Characterization of novel regulators of amino acid-sensitive mTORC1 signaling

描述（由申请人提供）：哺乳动物雷帕霉素靶点（mTOR）是丝氨酸-苏氨酸激酶和mTOR复合物1的催化亚基（mTORC1）。mTORC1是细胞生长和活力的主要调节因子，促进蛋白质合成、核糖体生物发生和线粒体生物发生，同时抑制自噬的分解代谢过程。不足为奇的是，这一途径在癌症中是失调的，许多特征明确的肿瘤抑制因子是mTORC1途径的负调节因子。这种广泛的合成代谢和分解代谢途径的调节需要mTORC1作为细胞健康状态的监测器。它的活性在细胞中受到氨基酸水平、生长因子水平、能量水平以及DNA损伤和其他细胞压力的严格调节。在所有这些输入中，氨基酸是独一无二的；它们利用一种独立的机制来激活激酶，并且是活性所必需的，即使在其他激活刺激存在的情况下。尽管越来越多的

DESCRIPTION (provided by applicant): Mammalian Target of Rapamycin (mTOR) is serine-threonine kinase and the catalytic subunit of mTOR Complex 1 (mTORC1). mTORC1 is a master regulator of cellular growth and viability, promoting protein synthesis, ribosome biogenesis, and mitochondrial biogenesis while inhibiting the catabolic process of autophagy. Not surprisingly, this pathway is dysregulated in cancer, and a number of well characterized tumor suppressors are negative regulators of the mTORC1 pathway. Regulation of such broad anabolic and catabolic pathways requires that mTORC1 acts as a monitor of the health status of the cell. Its activity is tightly regulated in cells by the levels of amino acids, growth factors, nd energy levels, as well as DNA damage and other cellular stresses. Of all of these inputs, amino acids are unique; they utilize an independent mechanism to activate the kinase and are absolutely required for activity, even in the presence of other activating stimuli. Despite growing knowledge of the upstream pathways that control mTORC1 activity, surprisingly little is known about how amino acids are sensed or how their levels signal to mTORC1. Elucidation of this crucial aspect of the mTORC1 pathway will be instrumental in providing new targets for pharmacological inhibition of mTORC1 while potentially minimizing toxic side effects. The goal of this project is to identify and characterize novel regulators of mTORC1 activity in response to amino acid levels. We have used a proteomic approach to identify proteins that interact with the machinery that activates mTORC1 in the presence of amino acids. In this proposal, we focus on the characterization of one particular interacting protein in the mTORC1 pathway. Additionally, this protein has been shown to be a cellular target of a Hepatitis B virus oncogene, possibly implicating a role for mTORC1 in both viral infection and Hepatitis B virus-associated hepatocellular carcinoma. To characterize this new protein, we propose to following aims: 1. Define the role of this new pathway member in the activation of mTORC1 by amino acids through its control of mTOR trafficking to the lysosome, where the kinase is activated, using knockdown experiments. 2. Identify proteins that interact with the new pathway member using immunoprecipitation followed by mass spectrometry in order to fully elucidate its function within the amino-acid sensing pathway. 3. Interrogate the role of the amino acid-sensitive mTORC1 pathway in Hepatitis B virus infection.