Mechanism of mTOR Signaling to Autophagy Machinery

描述（由申请人提供）：在压力或饥饿下，自噬是如何诱导的，目前尚不清楚。缺乏知识是细胞生物学和人类健康的一个重要问题，因为它阻碍了对与自噬失调相关的细胞异常的理解，而自噬失调会导致衰老和癌症。长期目标是了解哺乳动物雷帕霉素靶点（mTOR）如何调节自噬诱导，以及如何利用这种调节的知识来改善预防和治疗。当前应用的目的是确定蛋白激酶ULK1（秀丽隐杆线虫unc51样激酶1，Atg1的哺乳动物同源物）如何介导mTOR信号传导到自噬诱导机制。核心假设是mTOR通过磷酸化ULK1和抑制ULK1激活含atg14l的PI3 -激酶III类（PI3KC3）复合物的功能，负向调节自噬诱导。我们的假设是基于我们的初步数据，证明了一个由ULK1和Atg13组成的蛋白质复合物。我们确定该蛋白复合物是mTOR的靶标和mTOR活性对自噬机制的中介，定义了长期寻找的mTOR和自噬之间的分子联系。本研究的基本原理是，了解mTOR如何调节ULK1的功能将促进对自噬诱导机制的基本认识，并有助于开发目前缺乏的用于监测和调节特定自噬事件的分子工具。在强有力的初步数据的指导下，该假设将通过以下三个具体目标进行验证和完成：1)确定mTOR如何负调控ULK1-Atg13复合物；2)确定Atg13在诱导自噬中的作用；3)确定ULK1如何调控含atg14l的PI3KC3复合物。在第一个目标下，将鉴定作为mTOR靶点的ULK1的磷酸化位点，并表征其在自噬调节中的功能。在第二个目标下，将研究Atg13介导的蛋白-蛋白相互作用以及这种相互作用在调节含atg14l的PI3KC3复合物中的作用。第三个目标是鉴定ULK1底物Atg14L的磷酸化位点，并确定其磷酸化在诱导自噬中的作用。本应用程序中提出的研究具有高度创新性，因为它专注于先前未被探索的途径，填补了目前存在的将mTOR与自噬机制联系起来的空白。这项研究具有重要意义，因为它将使对自噬诱导机制的理解达到前所未有的详细水平。这是一系列研究中必不可少的第一步，有望开发出专门监测和操纵自噬活动的策略。

DESCRIPTION (provided by applicant): How autophagy is induced under stress or starvation remains poorly understood. The lack of knowledge represents an important problem for cell biology and human health because it prevents understanding of the cellular abnormalities associated with de-regulation of autophagy, which contribute to aging and cancer. The long-term goal is to understand how mammalian target of rapamycin (mTOR) regulates autophagy induction and how knowledge of this regulation can be utilized to improve prevention and therapy. The objective of the current application is to determine how the protein kinase ULK1 (C. elegans UNC51-like kinase 1, mammalian homologue of Atg1) mediates mTOR signaling to the autophagy induction machinery. The central hypothesis is that mTOR negatively regulates autophagy induction by phosphorylating ULK1 and inhibiting the function of ULK1 in activation of the Atg14L-containing PI 3- kinase class III (PI3KC3) complex. Our hypothesis has been formulated based on our preliminary data demonstrating a protein complex consisting of ULK1 and Atg13. We identified this protein complex as a target of mTOR and the mediator of mTOR activity to the autophagy machinery, defining the long-sought molecular link between mTOR and autophagy. The rationale for the proposed research is that understanding how mTOR regulates the function of ULK1 will advance the fundamental knowledge on the mechanism of autophagy induction and assist in the development of molecular tools that are currently lacking to monitor and modulate a specific autophagy event. Guided by strong preliminary data, the hypothesis will be tested and accomplished by pursing the following three specific aims: 1) Determine how mTOR negatively regulates the ULK1-Atg13 complex; 2) Determine the role of Atg13 in autophagy induction; 3) Determine how ULK1 regulates the Atg14L-containing PI3KC3 complex. Under the first aim, phosphorylation sites of ULK1 that is a target of mTOR will be identified and their function in the regulation of autophagy will be characterized. Under the second aim, the protein-protein interaction mediated by Atg13 and the role of the interaction in the regulation of the Atg14L-containing PI3KC3 complex will be studied. Under the third aim, phosphorylation sites of Atg14L that is a substrate of ULK1 will be identified and the function of the phosphorylation in autophagy induction will be determined. The research proposed in this application is highly innovative, because it focuses on a previously unexplored pathway that fills in the current existing gap that links mTOR with the autophagy machinery. The proposed research is significant, because it will transform understanding of the mechanism of autophagy induction to an unprecedented detailed level. This is the essential first step in a continuum of research that is expected to enable development of strategies that specifically monitor and manipulate autophagy activity.