Direct binding of CRKL to BCR-ABL is not required for BCR-ABL transformation.
CRKL has previously been shown to be a major tyrosine phosphorylated protein in neutrophils of patients with BCR-ABL+ chronic myelogenous leukemia and in cell lines expressing BCR-ABL CRKL and BCR-ABL form a complex as demonstrated by coimmunoprecipitation and are capable of a direct interaction in a yeast two-hybrid assay. We have mapped the site of interaction of CRKL and BCR-ABL to the amino terminal SH3 domain of CRKL with a proline rich region in the C-terminus of ABL. The proline-rich region was mutated and the effect of this deletion on BCR-ABL transforming function was assayed. Our data show that this deletion does not impair the ability of BCR-ABL to render myeloid cells factor independent for growth. In cells expressing the proline deletion mutation of BCR-ABL, CRKL is still tyrosine phosphorylated and forms a complex with BCR-ABL as demonstrated by coimmunoprecipitation. Our data suggest that the interaction between CRKL and the proline deletion mutant of BCR-ABL is an indirect interaction as CRKL does not interact directly with the proline deletion mutant of BCR-ABL in a gel overlay assay or in a yeast two-hybrid assay. Thus, a direct interaction of CRKL and BCR-ABL is not required for CRKL to become tyrosine phosphorylated by BCR-ABL and suggests that CRKL function may still be required for BCR-ABL function through an indirect interaction.
Heaney
C
C
Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland 97201, USA.
Kolibaba
K
K
Bhat
A
A
Oda
T
T
Ohno
S
S
Fanning
S
S
Druker
B J
BJ
D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D002455:Cell Division; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D016044:Fusion Proteins, bcr-abl