BCL-2 Function in Endoplasmic Reticulum

描述（由申请人提供）：Bcl-2是一种迷人的蛋白质，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细胞凋亡。 虽然对它在线粒体中的作用了解很多，但对其在ER中的作用知之甚少。 该提案研究了Bcl-2调节参与细胞周期进入和凋亡的细胞内Ca 2+信号的新概念。 这一概念是基于本实验室最近的发现，Bcl-2与肌醇1，4，5-三磷酸（IP 3）受体相互作用，并减少IP 3介导的Ca 2+从ER释放。 本提案的前两个目的是研究Bcl-2与IP 3受体相互作用并调节IP 3介导的Ca 2+释放的分子机制。 目的1采用GST-下拉技术测试体外直接相互作用和FRET分析测试体内直接相互作用。 无论相互作用是直接的还是间接的，目的2测试的假设，Bcl-2调节IP 3介导的Ca 2+释放的蛋白磷酸酶对接到IP 3受体，从而减少其磷酸化和降低其Ca 2+通道活性。 目的1和2的主要目标是鉴定Bcl-2突变，其消除Bcl-2对IP 3介导的Ca 2+释放的抑制作用。 目的3将使用这些Bcl-2突变体，沿着生理学方法，以确定Bcl-2对IP 3介导的Ca 2+释放的抑制作用是否有助于其在T细胞活化期间抑制细胞周期进入和凋亡的能力。 核心假设是通过减少IP 3介导的Ca 2+升高，Bcl- 2抑制T细胞活化中的两个近端步骤，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性激酶抑制剂p27 Kip 1的降解。 总的来说，这项提案的发现将为Bcl-2的作用机制提供新的见解。

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.