Regulation of apoptosis by PI 3-kinase/Akt signaling

描述(申请人提供)：拟议研究的总体目标是阐明PI3-激酶/Akt/GSK-3信号通路调节细胞生长和存活的机制。Akt和GSK-3都能磷酸化多种转录因子，但PI-3-K/Akt/GSK-3信号转导调控基因表达的整体程序尚不清楚。在过去的赠款期间，全球表达谱结合转录因子结合位点的计算/实验分析已经被用来表征PI 3-K/Akt/GSK-3信号的基因调控，无论是在生长因子刺激静止细胞之后，还是在维持在血清中的细胞的持续增殖期间。这些研究确定CREB是GSK-3在静止期细胞中靶向的关键转录因子，并进一步暗示AP-1、NFkB和NFAT转录因子家族成员是GSK-3的靶标。一组独特的基因，富含细胞程序性死亡的调节器，由增殖细胞中持续的PI 3-激酶信号控制。对上游调控区的计算分析结合染色质免疫沉淀分析表明，FOXO、NFkB和Myc/Mad/Max转录因子在这些基因的调控中发挥着重要作用。此外，对其mRNAs的3‘非翻译区的计算分析表明，mRNA的周转也参与了PI 3-K信号的基因调控。这些研究将继续下去，目的是阐明PI 3-K/Akt/GSK-3信号控制的基因调控程序，包括对mRNA稳定性的调控。拟议的实验将按照以下具体目标进行。1.静止期细胞中GSK-3调控转录程序的特征。AP-1、NFkB和NFAT转录因子的作用将通过染色质免疫沉淀和RNA干扰来研究。计算分析将扩展到确定预测的增强子和启动子区域中的转录因子结合部位，并将研究GSK-3在调节延迟初级反应和即刻早期基因中的作用。2.PI-3-K/Akt/GSK-3信号在增殖细胞中的转录调控RNA干扰和瞬时转染实验将被用来确定FOXO、NFkB和Myc/Mad/Max转录因子在增殖细胞中PI 3-激酶依赖基因表达中的作用。GSK-3在细胞增殖过程中的基因调控中的作用也将被研究。3.对mRNA稳定性的调控。通过检测PI 3-K/Akt/GSK-3信号对PI 3-K抑制后下调的mRNAs稳定性的影响，来研究PI 3-K对基因调控的作用。公共卫生相关性：影响PI3-激酶/Akt/GSK-3信号通路的突变是导致人类癌症发展的最常见的异常之一，该通路的组成部分是药物开发的活跃靶点。本提案试图进一步了解该途径调节细胞存活的机制。PI-3-K信号通路新靶点的发现可能为癌症治疗提供新的靶点。

DESCRIPTION (provided by applicant): The overall goal of the proposed research is to elucidate the mechanisms by which the PI 3-kinase/Akt/GSK-3 signaling pathway regulates cell growth and survival. Both Akt and GSK-3 phosphorylate multiple transcription factors, but the overall program of gene expression controlled by PI 3-kinase/Akt/GSK-3 signaling remains to be fully understood. During the past grant period, global expression profiling combined with computational/experimental analysis of transcription factor binding sites has been used to characterize gene regulation by PI 3-kinase/Akt/GSK-3 signaling, both following growth factor stimulation of quiescent cells and during the continuous proliferation of cells maintained in serum. These studies identified CREB as a key transcription factor targeted by GSK-3 in quiescent cells, and have further implicated members of the AP-1, NFkB and NFAT transcription factor families as GSK-3 targets. A distinct set of genes, enriched in regulators of programmed cell death, is controlled by continuous PI 3-kinase signaling in proliferating cells. Computational analysis of upstream regulatory regions combined with chromatin immunoprecipitation assays indicate that FOXO, NFkB and Myc/Mad/Max transcription factors play major roles in regulation of these genes. In addition, computational analysis of the 3' untranslated regions of their mRNAs suggests that mRNA turnover is also involved in gene regulation by PI 3-kinase signaling. These studies will be continued with the goal of elucidating the program of gene regulation controlled by PI 3-kinase/Akt/GSK-3 signaling, including regulation of mRNA stability. The proposed experiments will proceed according to the following specific aims. 1. Characterization of the transcriptional program regulated by GSK-3 in quiescent cells. The roles of AP-1, NFkB and NFAT transcription factors will be investigated by chromatin immunoprecipitation and RNA interference. Computational analysis will be expanded to identify transcription factor binding sites in predicted enhancers as well as promoter regions, and the role of GSK-3 in regulation of delayed primary response as well as immediate-early genes will be studied. 2. Transcriptional regulation by PI 3-kinase/Akt/GSK-3 signaling in proliferating cells. RNA interference and transient transfection assays will be used to determine the roles of FOXO, NFkB and Myc/Mad/Max transcription factors in PI 3-kinase-dependent gene expression in proliferating cells. The role of GSK-3 in gene regulation during cell proliferation will also be investigated. 3. Regulation of mRNA stability. The role of mRNA degradation in gene regulation by PI 3-kinase will be investigated by determining the effect of PI 3-kinase/Akt/GSK-3 signaling on the stability of mRNAs that are down-regulated following inhibition of PI 3-kinase. PUBLIC HEALTH RELEVANCE: Mutations affecting the PI 3-kinase/Akt/GSK-3 signaling pathway are among the most common abnormalities leading to the development of human cancer, and components of this pathway are active targets for drug development. The present proposal seeks to further understand the mechanism by which this pathway regulates cell survival. Discoveries of novel targets of PI 3-kinase signaling may provide new targets for cancer treatment.