title: Tyrosine 207 in CRKL is the BCR/ABL phosphorylation site.
abstract: BCR/ABL has a causal role in Philadelphia (Ph)-chromosome positive leukemia. The SH2/SH3 adapter protein CRKL is a major substrate of the deregulated BCR/ABL tyrosine kinase and is aberrantly tyrosine-phosphorylated in Ph-positive leukemia cells. In this study, experiments were pursued to identify and analyse the CRKL phosphorylation site(s). In an in vitro kinase assay, CRKL phosphorylation by the abl kinase was limited to a small region between the two CRKL SH3 domains. Within this region, mutation of tyrosine residue 207 yielded a mutant CRKL which could not be phosphorylated by BCR/ABL. Stable overexpression of CRKL or CRKL-Y207F did not transform NIH3T3 cells, while the Y207F mutation eliminated tyrosine-phosphorylation of CRKL. These studies indicate that Y207 in CRKL represents the major in vivo phosphorylation site. Phosphorylation of Y207 provides a binding site for the CRKL SH2 domain and potentially for other SH2-containing proteins. The Y207F mutation in CRKL did not enhance or decrease association with various target signalling proteins, including SOS or C3G, which interact specifically with the CRKL N-SH3 domain. These findings suggest that complex formation with cellular targets is not modulated by CRKL tyrosine-phosphorylation.
authors.affiliation: Department of Pathology, Childrens Hospital of Los Angeles, CA 90027, USA.
authors.forename: R
authors.initials: R
authors.lastname: de Jong
authors.forename: J
authors.initials: J
authors.lastname: ten Hoeve
authors.forename: N
authors.initials: N
authors.lastname: Heisterkamp
authors.forename: J
authors.initials: J
authors.lastname: Groffen
mesh_terms: D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl