AMPK Methods and Protocols

Chapter 10

Transient Expression of AMPK Heterotrimer Complexes

in Mammalian Cells

Jonathan S. Oakhill, John W. Scott, and Toby A. Dite

Abstract

Regulation of AMP-activated protein kinase (AMPK) signalling is complex and involves contributions from
adenine nucleotides, co-/posttranslational modifications, and isoform composition of the AMPK hetero-
trimer. It is becoming apparent that AMPK activation/inhibition by synthetic drugs involves similar levels
of complexity. Major advances in our understanding of these mechanisms have been gained from recombi-
nant expression systems that provide sufficient quantities of highly purified material for structure/function
studies. Here, we provide a detailed protocol for transient expression of affinity-tagged AMPK complexes in
mammalian cells. We have found this system to be optimal as a source of enzyme possessing regulatory
modifications found in vivo.

Key wordsRecombinant protein, Kinase, Mammalian cell, Transfection, Purification, Biochemistry,

Metabolism

1 Introduction

AMP-activated protein kinase (AMPK) is a phylogenetically con-

served Ser/Thr kinase that has emerged as an important integrator

of signals to control cellular energy balance [1]. At the whole-body

level, AMPK also integrates stress responses such as exercise, as well

as nutrient and hormonal signals, to control food intake, energy

expenditure, and substrate utilization. It is now widely thought that

this enzyme is a key component in mechanisms underlying the

health benefits of diet and exercise. There is also increasing evidence

for a role of AMPK in multiple common diseases including type

2 diabetes, cancer, obesity, and cardiovascular disease. Conse-

quently, a new age of AMPK drug development has emerged that

may have profound effects in the treatment of these metabolic

diseases in the future [2].

The AMPK complex is anαβγheterotrimer containing a cata-

lyticα-subunit and regulatoryβandγsubunits. Multiple isoforms

of each subunit exist in mammals (α1,α2,β1,β2,γ1,γ2,γ3) which

Dietbert Neumann and Benoit Viollet (eds.),AMPK: Methods and Protocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_10,©Springer Science+Business Media, LLC 2018

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