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（TSC 1）和2-（TSC 2）编码形成具有肿瘤抑制功能的复合体的蛋白质。TSC 1/TSC 2复合物的功能是将多种信号与哺乳动物雷帕霉素靶蛋白复合物1（mTORCI;一般称为mTOR）（蛋白质翻译的关键调节剂）整合。来自生长因子和能量储存的信号通过需要TSC 1/TSC 2复合物的过程调节mTOR，该过程涉及TSC 2蛋白磷酸化状态的变化。我们已经发现，TSC 1/TSC 2复合物是缺氧抑制mTOR所必需的，并且缺氧导致TSC 2蛋白中的磷酸酶敏感性迁移率改变。对这些数据的最简单解释是，响应于缺氧，激酶被激活，其磷酸化TSC 2，从而抑制mTOR。为了揭示负责低氧导致的TSC 2磷酸化的激酶，我们使用RNAi进行了无偏倚的遗传筛选。该筛选的目标是鉴定一种激酶，如TSC 2，当被敲低时，通过缺氧阻断mTOR抑制。由于RNAi的效率和较低的激酶组复杂性，我们试图在D.黑腹细胞。我们已经确定，在哺乳动物细胞中，在果蝇细胞中TOR响应于缺氧而被抑制，并且这种抑制依赖于完整的TSC-SC2复合物。已经进行了涉及638个冗余dsRNA的果蝇激酶组的RNAi筛选，并且我们已经选择了40个候选者用于进一步评估。该提议的具体目的是：（1）验证在初级RNAi筛选中鉴定的激酶，（2）定位和评估响应于缺氧的TSC 2上的磷酸化位点，以及（3）评估推定的缺氧信号传导激酶与TSC 2磷酸化之间的联系。缺氧激活的TSC 2激酶，调节mTOR的鉴定将有深远的影响，在我们的理解mTOR是如何在发展过程中和肿瘤的调节。

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.