BCL-2 Function in Endoplasmic Reticulum

BCL-2 在内质网中的功能

• 批准号: 6927609
• 负责人: CLARK W DISTELHORST
• 金额: $ 26.01万
• 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/6927609
• 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（由申请人提供）：BCL-2是一种引人入胜的蛋白质，可通过其在线粒体和内质网（ER）上的位置调节细胞周期的进入和凋亡。 尽管它在线粒体上的作用知之甚少，但对其在ER中的作用知之甚少。 该建议调查了Bcl-2调节细胞周期进入和凋亡中涉及的细胞内Ca2+信号的新概念。 该概念基于该实验室的最新发现，即Bcl-2与肌醇1,4,5-三磷酸（IP3）受体相互作用，并减少了从ER中IP3介导的Ca2+释放。 该提案的前两个目的研究了Bcl-2与IP3受体相互作用并调节IP3介导的Ca2+释放的分子机制。 AIM 1采用GST-Pulldown技术在体外和FRET分析中测试直接相互作用，以测试体内直接相互作用。 无论相互作用是直接还是间接的，AIM 2都通过将蛋白质磷酸酶停靠至IP3受体来调节IP3介导的Ca2+释放的假设，从而降低其磷酸化并降低其CA2+通道活性。 目标1和2的主要目标是鉴定Bcl-2突变，以消除Bcl-2对IP3介导的Ca2+释放的抑制作用。 AIM 3将使用这些Bcl-2突变体以及生理方法来确定Bcl-2对IP3介导的Ca2+释放的抑制作用是否有助于其在T细胞激活过程中抑制细胞周期进入和凋亡的能力。 中心假设是，通过减少IP3介导的Ca2+升高，BCl-2抑制了T细胞激活中的两个近端步骤，钙调蛋白介导的转录因子NFAT的去磷酸化和核转运NFAT以及CALPAIN介导的依赖Cyclin依赖性激酶依赖性激酶抑制剂的降解。 总体而言，该提案的发现将为BCL-2的作用机理提供新的见解。