BCR-ABL is a constitutively active tyrosine kinase that is the causative abnormality of chronic myelogenous leukemia (CML). A number of signaling proteins are activated by the association with and phosphorylation by BCR-ABL. Proteins known to associate directly with BCR-ABL include Grb2, c-Cbl, p62Dok and CrkL. It has been shown jhat individual mutations in BCR-ABL of the binding sites for Grb2, c-Cbl, p62Dok, and CfkL are able to abolish the direct interactions with these signaling proteins, but do not affect the transforming ability of BCR-ABL in myeloid cells. This may be because these provins are still able to co-immunoprecipitate with BCR-ABL via indirect associations. Thus, Jue to the complexity of BCR-ABL signaling and lack of a one to one correlation betweejn a direct binding site and a specific signaling protein, we made single, double and triple mutants of BCR-ABL for the Grb2, c-Cbl and CrkL binding sites. Stable myeloid cell lines (32D and BAF3) were generated expressing p210 BCR-ABL, p 185 BCR-ABL, single mutants, pairwise combinations of the double mutants and triple mutants. Cell proliferation assays were performed in the presence and absence of WeHI (an IL3 source) to assess growth factor requirements. Expression of the p210 or p 185 triple mutants in 32D cells does not confer growth factor independence, however BAF3 cells expressing the triple mutant are growth factor independent. Lysates from both of these cell lines were analyzed by immunoprecipitation and immunoblotting and demonstrated no association of BCRABL with c-Cbl, Grb2 or CrkL. Interestingly, Cbl remains highly tyrosine phosphorylated despite lack of direct interaction with BCR-ABL. Thus, our data demonstrates that Cbl phosphorylation is not dependent on a direct interaction with BCR-ABL and suggests that another kinase, activated by BCR-ABL, may be responsible for Cbl phosphorylation. Furthermore, phosphorylation of Cbl does not correlate with growth factor independence by BCR-A3L, and suggests that Cbl tyrosine phosphorylation is not required for growth factor independence. Analysis of other signaling proteins, such as STATS, p62Dok and PI3-kinase are ongoing. Evaluation of these mutants will be used to determine which associations, phosphorylation events, and pathways are required for BCR-ABL mediated transformation.
|Original language||English (US)|
|Issue number||11 PART I|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology