Chapter 16
Intact Cell Assays to Monitor AMPK and Determine
the Contribution of the AMP-Binding or ADaM Sites
to Activation
Simon A. Hawley, Fiona A. Fyffe, Fiona M. Russell,
Graeme J. Gowans, and D. Grahame Hardie
Abstract
AMP-activated protein kinase (AMPK) is extremely sensitive to cellular stress, so that nonphysiological
activation of the kinase can readily occur during harvesting of cells or tissues. In this chapter we describe
methods to harvest cells and tissues, and for kinase assays, that preserve the physiological activation status of
AMPK as far as possible. Note that similar care with methods of cell or tissue harvesting is required when
AMPK function is monitored by Western blotting, rather than by kinase assays. We also describe methods to
determine whether compounds that activate AMPK in intact cells do so indirectly by interfering with
cellular ATP synthesis or directly by binding to AMPK and, if the latter, whether this occurs by binding at
the AMP-binding sites on theγsubunit or at the ADaM site located between theαandβsubunits.
Key wordsAMP-activated protein kinase, AMPK, Kinase assay, Allosteric activation, Phosphoryla-
tion, Dephosphorylation1 Introduction
AMPK is mainly regulated by ligands that bind either to the ade-
nine nucleotide-binding sites on theγsubunit, with binding to site
3 appearing to be particularly critical [1], or to the ADaM site
located between N-lobe of the kinase domain on theαsubunit
and the carbohydrate-binding module on theβsubunit [2]. In
Chapter 5 we provided protocols for measuring the effects of
regulatory ligands on AMPK in cell-free assays. In this chapter we
describe methods to extract AMPK from intact cells to monitor
kinase activity and Thr172 phosphorylation and also describe spe-
cially constructed cell lines that allow the investigator to determine
whether an activating compound is acting either: (1) indirectly, by
inhibiting cellular ATP production and increasing cellular
AMP/ATP and/or ADP/ATP ratios (e.g., phenformin,Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_16,©Springer Science+Business Media, LLC 2018
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