Illuminating Spatiotemporal Regulation of AMPK with a Genetically Encoded Excitation‐Ratiometric Biosensor for AMPK

Abstract

AMP activated protein kinase (AMPK) is a heterotrimeric protein responsible for moderating cellular energy homeostasis. In cancer, AMPK has been implicated as a context‐dependent tumor promoter or suppressor. To illuminate spatiotemporal regulation of AMPK, which is critical for context dependent roles of AMPK, we developed a single‐fluorophore excitation‐ratiometric AMPK activity reporter (ExRai AMPKAR), which exhibits greater sensitivity and dynamic range compared to our previous AMPK biosensor. We used pharmacological perturbations to activate AMPK through both nucleotide‐dependent (2‐deoxyglucose) and ‐independent (MK‐8722) mechanisms, finding context‐dependent AMPK activity throughout the cell and fast AMPK activity dynamics around membranous organelles. A 3D computational model suggests high AMPK activity around organelles such as the lysosome are due to active transport across the lysosomal membrane. Furthermore, we observed robust AMPK activation in the nucleus following various pharmacological stimulation. To further investigate the mechanism regulating nuclear AMPK activity, we used fluorescence recovery after photobleaching experiments which showed that a catalytic subunit of AMPK, AMPK‐2, had no obvious change in subcellular location following pharmacological stimulation. Nuclear AMPK activity is enhanced upon LKB1 expression in LKB1‐null cells, indicating this important upstream regulator of AMPK is involved in nuclear AMPK activity. Altogether, using ExRai AMPKAR with complimentary computational and biophysical methods, we have characterized subcellular AMPK activity around membranous organelles, and unveiled a resident population of AMPK in the nucleus, which could be acutely controlled to provide fast regulation of transcription. Beyond the current study, our new AMPK activity reporter has the potential to provide further insight into localized AMPK activity in many cellular contexts.