Jak2 involvement in Bcr-Abl oncogenic transformation

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DESCRIPTION (provided by applicant): The Janus kinase (Jak) family has a critical role in cytokine-stimulated activities. Jak2 is activated by ligand binding to the IL-3/GM-CSF receptors. IL-3 dependent cell lines when transduced with Bcr-Abl can yield cell clones that no longer require IL-3 for growth and survival. We have investigated Jak2 interaction with Bcr-Abl and are studying the effects of Jak2 kinase activation with regards to the oncogenic effects of Bcr-Abl. Our findings indicate that Bcr-Abl activates Jak2 by phosphorylation of tyrosine 1007, a residue required for activation of the Jak2 tyrosine kinase. A kinase-inactive form of Jak2 interferes with the oncogenic effects of Bcr-Abl. To determine the signal transduction pathways involved with Jak2 activation by Bcr-Abl, we searched for other proteins in the Jak2/Bcr-Abl complex from mouse myeloid 32Dp210 cells, which are rendered IL-3 independent by means of Bcr-Abl expression. Jak2 antibody immunoprecipitation/Western blotting studies detected several other proteins in these Jak2/Bcr-Abl complexes. They include SH2-Bbeta, p56 DOK2, p56 LYN and CrkL; these proteins are tyrosine-phosphorylated as well. Surprisingly, no Stat proteins were detected in these complexes. Importantly, the common a chain of the IL-3/GM-CSF receptors was also tyrosine-phosphorylated in 32Dp210 cells. These receptors are known to be involved in elevating c-Myc expression. In this regard we found that the Jak2 kinase was required for enhancing the expression of the c-Myc protein in studies using a Jak2 kinase inhibitor (AG490) and dominant negative forms of Jak2 and SH2-Bbeta. Since c-Myc is required for the oncogenic effects of Bcr-Abl, we hypothesize that the Jak2/Bcr-Abl complex is critical for enhancing the expression of c- Myc by activating the IL-3/GM-CSF receptors in the absence cytokines. The specific aims are: 1) Investigate the requirement of Jak2 for the induction of c-Myc by Bcr-Abl; 2) Investigate the down-stream pathways involved in c-Myc induction through Jak2/Bcr-Abl; 3) Investigate the role of the IL-3/GM-CSF receptors in the conversion of NIH 3T3 cells to a permissive phenotype for oncogenic transformation by BCR-ABL; NIH 3T3 cells are resistant to foci-formation caused by Bcr-Abl; we will investigate the role alpha/beta receptor chains in Bcr-Abl induced foci-formation and c-Myc induction by mutagenesis of the receptor chains; 4) Characterize the proteins that co-immunoprecipitate with Jak2 and Bcr-Abl by immunological and structural studies including proteomic methods; explore the functional roles of the Jak2/Bcr-Abl associated proteins (e.g. use of DN SH2-Bbeta) in Bcr-Abl-induced oncogenic effects. These studies will provide important new information on mechanism of Bcr-Abl positive leukemia, and will provide new strategies for therapy of these leukemias.