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

描述(申请人提供)：结节性硬化症复合体基因(TSC1)和2-(TSC2)编码的蛋白质形成具有肿瘤抑制功能的复合体。TSC1/TSC2复合体的功能是将多种信号与哺乳动物靶标雷帕霉素复合体1(mTORCI；统称为mTOR)整合，mTOR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复合体的过程来调节mTOR，该过程涉及TSC2蛋白的磷酸化状态的变化。我们已经发现，TSC1/TSC2复合体是低氧抑制mTOR所必需的，并且低氧导致TSC2蛋白对磷酸酶敏感的迁移率改变。对这些数据最简单的解释是，在低氧条件下，一种激酶被激活，它使TSC2磷酸化，从而抑制mTOR。为了揭示低氧导致TSC2磷酸化的激酶，我们使用RNAi进行了无偏见的遗传筛选。这一筛选的目的是确定一种类似于TSC2的激酶，当被击倒时，可以阻断缺氧对mTOR的抑制。由于RNAi的效率和基因组的较低复杂性，我们试图在黑腹果蝇细胞中进行这一筛选。我们已经证实，在哺乳动物细胞中，在果蝇细胞中，TOR在低氧反应中受到抑制，这种抑制依赖于完整的TSCirrSC2复合体。我们已经对果蝇基因组进行了涉及638个冗余dsRNAs的RNAi筛选，并选择了40个候选基因进行进一步评估。这一建议的具体目的是：(1)验证初步RNAi筛选中识别的激酶；(2)定位和评估TSC2上低氧响应的磷酸化位点(S)；(3)评估可能的低氧信号通路(S)与TSC2磷酸化之间的联系。低氧激活的调节mTOR的TSC2激酶的发现将对我们理解mTOR在发育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.