Signal Transduction to p70 S6 Kinase 1

描述(由申请人提供)：许多人类疾病是由于对细胞生长(细胞质量和大小的增加)、增殖、迁移、存活和死亡的不当调控造成的。这些过程受到一种由哺乳动物雷帕霉素靶标(MTOR)、Lst8和Raptor组成的复合体的严格调控，称为mTORC1复合体。S6蛋白激酶(S6K)是mTORC1的主要效应因子。MTORC1/S6K信号系统是细胞多种动态平衡输入的中心集成点，感知生长因子的可获得性、能量水平和氨基酸充分性。MTORC1/S6K信号的过度激活是几乎所有人类癌症的共同特征。MTORC1抑制剂，如雷帕霉素及其类似物，目前正在进行癌症治疗的临床评估。虽然这些抑制剂显示出了一些希望，但雷帕霉素不敏感的mTOR信号也影响肿瘤的发生，并且存在着在雷帕霉素治疗后上调生存路径的反馈环。因此，需要针对这一途径的其他成分的更多治疗剂。通过采用系统范围的方法来定义mTORC1/S6K通路调节以及该信号系统调节各种生物过程的机制，我们希望提供见解，从而识别治疗依赖mTORC1/S6K的癌症和其他代谢疾病的新治疗策略。 目标1将通过S6K1特异的相互作用蛋白Skar来研究S6K1信号、基因表达和细胞生长调控之间的联系。描述了研究SKAR和S6K1在调节mRNA生物发生和蛋白质翻译中的作用的方法。这一目标也为我们如何处理与目标2中确定的mTORC1/S6K信号系统相关联的所有mRNA结合蛋白奠定了基础。 AIM 2中概述的方法将各种生化纯化方法与质谱分析相结合，以识别和验证S6K信号系统的一组常见的上游调控因子和下游效应因子。使用多条汇聚的研究路线将使我们的努力集中在该途径最关键的组成部分上，使我们能够剖析S6K是如何调控如此多不同的细胞过程的。 目的3利用基于RNAi的遗传学方法阐明mTORC1/S6K途径的动态平衡调节机制。为此，我们开发了一种灵敏的、高通量的、基于图像的筛选策略来监测体内S6K的活性。我们建议利用这一独特的方法来广泛地询问mTORC1/S6K途径中有丝分裂原和营养调节的输入。公共卫生相关性：我们希望这项提案中概述的研究将加深我们对mTORC1/S6K信号缺陷的理解，这些缺陷导致癌症进展和细胞生长相关疾病，如儿童癌症易感综合征结节性硬化症。这些研究还将影响我们对其他与S6K相关的代谢性疾病的理解，如糖尿病和肥胖症。我们相信这种机械性的洞察力将为识别新的治疗策略打开大门，以抑制mTORC1/S6K信号网络的生长因子和/或氨基酸传感臂。

DESCRIPTION (provided by applicant): Many human diseases result from improper regulation of cell growth (an increase in cell mass and size), proliferation, migration, survival and death. These processes are critically regulated by a complex containing the mammalian target of rapamycin (mTOR), Lst8 and Raptor, called the mTORC1 complex. The S6 protein kinases (S6K) are major effectors of mTORC1. The mTORC1/S6K signaling system is the cell's central integration point for multiple homeostatic inputs, sensing growth factor availability, energy levels, and amino acid sufficiency. Hyperactivation of mTORC1/S6K signaling is a common feature of nearly all human cancers. mTORC1 inhibitors, such as rapamycin and its analogs, are currently being clinically evaluated for the treatment of cancer. While the inhibitors have exhibited some promise, rapamycin- insensitive mTOR signaling also influences tumorigenesis, and feedback loops exist that up- regulate survival pathways following rapamycin treatment. Thus, additional therapeutic agents targeting other components of this pathway are needed. By taking a systems-wide approach towards defining mTORC1/S6K pathway regulation and the mechanisms by which this signaling system modulates various biological processes, we hope to provide insights that will lead to the identification of novel therapeutic strategies for the treatment of mTORC1/S6K- dependent cancers and other metabolic disorders. Aim 1 will focus on the connection between S6K1 signaling, gene expression and cell growth control through an S6K1-specific interacting protein SKAR. Approaches are described that investigate the role of SKAR and S6K1 in the regulation of mRNA biogenesis and protein translation. This aim also sets the foundation for how we will approach all mRNA binding proteins linked to the mTORC1/S6K signaling system identified in Aim 2. The approach outlined in Aim 2 combines a variety of biochemical purification approaches with mass spectrometry analysis to identify and validate a common set of proximal upstream regulators and downstream effectors of the S6K signaling system. The use of multiple converging lines of investigation will focus our efforts on the most critical components of the pathway, allowing us to dissect how S6K regulates so many disparate cellular processes. Aim 3 utilizes RNAi-based genetic approaches to elucidate the mechanism of homeostatic regulation of the mTORC1/S6K pathway. To this end, we have developed a sensitive, high- throughput, image-based screening strategy for monitoring S6K activity in vivo. We propose to utilize this unique assay to broadly interrogate mitogen- and nutrient-regulated inputs into the mTORC1/S6K pathway. PUBLIC HEALTH RELEVANCE: We hope that the studies outlined in this proposal will deepen our understanding of the defects in mTORC1/S6K signaling that are responsible for cancer progression and cell growth-associated diseases, such as the childhood cancer predisposition syndrome Tuberous Sclerosis. These studies will also impact our understanding of other metabolic diseases linked to S6K, such as diabetes and obesity. We believe such mechanistic insight will open the door to the identification of novel therapeutic strategies for inhibiting the growth factor and/or amino acid sensing arms of the mTORC1/S6K signaling network.