Regulation of apoptosis by PI 3-kinase/Akt signaling

描述（由申请人提供）：拟议研究的总体目标是阐明PI 3-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都能磷酸化多个转录因子，但PI 3-激酶/Akt/GSK-3信号通路控制基因表达的整体程序尚不完全清楚。在过去的资助期间，全球表达谱结合转录因子结合位点的计算/实验分析已被用于表征PI 3-激酶/Akt/GSK-3信号通路的基因调控，无论是在生长因子刺激静止细胞后还是在血清中维持细胞的持续增殖期间。这些研究发现CREB是GSK-3在静止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的靶点。一组独特的基因，富集程序性细胞死亡的调节因子，是由增殖细胞中连续的PI 3-激酶信号传导控制的。上游调控区域的计算分析结合染色质免疫沉淀试验表明，FOXO、NFkB和Myc/Mad/Max转录因子在这些基因的调控中起主要作用。此外，对其mRNA的3'非翻译区进行计算分析表明，mRNA的周转也参与了PI 3-激酶信号的基因调控。这些研究将继续进行，目的是阐明PI 3-激酶/Akt/GSK-3信号控制的基因调控程序，包括mRNA稳定性的调控。拟议的实验将根据以下具体目标进行。1. 静止细胞中GSK-3调控的转录程序的表征。AP-1、NFkB和NFAT转录因子的作用将通过染色质免疫沉淀和RNA干扰进行研究。计算分析将扩大到确定预测增强子和启动子区域的转录因子结合位点，GSK-3在调节延迟初级反应和立即早期基因中的作用将被研究。2. PI 3-激酶/Akt/GSK-3信号在增殖细胞中的转录调控。RNA干扰和瞬时转染试验将用于确定FOXO， NFkB和Myc/Mad/Max转录因子在增殖细胞中PI 3激酶依赖性基因表达中的作用。GSK-3在细胞增殖过程中基因调控的作用也将被研究。3. mRNA稳定性的调控。通过确定PI 3-激酶/Akt/GSK-3信号通路对PI 3-激酶抑制后下调mRNA稳定性的影响，研究mRNA降解在PI 3-激酶基因调控中的作用。公共卫生相关性：影响PI 3-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异常之一，该通路的组分是药物开发的活性靶点。目前的建议旨在进一步了解该途径调节细胞存活的机制。发现新的PI 3-激酶信号靶点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.