Mechanism of mTOR signaling to autophagy machinery

描述(由申请人提供)：自噬是如何在压力或饥饿下诱导的，目前仍知之甚少。缺乏知识对细胞生物学和人类健康来说是一个重要的问题，因为它阻碍了对与自噬去调节相关的细胞异常的理解，自噬的去调节导致衰老和癌症。长期目标是了解哺乳动物雷帕霉素靶标(MTOR)如何调控自噬诱导，以及如何利用这一调控知识来改进预防和治疗。目前应用的目的是确定蛋白激酶ULK1(秀丽隐杆菌类蛋白激酶1，Atg1的哺乳动物同源物)是如何介导mTOR信号到自噬诱导机制的。核心假设是mTOR通过磷酸化ULK1并抑制ULK1在含有Atg14L的PI3KC3复合体(PI3KC3)激活中的功能来负向调节自噬诱导。我们的假设是基于我们的初步数据证明了一个由ULK1和ATG13组成的蛋白质复合体。我们确定这个蛋白质复合体是mTOR的靶标，也是mTOR活性对自噬机制的中介，从而确定了长期以来寻找的mTOR和自噬之间的分子联系。这项研究的基本原理是，了解mTOR如何调控ULK1的功能将促进对自噬诱导机制的基础知识，并有助于开发目前缺乏的监测和调节特定自噬事件的分子工具。在强大的初步数据的指导下，该假说将通过追求以下三个具体目标来检验和实现：1)确定mTOR如何负面调节ULK1-Atg13复合体；2)确定Atg13在自噬诱导中的作用；3)确定ULK1如何调节含有Atg14L的PI3KC3复合体。在第一个目标下，将确定作为mTOR靶标的ULK1的磷酸化位点，并表征它们在自噬调节中的功能。在第二个目标下，将研究Atg13介导的蛋白质-蛋白质相互作用以及这种相互作用在含有Atg14L的PI3KC3复合体调节中的作用。在第三个目标下，将确定作为ULK1底物的Atg14L的磷酸化位点，并确定该磷酸化在自噬诱导中的作用。本申请中提出的研究具有很高的创新性，因为它专注于一条以前未被探索的途径，填补了目前存在的将mTOR与自噬机制联系在一起的空白。这项拟议的研究意义重大，因为它将把对自噬诱导机制的理解转变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，预计这一研究将有助于制定具体监测和操纵自噬活动的策略。 公共卫生相关性：拟议的研究与公共健康相关，因为已确定的机制最终将促进对自噬调控的了解，自噬是一种进化保守的细胞降解过程，在癌症、衰老和与衰老相关的疾病中经常被放松调控。该项目与美国国立卫生研究院的使命有关，该使命涉及发展基础知识，以增强我们治疗或预防与放松自噬管制相关的人类疾病的能力。

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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the identified mechanism will ultimately advance knowledge on the regulation of autophagy, the evolutionarily-conserved cellular degradation process that is often deregulated in cancer, aging and aging-related diseases. The project is relevant to NIH's mission that pertains to developing fundamental knowledge that will enhance our ability to treat or prevent the human diseases associated with deregulation of autophagy.