Jak2 involvement in Bcr-Abl oncogenic transformation

描述（由申请人提供）：Janus激酶（Jak）家族在细胞因子刺激活动中起关键作用。Jak2通过与IL-3/GM-CSF受体结合的配体激活。当用Bcr-Abl转导IL-3依赖性细胞系时，可以产生不再需要IL-3来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。我们的研究结果表明，Bcr-Abl通过酪氨酸1007的磷酸化激活Jak2，酪氨酸1007是Jak2酪氨酸激酶激活所需的残基。激酶失活形式的Jak2干扰Bcr-Abl的致癌作用。为了确定Bcr-Abl激活Jak2的信号转导途径，我们从小鼠骨髓32Dp210细胞中寻找Jak2/Bcr-Abl复合物中的其他蛋白，这些蛋白通过Bcr-Abl的表达而不依赖于IL-3。Jak2抗体免疫沉淀/Western blotting研究在这些Jak2/Bcr-Abl复合物中检测到其他几种蛋白质。它们包括SH2-Bbeta、p56 DOK2、p56 LYN和CrkL；这些蛋白质也是酪氨酸磷酸化的。令人惊讶的是，在这些复合物中没有检测到Stat蛋白。重要的是，在32Dp210细胞中，IL-3/GM-CSF受体的共同a链也被酪氨酸磷酸化。已知这些受体参与c-Myc表达的升高。在这方面，我们发现在使用Jak2激酶抑制剂（AG490）和Jak2和sh2 - β的显性阴性形式的研究中，Jak2激酶是增强c-Myc蛋白表达所必需的。由于c-Myc是Bcr-Abl的致癌作用所必需的，我们假设Jak2/Bcr-Abl复合物在缺乏细胞因子的情况下通过激活IL-3/GM-CSF受体来增强c-Myc的表达是至关重要的。具体目的是：1)研究Jak2对Bcr-Abl诱导c-Myc的要求；2)通过Jak2/Bcr-Abl研究参与c-Myc诱导的下游通路；3)研究IL-3/GM-CSF受体在NIH 3T3细胞转化为BCR-ABL致瘤转化的许可表型中的作用；NIH 3T3细胞对Bcr-Abl引起的病灶形成具有抗性；我们将研究α / β受体链在Bcr-Abl诱导的病灶形成和通过受体链诱变诱导c-Myc中的作用；4)通过免疫学和结构研究（包括蛋白质组学方法）表征与Jak2和Bcr-Abl共免疫沉淀的蛋白；探索Jak2/Bcr-Abl相关蛋白（例如使用DN SH2-Bbeta）在Bcr-Abl诱导的致癌作用中的功能作用。这些研究将为Bcr-Abl阳性白血病的发生机制提供重要的新信息，并为这些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.