Role of the ULK1 Complex in Autophagy

描述(申请人提供)：这项拟议研究的目标是了解ULK1复合体--自噬途径的重要上游组件--感知营养信号并将信号传递给下游自噬机制的机制。自噬是一种分解代谢的细胞过程，由溶酶体活性和细胞内膜的运输和重组介导。它的功能是降解长寿命的蛋白质和庞大的细胞器，以维持细胞的动态平衡，促进在应激条件下的生存。自噬在所有真核细胞中都是保守的，对正常发育和细胞生长至关重要。自噬的解除管制涉及人类疾病，如癌症、神经退行性疾病、传染病和心脏疾病。虽然许多自噬基因(ATG)已经被发现，但在哺乳动物中，自噬是如何被诱导和调控的，以及它是如何调控各种生物事件的，还不完全清楚。我们和其他人以前发现ULK1-ATG13-FIP200蛋白激酶复合体(简称ULK1复合体)是自噬途径中营养敏感激酶mTOR的直接中介。MTOR复合体-1(MTORC1)通过磷酸化蛋白激酶ULK1及其调节蛋白ATG13来抑制自噬，但从机制上讲，mTOR驱动的磷酸化如何抑制ULK1复合体的自噬活性尚不清楚。此外，尽管ULK1复合体被认为是自噬途径最上游的组成部分，但新的证据表明，它也在自噬膜融合的后期阶段发挥作用。此外，由于ULK1的蛋白激酶活性是其自噬活性所必需的，因此需要鉴定ULK1的细胞蛋白底物，以了解ULK1复合体如何与下游ATG蛋白通信。最近，我们获得了一系列初步结果，为这些问题提供了见解。在这些初步结果的基础上，在本提案中，我们将通过以下几个方面确定ULK1复合体自噬功能的分子基础：(1)确定营养调节的ATG13磷酸化在调节ULK1复合体自噬活性中的作用；(2)鉴定蛋白激酶ULK1的细胞底物并研究其潜在的自噬功能；以及(3)确定ULK1复合体是否调节下游自噬膜融合，如果是，则确定支撑机制。为了实现这些目标，我们将结合使用多种方法，包括传统的细胞生物/生化方法和更先进的技术，如化学遗传学、活细胞成像和基于SILAC的蛋白质组学。这项研究的成功将阐明哺乳动物自噬的分子基础，这是一个涉及正常生理和各种疾病的关键细胞过程。

DESCRIPTION (provided by applicant): The goal of this proposed research is to understand the mechanisms by which the ULK1 complex, an essential upstream component of the autophagy pathway, senses the nutrient signal and relays the signal to the downstream autophagy machinery. Autophagy is a catabolic cellular process mediated by lysosomal activity and intracellular membrane trafficking and reorganization. It functions to degrade long-lived proteins and bulky organelles in order to maintain cellular homeostasis and to promote survival under stressful conditions. Autophagy is conserved in all eukaryotic cells and crucial for normal development and cell growth. Deregulation of autophagy is involved in human diseases such as cancer, neurodegenerative disorders, infectious diseases and cardiac diseases. Although many autophagy genes (ATG) have been identified, in mammals, how autophagy is induced and regulated, and how it modulates various biological events are not fully understood. We and others previously identified the ULK1-ATG13-FIP200 protein kinase complex (abbreviated as the ULK1 complex) as the direct mediator of the nutrient-sensing kinase mTOR in the autophagy pathway. The mTOR complex-1 (mTORC1) inhibits autophagy by phosphorylating both the protein kinase ULK1 and its regulatory protein ATG13, but mechanistically how mTOR-driven phosphorylation suppresses the autophagic activity of the ULK1 complex is not known. In addition, although the ULK1 complex is considered to be the most upstream component of the autophagy pathway, new evidence suggests that it also plays a role at the later autophagic membrane fusion stages. Further, because the protein kinase activity of ULK1 is essential for its autophagic activity, identification of cellular protein substrates of ULK1 is required for understanding how the ULK1 complex communicates with downstream ATG proteins. Recently, we have obtained a series of preliminary results that have provided insights into these questions. Built upon these preliminary results, in this proposal we will determine the molecular basis underlying the autophagic function of the ULK1 complex by (1) defining the role of nutrient-modulated ATG13 phosphorylation in regulating the autophagic activity of the ULK1 complex; (2) identifying cellular substrates of the protein kinase ULK1 and investigating their potential autophagy function; and (3) determining whether the ULK1 complex regulates downstream autophagic membrane fusion, and if so, the underpinning mechanism. To achieve these aims, we will employ a combination of approaches including both conventional cell biological/biochemical methods and more advanced techniques such as chemical genetics, live-cell imaging, and SILAC-based proteomics. Success of this study will elucidate the molecular basis of mammalian autophagy, a critical cellular process involved in normal physiology and various diseases.