An RNAi Screen in Drosophila Cells for Kinases Involved in TOR Regulation

描述（由申请人提供）：结节性硬化症复合体基因（TSC1）和2-(TSC2)编码形成具有肿瘤抑制功能复合体的蛋白质。TSC1/TSC2复合体的功能是将多种信号整合到哺乳动物雷帕霉素靶蛋白复合体1 （mTORCI，一般称为mTOR）中，后者是蛋白质翻译的关键调节因子。来自生长因子和能量储存的信号通过一个需要TSC1/TSC2复合物的过程调节mTOR，该过程涉及TSC2蛋白磷酸化状态的变化。我们已经发现TSC1/TSC2复合体是缺氧抑制mTOR所必需的，并且缺氧导致TSC2蛋白中磷酸酶敏感的迁移转移。对这些数据最简单的解释是，在对缺氧的反应中，一种激酶被激活，使TSC2磷酸化，从而抑制mTOR。为了揭示缺氧导致TSC2磷酸化的激酶，我们使用RNAi进行了无偏倚的遗传筛选。这项筛选的目的是确定一种激酶，它像TSC2一样，当被敲除时，会阻断缺氧对mTOR的抑制。由于RNAi的效率和较低的基因组复杂性，我们试图在D. melanogaster细胞中进行这种筛选。我们已经确定，在哺乳动物细胞中，果蝇细胞的TOR在缺氧时受到抑制，这种抑制依赖于完整的TSCirrSC2复合体。我们对涉及638个冗余dsrna的果蝇基因组进行了RNAi筛选，并选择了40个候选基因进行进一步评估。本提案的具体目的是：(1)验证在初级RNAi筛选中鉴定的激酶，(2)绘制和评估缺氧时TSC2磷酸化的位点，(3)评估假定的缺氧信号激酶与TSC2磷酸化之间的联系。低氧激活的TSC2激酶调节mTOR的鉴定将对我们理解mTOR在发育过程和肿瘤中是如何调节的具有深远的意义。

DESCRIPTION (provided by applicant): The tuberous sclerosis complex genes (TSC1) and 2-(TSC2) encode proteins that form a complex with tumor suppressor function. The TSC1/TSC2 complex functions to integrate a variety of signals with mammalian target of rapamycin complex 1 (mTORCI; generically referred to as mTOR), a critical regulator of protein translation. Signals from growth factors and energy stores regulate mTOR through a process that requires the TSC1/TSC2 complex and which involves changes in the phosphorylation status of the TSC2 protein. We have discovered that the TSC1/TSC2 complex is required for mTOR inhibition by hypoxia and that hypoxia results in a phosphatase-sensitive mobility shift in the TSC2 protein. The simplest explanation for these data is that in response to hypoxia a kinase is activated, which phosphorylates TSC2 and thereby inhibits mTOR. To uncover the kinase responsible for TSC2 phosphorylation by hypoxia, we have undertaken an unbiased genetic screen using RNAi. The goal of this screen is to identify a kinase that like TSC2, when knocked down, blocks mTOR inhibition by hypoxia. Because of the efficiency of RNAi and lesser complexity of the kinome we sought to conduct this screen in D. melanogaster cells. We have established that as in mammalian cells, in Drosophila cells TOR is inhibited in response to hypoxia and that this inhibition depends upon an intact TSCirrSC2 complex. An RNAi screen of the Drosophila kinome involving 638 redundant dsRNAs has been conducted and we have selected 40 candidates for further evaluation. The specific aims for this proposal are: (1) to validate of kinases identified in primary RNAi screen, (2) to map and to evaluate the site(s) of phosphorylation on TSC2 in response to hypoxia, and (3) to evaluate of the link between the putative hypoxia signaling kinase(s) and TSC2 phosphorylation. The identification of a hypoxia activated TSC2 kinase that regulates mTOR would have profound implications in our understanding of how mTOR is regulated during development and in tumors.