Spatial regulation of AMPK activity under oxidative stress requires LKB1

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) and calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2). 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. Although oxidative stress is known to stimulate AMPK activity, 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 across all locations measured, phosphorylation of AMPK by LKB1 is required for AMPK activity. Using a multi- omics approach, we discover that in response to oxidative stress, AMPK mediates significant metabolic and gene expression changes. These findings support a role for AMPK in regulating adaptive responses 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.