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

描述(由申请人提供)：我们广泛的、长期的目标是在分子水平上了解抗癌药物的作用机制，总体目标是改进目前的化疗并确定新的靶点和药物。Bcl2蛋白在微管抑制物诱导的细胞凋亡中所起的作用是人们特别感兴趣的。BCL-2蛋白受到复杂的调控，其中最显著的翻译后修饰之一是微管抑制剂对BCL-XL和BCL-2的磷酸化。然而，这些修饰和负责的激酶(S)的作用仍然很不清楚，尽管它们作为微管功能障碍和诱导细胞凋亡之间的机制联系具有潜在的重要性。用KB-3细胞进行的初步研究表明，长春花碱诱导的Bclxl和Bcl2的磷酸化是由同一个激酶催化的协同事件，它们的去磷酸化与细胞凋亡的启动有关。我们的假设是，Bclxl/Bcl2磷酸化是控制抗分裂药物诱导细胞凋亡的关键事件，并且是由微管功能障碍时激活的一种新的激酶催化的。在特定目的1中，将通过蛋白质纯化、胰酶消化和质谱分析来确定长春新碱作用于KB-3细胞后，Bc l-xl和Bc l-2的磷酸化位点。在特定的目标2中，将通过表达磷酸化缺陷或磷酸化模拟分子和检测细胞对长春花碱的敏感性来评估Bclxl/Bcl2磷酸化在长春花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Bclxl或Bcl2亚细胞定位或蛋白质/蛋白质相互作用。在特定的目标4中，将纯化和鉴定长春花碱激活的Bclxl/Bcl2激酶，长期目标是了解其在细胞对微管损伤的反应中的功能和调节。本研究将对长春新碱和其他抗有丝分裂药物的作用机制提供新的见解，并为研究Bcl2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更好的化疗药物、新的药物靶点和克服肿瘤细胞对这些药物的耐药性的方法。

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.