BcL-2 Proteins in Mechanism of Anti-mitotic Drug Action

描述（由申请人提供）：我们广泛的长期目标是在分子水平上了解抗癌药物的作用机制，总体目标是改善目前的化疗和鉴定新的靶点和药物。Bcl-2蛋白在微管相关蛋白诱导的细胞凋亡中所起的作用特别令人感兴趣。Bcl-2蛋白受到复杂的调节，并且最突出的翻译后修饰之一是响应微管抑制剂而发生的Bcl-xL和Bcl-2的磷酸化。然而，这些修饰和负责的激酶的作用在很大程度上仍然是模糊的，尽管它们作为微管功能障碍和凋亡诱导之间的机制联系的潜在重要性。使用KB-3细胞的初步研究已经表明，长春碱诱导的Bcl-xL和Bcl-2的磷酸化是由相同的激酶催化的协调事件，并且它们的去磷酸化与凋亡起始相关。我们的假设是Bcl-xL/Bcl-2磷酸化是控制抗有丝分裂药物诱导凋亡的关键事件，并由响应微管功能障碍而激活的新型激酶催化。在特定目标1中，将通过蛋白质纯化、胰蛋白酶消化和质谱分析来鉴定响应于KB-3细胞的长春碱处理的Bcl-xL和Bcl-2中的磷酸化位点。在特定目标2中，将通过表达磷酸化缺陷或磷酸化模拟分子并检查细胞对长春碱的敏感性来评价Bcl-xL/Bcl-2磷酸化在长春碱诱导的细胞凋亡中的作用。在具体目标3中，将进行机制研究以确定磷酸化是否影响Bcl-xL或Bcl-2亚细胞定位或蛋白质/蛋白质相互作用。在具体目标4中，将对长春碱激活的Bcl-xL/Bcl-2激酶进行纯化和表征，其长期目标是了解其在微管损伤细胞反应中的功能和调节。本研究将为Bcl-xL/Bcl-2磷酸化在长春碱和其他抗有丝分裂药物作用机制中的作用提供新的见解，并为Bcl-2蛋白功能的调控机制提供重要的基础信息。这些发现反过来将有助于寻找上级化疗药物、新的药物靶点和克服肿瘤细胞对这些药物耐药性的方法。

DESCRIPTION (provided by applicant): Our broad, long-term objective is to understand on a molecular level the mechanism of action of anticancer drugs with an overall goal of improving current chemotherapy and identification of novel targets and drugs. The role that Bcl-2 proteins play in microtubule inhibitor-induced apoptosis is of special interest. Bcl-2 proteins are subject to complex regulation, and one of the most prominent post-translational modifications is the phosphorylation of Bcl-xL and Bcl-2 occurring in response to microtubule inhibitors. However, the role of these modifications and the kinase(s) responsible have remained largely obscure, despite their potential importance as a mechanistic link between microtubule dysfunction and apoptosis induction. Preliminary studies using KB-3 cells have indicated that vinblastine-induced phosphorylation of Bcl-xL and Bcl-2 are coordinated events catalyzed by the same kinase and that their dephosphorylation correlates with apoptosis initiation. Our hypothesis is that Bcl-xL/Bcl-2 phosphorylation is a key event controlling apoptosis induction by antimitotic drugs and is catalyzed by a novel kinase activated in response to microtubule dysfunction. In Specific Aim 1, the sites of phosphorylation in Bcl-xL and Bcl-2 in response to vinblastine treatment of KB-3 cells will be identified by protein purification, trypsin digestion, and mass spectrometry analysis. In Specific Aim 2, the role of Bcl-xL/Bcl-2 phosphorylation in vinblastine-induced apoptosis will be evaluated by expressing phosphorylation-defective or phosphorylation-mimic molecules and examining cellular sensitivity to vinblastine. In Specific Aim 3, mechanistic studies will be performed to determine whether phosphorylation affects Bcl-xL or Bcl-2 subcellular localization or protein/protein interactions. In Specific Aim 4, the vinblastine-activated Bcl-xL/Bcl-2 kinase will be purified and characterized, with a long-term goal of understanding its function and regulation in the cellular response to microtubule damage. This study will provide novel insight into the role of Bcl-xL/Bcl-2 phosphorylation in the mechanism of action of vinblastine and other antimitotic drugs and provide important basic information on regulatory mechanisms of Bcl-2 protein function. These findings in turn will aid in the search for superior chemotherapeutic drugs, new drug targets, and methods to overcome tumor cell resistance to these agents.