Role of the ULK1 Complex in Autophagy

描述（由申请人提供）：这项拟议研究的目标是了解ULK 1复合物（自噬途径的重要上游组分）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内膜运输和重组介导的分解代谢细胞过程。它的功能是降解长寿命的蛋白质和庞大的细胞器，以维持细胞的稳态，并促进在压力条件下的生存。自噬在所有真核细胞中是保守的，并且对正常发育和细胞生长至关重要。自噬的失调涉及人类疾病，如癌症、神经退行性疾病、传染病和心脏病。虽然许多自噬基因（ATG）已被确定，在哺乳动物中，自噬是如何诱导和调节的，以及它如何调节各种生物事件还没有完全了解。我们和其他人先前鉴定了ULK 1-ATG 13-FIP 200蛋白激酶复合物（缩写为ULK 1复合物）作为自噬途径中营养感应激酶mTOR的直接介导剂。mTOR复合物-1（mTORC 1）通过磷酸化蛋白激酶ULK 1及其调节蛋白ATG 13来抑制自噬，但mTOR驱动的磷酸化如何抑制ULK 1复合物的自噬活性尚不清楚。此外，虽然ULK 1复合物被认为是自噬途径的最上游组分，但新的证据表明，它在后期的自噬膜融合阶段也发挥作用。此外，由于ULK 1的蛋白激酶活性对其自噬活性是必不可少的，因此需要鉴定ULK 1的细胞蛋白底物以了解ULK 1复合物如何与下游ATG蛋白通讯。最近，我们已经获得了一系列的初步结果，提供了这些问题的见解。基于这些初步结果，在本研究中，我们将确定ULK 1复合物自噬功能的分子基础，包括：（1）确定营养素调节的ATG 13磷酸化在调节ULK 1复合物自噬活性中的作用;（2）鉴定蛋白激酶ULK 1的细胞底物并研究其潜在的自噬功能;和（3）确定ULK 1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/生物化学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，自噬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.