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是一种有趣的蛋白质，通过其在线粒体和内质网（ER）上的位置调节细胞周期进入和凋亡。虽然人们对其在线粒体中的作用了解甚多，但对其在内质网中的作用知之甚少。本研究探讨了Bcl-2调控细胞内Ca2+信号参与细胞周期进入和凋亡的新概念。这一概念是基于该实验室最近发现的Bcl-2与肌醇1,4,5-三磷酸（IP3）受体相互作用并减少IP3介导的内质网Ca2+释放。本研究的前两个目的是研究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。目的1采用gst -pull - down技术检测体外直接相互作用，采用FRET分析检测体内直接相互作用。无论相互作用是直接的还是间接的，Aim 2验证了Bcl-2通过将蛋白磷酸酶与IP3受体对接，从而减少其磷酸化并降低其Ca2+通道活性，从而调节IP3介导的Ca2+释放的假设。目的1和目的2的一个主要目标是鉴定Bcl-2突变，该突变消除了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，以及生理学方法，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细胞活化过程中抑制细胞周期进入和凋亡的能力。核心假设是，通过降低ip3介导的Ca2+升高，Bcl- 2抑制了T细胞激活的两个近端步骤，钙调磷酸酶介导的转录因子NFAT的去磷酸化和核易位，以及钙调磷酸酶介导的细胞周期蛋白依赖性激酶抑制剂p27Kip1的降解。总之，本研究结果将为Bcl-2的作用机制提供新的见解。