LKB1 Spatially Regulates AMPK Activity Coordinate Cellular Oxidative Stress Response

AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis, with over 100 identified downstream targets throughout the cell. In response to cellular stress, including energetic stress, AMPK is activated via binding of AMP and phosphorylation by upstream kinases, including liver kinase B1 (LKB1). We and others have found that the activation of AMPK in response to cellular stress has distinct subcellular mechanisms, indicating compartmentalized regulation of AMPK signaling. Oxidative stress is known to stimulate AMPK activity, but how AMPK is spatially regulated by oxidative stress is underexplored. Using a single-fluorophore excitation-ratiometric AMPK activity reporter (ExRai AMPKAR), we find that oxidative stress induced by hydrogen peroxide (H2O2) results in AMPK activity with distinct spatiotemporal dynamics. We found that in the cytoplasm, nucleus, outer mitochondrial membrane, and cytosolic lysosomal surface, phosphorylation of AMPK by LKB1 is required for AMPK activity. Using a biosensor for ATP, we found at the cytoplasm and lysosome local ATP depletion dictates kinetics of AMPK activity. Using a multi- omics approach, we discover that in response to oxidative stress, AMPK mediates significant metabolic and gene expression changes, including upregulation of oxidative stress response through nuclear factor erythroid 2-related factor 2 (NRF2). Expanding on this identified mechanism, we find that non-small cell lung cancers harboring Kelch-like ECH-associated protein 1 (KEAP1) mutations have a functionally deficient LKB1-AMPK signaling network in response to oxidative stress. Altogether, this work provides new insights into how the subcellular environment influences localized AMPK activity, and identifies how AMPK regulates the cellular response to oxidative stress.