AMPK Methods and Protocols

Chapter 25

Determining AMPK Activation via the Lysosomal

v-ATPase-Ragulator-AXIN/LKB1 Axis

Chen-Song Zhang, Mengqi Li, Yue Zong, and Sheng-Cai Lin

Abstract

Recent studies have revealed how AMPK is activated inside the cell and animal tissues: in response to low
glucose, AXIN tethers LKB1, by virtue of their constitutive association, to AMPK located on the surface of
late endosome/lysosome. Importantly, the lysosomal v-ATPase (vacuolar ATPase)-Ragulator complex,
when primed by glucose starvation or concanamycin A, facilitates AXIN/LKB1 to interact with AMPK.
Here, we describe the experimental procedures of the assays for detecting the translocation of AXIN/LKB1
or the assembly of the AXIN-based AMPK-activating complexes on the late endosome/lysosome. The
methods in this chapter will be useful for determining whether various metabolic stresses or pharmacologi-
cal stimuli activate AMPK via the v-ATPase-Ragulator-AXIN/LKB1 axis, which also concomitantly inacti-
vates mTORC1. Detailed protocols for determining the levels of adenylates are also described.

Key wordsAMPK, LKB1, AXIN, Ragulator, v-ATPase, Lysosome, Detergent-resistant membrane,

Light organelles

1 Introduction

AMPK is a heterotrimer, comprised of a catalyticαsubunit and

regulatoryβandγsubunits [1, 2]. The adenylates AMP, ADP, and

ATP can all bind to theγsubunit and allosterically modulate AMPK

activity [3–7]. AMP binding leads to AMPK activation through

promoting the upstream kinase LKB1 to phosphorylate threonine

172 of theαsubunit of AMPK (Thr-172) and subsequently serine

79 of ACC (Ser-79), which is a “gold standard” for evaluating the

activity of AMPK [8–11]. Binding of ADP as well as AMP protects

p-Thr-172 from being dephosphorylated, whereas ATP binding

inhibits AMPK activation [7, 11–14]. However, it is now clear

that activation of AMPK in vivo entails much more complex steps

and involves many more factors. It has been recently shown that

AMPK activation in response to glucose starvation or after treat-

ment of metformin occurs on the surface of late endosome/lyso-

some (hereafter only lysosome is referred to) [15, 16]. This process

Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_25,©Springer Science+Business Media, LLC 2018

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