Spatiotemporal Regulation of AMPK Revealed by a Sensitive Kinase Activity Reporter

AMP activated protein kinase (AMPK) is a heterotrimeric kinase essential to the maintenance of cellular energy homeostasis. AMPK has downstream substrates throughout the cell, and spatially compartmentalized AMPK activity is hypothesized to confer specificity to AMPK signaling. However, the specific spatial organization of AMPK activity and the underlying regulatory mechanisms are not clear. To monitor the compartmentalized activity of AMPK, we developed a single‐fluorophore excitation‐ratiometric AMPK activity reporter (ExRai AMPKAR) which exhibits an excitation wavelength change when phosphorylated by AMPK, providing a specific readout of AMPK activity in single living cells. Using subcellularly localized ExRai AMPKAR, we observed distinct AMPK activity dynamics at the lysosome and mitochondria. In complement with computational modeling approaches, we discovered that the kinetics of local ATP depletion influence AMPK activity at the mitochondria. In the nucleus, the occurrence and regulation of AMPK activity has been controversial. Remarkably, using nuclear‐localized ExRai AMPKAR we found AMPK activity robustly accumulates in the nucleus following pharmacological stimulation, which relies on the ability of AMPK to shuttle between the cytosol and the nucleus. Through the development of a new reporter for AMPK activity, ExRai AMPKAR, with computational and biophysical methods, we have characterized subcellular AMPK dynamics and unveiled a mechanism for nuclear AMPK activity. This work provides a greater understanding of the compartmentalized regulation of AMPK signaling networks within the cell.