PROJECT SUMMARY Loss of energy homeostasis is a key driver in human disease. Failure to maintain cellular energy balance results in many disease states, including diabetes, cancer, and neurological diseases. AMP-activated kinase (AMPK) is a highly conserved kinase responsible for maintaining cellular energy balance. Under conditions of low nutrients and low energy, AMPK controls the metabolic switch from an energy-consuming anabolic state to an energy-producing catabolic state at both the cellular and organismal level. Upon activation by energetic stress, AMPK phosphorylates downstream targets to arrest cell growth, regulate transcription, inhibit protein synthesis, and reprogram cellular metabolism. One of the most well-established downstream signaling pathway inhibited by AMPK is the pro-growth mechanistic Target of Rapamycin complex 1 (mTORC1) pathway. However, the functional role that mTORC1 inhibition plays in cellular metabolic reprogramming and maintaining energy homeostasis upon AMPK activation is controversial, as both AMPK-independent inhibition of mTORC1 and mTORC1-independent regulation of metabolic pathways have been described. The long-term scope of my research program is to elucidate the mechanisms that regulate energy homeostasis and refine the current understanding of how pathways exert control of metabolic reprogramming. To accomplish this long-term goal, my research group will address two major knowledge gaps in our current understanding of how mTORC1 inhibition interacts with AMPK activity to drive cellular metabolism and growth: 1) Is mTORC1 inhibition necessary for cellular metabolic reprogramming by AMPK? What aspects of cellular metabolic reprogramming require mTORC1 inhibition by AMPK? 2) How does mTORC1 inhibition and AMPK activation coordinate the regulation of processes involved in metabolic reprogramming? My extensive expertise in defining molecular mechanisms of cellular processes and using innovative mouse models makes my recently established independent laboratory an ideal environment to undertake these efforts toward a more complete understanding of AMPK signaling. We will employ novel point-mutant mice harboring specific serine-to-alanine mutations at the phosphorylation sites required for AMPK-dependent inhibition of mTORC1. Using both mouse tissues and primary cells derived from these mice, we will perform functional genomic and biochemical analyses to uncover the precise role of mTORC1 inhibition induced by AMPK activation in regulating gene expression, autophagy, and lipid synthesis, which are critical for cellular metabolic reprogramming. Together, these experiments will provide conceptual advances in how we think about AMPK and its relationship with mTORC1 and will offer mechanistic insight into how dysregulation of these pathways can lead to metabolic dysfunction and disease, which will allow us to identify new therapeutic strategies for treatment of metabolically-linked human disease.