Autophagy is a conserved catabolic process through which cytoplasmic components are targeted to the lysosome for degradation, via a series of intracellular vesicular formation and fusion events. Autophagy plays a vital role in maintaining cellular homeostasis under stressful conditions, such as nutrient starvation. As such, it is implicated in a plethora of human diseases, including cancer and neurodegenerative disorders. Molecularly, many autophagy-related (Atg) proteins are required for autophagy, with the Ulk1-Atg13- FIP200 complex functioning at the top of the hierarchy. Ulk1 is a protein kinase mediating the function of the nutrient-sensing kinase mTOR. Atg13 is a regulatory protein of Ulk1. The phosphorylation status of Atg13 is thought to control the induction of autophagy. However, the detailed mechanism by which Atg13 modulates Ulk1 and autophagy has not been defined. Furthermore, despite the requirement of Ulk1 kinase activity to stimulate autophagy, its physiological protein substrates have not been identified. The long-term goals of this proposal are to understand the upstream molecular mechanisms that regulate the induction of mammalian autophagy and understand how this signal is transduced to the downstream, core machinery of the pathway. The first specific aim is to identify nutrient-sensitive phosphorylation sites of mammalian Atg13 and determine their function in regulating the induction of autophagy. To achieve this goal, I will purify in vivo phosphorylated Atg13 and map phospho-acceptor residues by mass spectrometry (MS). Phospho-defective and mimetic mutants will be used in functional assays to determine how Atg13 phosphorylation regulates the autophagic function of Ulk1, and the eventual outcome, autophagy. The second specific aim is to investigate the mechanism by which the Ulk1 complex transduces the autophagy induction signal downstream. Ulk1 substrates will be identified using a chemical genetic approach in combination with affinity purification and MS. Subsequently, phospho-defective mutants will be used to determine which protein substrates of Ulk1 regulate autophagy in a phosphorylation-dependent manner. Taken together, the findings of this research will identify novel molecular mechanisms underlying mammalian autophagy, and inform the development of approaches to target autophagy for therapeutic benefit.
This project is to elucidate the molecular mechanisms that regulate mammalian autophagy, a cellular process essential for multiple biological events and involved in various human diseases. As such, success of this research will provide novel insights into the fundamental biology of autophagy and have relevant therapeutic implications.