Chapter 5
Cell-Free Assays to Measure Effects of Regulatory
Ligands on AMPK
Fiona A. Fyffe, Simon A. Hawley, Alexander Gray, and D. Grahame Hardie
Abstract
AMP-activated protein kinase (AMPK) is an energy sensor that is activated by increases in the cellular
AMP/ATP and ADP/ATP ratios by three mechanisms: (1) allosteric activation, (2) promotion of phos-
phorylation at Thr172 on theαsubunit by upstream kinases, and (3) inhibition of dephosphorylation of
Thr172 by protein phosphatases. All of these effects are triggered by the binding of AMP or ADP at one or
more of three sites on theγsubunit, where they displace ATP. AMPK is also activated by ligands that bind in
the ADaM site, which is located between theαandβsubunits. In this chapter we describe cell-free assays
that can be used to study these varied activation mechanisms.
Key wordsAMP-activated protein kinase, AMPK, Kinase assay, Allosteric activation, Phosphoryla-
tion, Dephosphorylation1 Introduction
AMP-activated protein kinase (AMPK) is a protein kinase that exists
universally as heterotrimeric complexes containing catalyticαsub-
units and regulatoryβandγsubunits [1]. Each subunit is encoded
by at least two genes (PRKAA1/PRKAA2encodingα1 andα2,
PRKAB1/PRKAB2 encoding β1 and β2, and PRKAG1/
PRKAG2/PRKAG3encodingγ1,γ2, andγ3), generating at least
12 heterotrimeric combinations [2]. The regulatory nucleotides
AMP, ADP, and ATP bind to two or three sites located between
the two pairs of CBS repeat motifs on theγsubunit, with one site
(involving CBS2) remaining unoccupied [3–6]. AMPK is normally
only active when phosphorylated at a conserved threonine residue
(usually termed Thr172 [7]) located in the activation loop of the
kinase domain on theαsubunit, which is phosphorylated by the
upstream kinases LKB1 and CaMKK2. AMPK acts as a sensor of
cellular energy status, being activated by increases in cellular
AMP/ATP or ADP/ATP ratio by three complementary mechan-
isms: (1) allosteric activation, caused by binding of AMP only,Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_5,©Springer Science+Business Media, LLC 2018
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