Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter

Abstract

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.

Rapid metabolic reprogramming mediated by the AMP-activated protein kinase during the lytic cycle of Toxoplasma gondii

“…But the underlying mechanisms are largely unknown. It is also not clear whether the overexpressed TgAMPKα in the absence of TgAMPKγ used the same mechanism to regulate parasite metabolism and activities as native TgAMPKα (such as that in wildtype parasites) in the presence of TgAMPKγ, because in other model organisms, the activation and subsequent activities of AMPK may be modulated differently by different degree and types of stress or metabolic fluctuations 44 . Nonetheless, TgAMPKγ depletion leads to growth arrest but does not seem to cause dramatic alteration in gene transcription.…”

The Inositol Phosphate System—A Coordinator of Metabolic Adaptability

“…As glucose levels decline, the glycolytic metabolite F(1,6)BP decreases, ultimately detaching from aldolase through a mass-action effect and producing a conformational change in the signaling complex that favors the association of AXIN:LKB1 with the Ragulator component LAMTOR1. This promotes the formation of the lysosomal AMPK activation complex, bridging LKB1 with lysosomal AMPK for activation [ 70 , 73 ]. Inositol phosphate signaling has been observed to participate in AMPK and mTOR regulation, not only by providing signaling molecules, but also through scaffolding functions of inositol phosphate multikinase (IPMK).…”

Sensing local energetics to acutely regulate mitophagy in skeletal muscle

“…In some cell culture systems, Ulk1 has been shown to localize to mitochondria (Wu et al, 2014;Tian et al, 2015), which is associated with mitophagy, but some evidence suggest that Ulk1 is dephosphorylated at S555 by the time it localizes to mitochondria (Hung et al, 2021). The continued advent of novel fluorescent activity reporters (Schmitt et al, 2022) will hopefully be able to shed new light on this area in the context of skeletal muscle and exercise.…”

“…When activated in response to energetic stress, such as exercise, degradation through mitophagy occurs in spatially distinct domains (Glancy et al, 2017;Laker et al, 2017;Drake et al, 2021), suggesting autophagosomes and lysosomes may be synthesized in the vicinity of the mitochondrial region identified for degradation. Recent discoveries have suggested that the spatial specificity of mitophagy may be governed through locally distinct mechanisms at mitochondria, possibly in response to the energetic microenvironment (Miyamoto et al, 2015;Glancy et al, 2017;Zong et al, 2019;Drake et al, 2021;Schmitt et al, 2022). In this review, we briefly summarize the production of cellular energy by mitochondria and the biochemical and molecular consequences of energetic stress, namely acute exercise, in relation to mechanisms that locally monitor mitochondrial energetics to promote mitophagy and identify unresolved questions.…”