Methods to Study Lysosomal AMPK Activation

The AMP-activated protein kinase (AMPK) is a master regulator of metabolic homeostasis. It is activated by the upstream kinase LKB1 (liver kinase B1) when the AMP/ATP ratio is increased during starvation or heightened exercises. Based on reconstitution experiments using purified individual proteins, AMPK was demonstrated to be directly phosphorylated on its conserved residue Thr172 by LKB1, which was promoted by increased levels of AMP. However, recent studies have engendered a paradigm shift for how AMPK is activated inside the cell or animal tissues, unraveling that AXIN binds to LKB1 and tethers it to AMPK located on the surface of late endosome and lysosome (hereafter, only lysosome is discussed) in response to glucose starvation. Moreover, AXIN extends its interaction with the v-ATPase-Ragulator complex, which is paradoxically also required for activation of mTORC1 despite the fact that the two kinases AMPK and mTORC1 are inversely activated. Here, we summarize the experimental procedures of the assays for translocation of AXIN/LKB1 to the detergent-resistant lipid fractions of lysosomal membrane and the assembly of AMPK-activating complexes thereon. These methods will be useful for determining whether AMPK activation induced by various metabolic stresses or by pharmacological stimuli is mediated by the v-ATPase-Ragulator-AXIN/LKB1 axis.