Role of the ULK1 Complex in Autophagy

描述（由申请人提供）：本研究的目的是了解ULK1复合物（自噬途径的重要上游成分）感知营养信号并将信号传递给下游自噬机制的机制。自噬是由溶酶体活性和细胞膜内运输和重组介导的细胞分解代谢过程。它的功能是降解长寿命的蛋白质和笨重的细胞器，以维持细胞内稳态，促进应激条件下的生存。自噬在所有真核细胞中都是保守的，对细胞的正常发育和生长至关重要。人类疾病如癌症、神经退行性疾病、传染病和心脏病都涉及自噬的失调。虽然许多自噬基因（ATG）已经被鉴定出来，但在哺乳动物中，自噬是如何被诱导和调节的，以及它如何调节各种生物事件还不完全清楚。我们和其他人之前发现ULK1- atg13 - fip200蛋白激酶复合物（简称ULK1复合物）是自噬途径中营养感应激酶mTOR的直接介质。mTOR复合物-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mTOR驱动的磷酸化如何抑制ULK1复合物的自噬活性的机制尚不清楚。此外，尽管ULK1复合物被认为是自噬途径的最上游组分，但新的证据表明，它在自噬膜融合后期也发挥作用。此外，由于ULK1的蛋白激酶活性对其自噬活性至关重要，因此需要鉴定ULK1的细胞蛋白底物，以了解ULK1复合物如何与下游ATG蛋白进行通信。最近，我们获得了一系列初步结果，对这些问题提供了一些见解。基于这些初步结果，在本提案中，我们将通过以下方式确定ULK1复合物自噬功能的分子基础：(1)定义营养调节的ATG13磷酸化在调节ULK1复合物自噬活性中的作用；(2)鉴定蛋白激酶ULK1的细胞底物并研究其潜在的自噬功能；(3)确定ULK1复合物是否调节下游自噬膜融合，如果是，则确定其基础机制。为了实现这些目标，我们将采用多种方法，包括传统的细胞生物学/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，这是一个参与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.