all cellular proteins depend on Pro at P+3 for Abl recogni tion. However, phosphorylation of Crk by Y569W Abl in these cells is marked ly reduced relative to wild-type Abl. A truncated form of Abl lacking the C -terminal polyproline region is not able to phosphorylate Crk in these assa y conditions. Thus, proper phosphorylation of Crk by Abl depends not only o n the interaction of the Crk SH3 domain with the Abl polyproline region, bu t also on the recognition of amino acids surrounding tyrosine by the Abl ca talytic domain. (C) 2000 Elsevier Science Inc. All rights reserved. (C) 200 0 Elsevier Science Inc. All rights reserved.