Jak2 involvement in Bcr-Abl oncogenic transformation

描述(由申请人提供)：Janus Kinase(JAK)家族在细胞因子刺激的活动中起着关键作用。JAK2通过与IL-3/GM-CSF受体的配体结合而激活。IL-3依赖的细胞系在转导bcr-Abl后，可以产生不再需要IL-3生长和存活的细胞克隆。我们已经研究了JAK2与BCR-Abl的相互作用，并正在研究JAK2激酶激活对BCR-Abl致癌作用的影响。我们的发现表明，BCR-Abl通过酪氨酸1007的磷酸化来激活JAK2，酪氨酸1007是激活JAK2酪氨酸激酶所需的残基。JAK2的一种激酶失活形式干扰了bcr-abl的致癌作用。为了确定与BCR-Abl激活JAK2相关的信号转导途径，我们从小鼠髓系32Dp210细胞中寻找JAK2/BCR-Abl复合体中的其他蛋白质，这些蛋白质通过BCR-Abl的表达而使IL-3独立。JAK2抗体免疫沉淀/Western blotting研究在这些JAK2/bcr-Abl复合体中检测到其他几种蛋白质。它们包括SH2-Bbeta、p56DOK2、p56Lyn和CrkL；这些蛋白也是酪氨酸磷酸化的。令人惊讶的是，在这些复合体中没有检测到Stat蛋白。重要的是，32Dp210细胞中常见的IL-3/GM-CSF受体a链也被酪氨酸磷酸化。已知这些受体参与上调c-Myc的表达。在这方面，我们发现在使用JAK2激酶抑制剂(AG490)和显性阴性形式的JAK2和SH2-Bβ的研究中，JAK2激酶是增强c-Myc蛋白表达所必需的。由于c-Myc是BCR-Abl致癌所必需的，我们推测JAK2/BCR-Abl复合体在缺乏细胞因子的情况下通过激活IL-3/GM-CSF受体而增强c-Myc的表达。其具体目的是：1)研究JAK2对bcr-Abl诱导c-Myc的需求；2)研究JAK2/bcr-Abl诱导c-Myc的下游途径；3)研究IL-3/GM-CSF受体在BCR-Abl诱导NIH3T3细胞向允许表型转化致癌过程中的作用；NIH3T3细胞对BCR-Abl诱导的病灶形成具有抵抗力；我们将探讨α/β受体链在BCR-Abl诱导的病灶形成和通过受体链突变诱导c-Myc中的作用；4)通过包括蛋白质组学方法在内的免疫学和结构研究，鉴定与JAK2和BCR-Abl共沉淀的蛋白质；探索JAK2/BCR-Abl相关蛋白(如使用DNSH2-Bbeta)在BCR-Ab1诱导的致癌效应中的功能作用。这些研究将为研究bcr-Abl阳性白血病的发病机制提供重要的新信息，并将为这类白血病的治疗提供新的策略。

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.