A variety of different signals induce specific responses through a common, extracellular-signal regulated kinase (ERK)-dependent cascade. It has been suggested that signaling specificity can be achieved through precise temporal regulation of ERK activity. Given the wide distrubtion of ERK susbtrates across different subcellular compartments, it is important to understand how ERK activity is temporally regulated at specific subcellular locations. To address this question, we have expanded the toolbox of Förster Resonance Energy Transfer (FRET)-based ERK biosensors by creating a series of improved biosensors targeted to various subcellular regions via sequence specific motifs to measure spatiotemporal changes in ERK activity. Using these sensors, we showed that EGF induces sustained ERK activity near the plasma membrane in sharp contrast to the transient activity observed in the cytoplasm and nucleus. Furthermore, EGF-induced plasma membrane ERK activity involves Rap1, a noncanonical activator, and controls cell morphology and EGF-induced membrane protrusion dynamics. Our work strongly supports that spatial and temporal regulation of ERK activity is integrated to control signaling specificity from a single extracellular signal to multiple cellular processes.
AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But whether AMPK activity is regulated at different subcellular locations to provide precise spatial and temporal control over metabolism is unclear. Genetically encoded AMPK activity reporters (AMPKAR) have provided a window into spatial AMPK activity, but the limited dynamic range of current AMPKARs hinders detailed study. To monitor the dynamic activity of AMPK with high sensitivity, we developed a single-fluorophore AMPK activity reporter (ExRai AMPKAR) that exhibits an excitation ratiometric fluorescence change upon phosphorylation by AMPK, with over 3-fold greater response compared to previous AMPKARs. Using subcellularly localized ExRai AMPKAR, we found that the activity of AMPK at the lysosome and mitochondria are differentially regulated. While different activating conditions, irrespective of their effects on ATP, robustly yet gradually increase mitochondrial AMPK activity, lysosomal AMPK activity accumulates with much faster kinetics. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) resulted in slower AMPK activity at lysosomes but did not affect the response amplitude at either location, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further discovered AMPK activity in the nucleus, which resulted from LKB1-mediated cytoplasmic activation of AMPK followed by nuclear shuttling. Thus, a new, sensitive reporter for AMPK activity, ExRai AMPKAR, in complement with mathematical and biophysical methods, captured subcellular AMPK activity dynamics in living cells and unveiled complex regulation of AMPK signaling within subcellular compartments.
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.
Summary Fluorescent protein (FP)-based kinase activity biosensors are powerful tools for probing the spatiotemporal dynamics of signaling pathways in living cells. Yet, the limited sensitivity of most kinase biosensors restricts their reliable application in high-throughput detection modalities. Here, we report a protocol for using an ultrasensitive excitation-ratiometric PKA activity reporter, ExRai-AKAR2, to detect live-cell PKA activity via fluorescence microplate reading and epifluorescence microscopy. The high sensitivity of ExRai-AKAR2 is well suited to these high-throughput applications. For complete details on the use and execution of this protocol, please refer to Mehta et al. (2018) and Zhang et al., 2021a ) .
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.