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.

Public Health Relevance

The goal of this research is to understand the molecular mechanisms and regulation of autophagy in mammalian cells, a cellular process essential for normal physiology and involved in multiple human diseases. This study will elucidate the molecular basis of mammalian autophagy, in particular its upstream molecular signaling and regulatory events. This study will also provide insights into development of novel therapeutic approaches to treat autophagy-related diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM113013-03
Application #
9204838
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Maas, Stefan
Project Start
2015-02-01
Project End
2019-01-31
Budget Start
2017-02-01
Budget End
2018-01-31
Support Year
3
Fiscal Year
2017
Total Cost
$347,765
Indirect Cost
$150,059
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
Research Institutes
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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