BCL-2 Function in Endoplasmic Reticulum

BCL-2 在内质网中的功能

• 批准号: 7031020
• 负责人: CLARK W DISTELHORST
• 金额: $ 25.4万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-03 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031020
• 关键词: BCL2 gene /protein; T cell receptor; apoptosis; calcineurin; calcium channel; calcium flux; carbohydrate receptor; cell cycle proteins; cyclin dependent kinase; endoplasmic reticulum; enzyme inhibitors; fluorescence resonance energy transfer; gene expression; genetically modified animals; inositol phosphates; intracellular transport; laboratory mouse; phosphoprotein phosphatase; phosphorylation; protein degradation; protein protein interaction; protein structure function; protein transport; receptor expression; transcription factor

DESCRIPTION (provided by applicant): Bcl-2 is a fascinating protein that regulates both cell cycle entry and apoptosis through its location on the mitochondria and the endoplasmic reticulum (ER). Although much is known about its role on mitochondria, little is known about its role on the ER. This proposal investigates the novel concept that Bcl-2 regulates intracellular Ca2+ signals involved in cell cycle entry and apoptosis. This concept is based on the recent discovery in this laboratory that Bcl-2 interacts with inositol 1,4,5- trisphosphate (IP3) receptors and reduces IP3-mediated Ca2+ release from the ER. The first two aims of this proposal investigate the molecular mechanism by which Bcl-2 interacts with IP3 receptors and regulates IP3-mediated Ca2+ release. Aim 1 employs the GST-pulldown technique to test for direct interaction in vitro and FRET analysis to test for direct interaction in vivo. Regardless of whether the interaction is direct or indirect, Aim 2 tests the hypothesis that Bcl-2 regulates IP3 mediated Ca2+ release by docking a protein phosphatase to IP3 receptors, thereby reducing their phosphorylation and decreasing their Ca2+ channel activity. A major goal in Aims 1 & 2 is to identify Bcl-2 mutations that abrogate the inhibitory effect of Bcl-2 on IP3-mediated Ca2+ release. Aim 3 will use these Bcl-2 mutants, along with physiological approaches, to determine if the inhibitory effect of Bcl-2 on IP3-mediated Ca2+ release contributes to its ability to inhibit cell cycle entry and apoptosis during T cell activation. The central hypothesis is that by reducing IP3-mediated Ca2+ elevation, Bcl- 2 inhibits two proximal steps in T cell activation, calcineurin-mediated dephosphorylation and nuclear translocation of the transcription factor NFAT, and calpain-mediated degradation of the cyclin dependent kinase inhibitor p27Kip1. Overall, the findings of this proposal will provide novel insight into the mechanism of action of Bcl-2.

描述(申请人提供)：bcl2是一种引人入胜的蛋白质，通过其在线粒体和内质网(ER)上的位置来调节细胞周期进入和细胞凋亡。尽管人们对其在线粒体上的作用知之甚少，但对其在内质网中的作用却知之甚少。这项建议探讨了一个新的概念，即Bcl-2调节细胞内钙信号，参与细胞周期进入和细胞凋亡。这一概念是基于本实验室最近发现的，即Bcl-2与三磷酸肌醇(IP3)受体相互作用，减少IP3介导的内质网钙释放。这一建议的前两个目的是研究Bcl2与IP3受体相互作用并调节IP3介导的钙释放的分子机制。目的1采用GST-Pull-Down技术检测体外的直接相互作用，用FRET分析检测体内的直接相互作用。无论这种相互作用是直接的还是间接的，Aim 2都验证了这样的假设：通过将蛋白磷酸酶对接到IP3受体上，从而减少其磷酸化并降低其钙通道活性，从而调节由IP3介导的钙离子释放。AIMS 1和AIMS 2的一个主要目标是确定可以消除Bcl2对IP3介导的钙释放的抑制作用的Bcl2突变。目的3将利用这些突变体，结合生理学方法，确定是否通过抑制IP3介导的钙释放，从而抑制细胞周期进入和T细胞活化过程中的细胞凋亡。中心假设是，通过降低IP3介导的钙升高，Bcl-2抑制T细胞激活的两个近端步骤，即钙调神经磷酸酶介导的转录因子NFAT的去磷酸化和核转位，以及钙蛋白酶介导的细胞周期蛋白依赖的激酶抑制物p27Kip1的降解。总体而言，该提案的发现将为了解Bc l-2的作用机制提供新的见解。