Non-canonical roles for AMPK and TBK1 in amino acid sensing by mTOR - Abstract Cellular and organismal homeostasis requires the integration of diverse environmental cues by cell signaling networks, the disruption of which contributes to pathological conditions. The kinase mTOR, which comprises the catalytic core of two distinct complexes (mTORC1 and mTORC2), responds to nutrients (amino acids (AAs); glucose), growth factors (EGF), hormones (insulin), and energetic stress to control fundamental cellular processes that impact cell metabolism, growth, proliferation, and survival. Not surprisingly, aberrant mTOR signaling contributes to diverse pathologic states including type II diabetes, cancer, and immune and neurodegenerative disorders. Despite the physiological importance of mTOR, major gaps exist in our understanding of mTOR regulation and function, in particular how mTOR communicates with other regulatory systems. Our research focuses on how non-canonical functions of the energy sensing kinase AMPK and the innate immune kinase TBK1 control mTOR signaling and function in cells and in mice in vivo. While it is well- established that AMPK inhibits mTORC1 during energetic stress, our prior work revealed that AMPK paradoxically i) phosphorylates mTOR on a site that promotes mTORC1 signaling and ii) supports mTORC1 signaling during prolonged amino acid (AA) deprivation, a stress condition that induces autophagy and liberates AAs, a nutrient that supports mTORC1 signaling in response to growth factors and hormones. These results reveal AMPK as a previously unknown sensor of cellular AA levels. Thus, this proposal will define how AMPK supports mTORC1 signaling and function in specific cellular contexts rather than universally opposing mTORC1, which would invoke a paradigm shift to the AMPK and mTOR fields. While TBK1 is best known to initiate host defense responses against microbial pathogens, TBK1 also contributes to tumorigenesis and glycemic control during obesity. How TBK1 mediates these non-canonical processes remains poorly defined, however. Our prior work revealed that i) TBK1 phosphorylates mTOR to promote mTORC1 and mTORC2 signaling in response to microbial-derived signals and insulin and ii) the TBK1-mTOR axis protects against hyperglycemia and insulin resistance in obese but not lean mice. Our preliminary results also indicate that AAs increase TBK1 activity, revealing TBK1 as another previously unknown sensor of cellular AA levels. Thus, this proposal will also determine how AAs increase TBK1 activity to promote mTORC1 signaling and better define how the TBK1- mTOR axis promotes glucose homeostasis and insulin sensitivity in specific physiological contexts, i.e., in the obese but not lean state. This research will provide conceptual advance, as it will shift how we think about AMPK and TBK1 in metabolic control and their relationship to mTORC1. Molecular understanding of mTOR network regulation and how mTOR engages in crosstalk with other signaling systems will provide critical insights that will advance our understanding of mTOR in health and disease, which may identify new therapeutic targets for treatment of mTOR-linked disorders.
