title: The substrate specificity of the catalytic domain of Abl plays an important role in directing phosphorylation of the adaptor protein Crk.
abstract: c-Abl preferentially phosphorylates peptide substrates that contain proline at the P+3 site (relative to the phosphorylated tyrosine). We previously described a mutant form of the Abl catalytic domain (Y569W) with altered substrate specificity at the P+3 position, as measured using synthetic peptides. In this study, we examine the phosphorylation of Crk, a protein substrate of Abl that is phosphorylated in the sequence Tyr221-Ala-Gln-Pro. In vitro, 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, indicating that not all cellular proteins depend on Pro at P+3 for Abl recognition. However, phosphorylation of Crk by Y569W Abl in these cells is markedly 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 assay conditions. Thus, proper phosphorylation of Crk by Abl depends not only on the interaction of the Crk SH3 domain with the Abl polyproline region, but also on the recognition of amino acids surrounding tyrosine by the Abl catalytic domain.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, USA.
authors.forename: M
authors.initials: M
authors.lastname: Amoui
authors.forename: W T
authors.initials: WT
authors.lastname: Miller
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002461:Cell Line, Transformed; D002645:Chickens; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D013379:Substrate Specificity
chemical_list: D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011494:Protein Kinases; D016315:Proto-Oncogene Proteins c-abl