AMPK Methods and Protocols

Chapter 14

Kinase Activity Determination of Specific AMPK Complexes/

Heterotrimers in the Skeletal Muscle

Jesper B. Birk and Jørgen F. P. Wojtaszewski

Abstract

Measuring the kinase activity of the 5^0 -AMP-activated protein kinase (AMPK) is an essential part of
understanding the regulation of this metabolic master switch. The AMPK heterotrimer can exist in
12 different constellations with potentially diverse activation patterns. It is therefore important to be able
to measure heterotrimer-specific activity to discriminate between these patterns. In this chapter we describe
how to measure the AMPK activity of specific heterotrimeric complexes by consecutive immunoprecipita-
tions and how the assay can be performed in a medium throughput fashion using 96-well plates.

Key wordsImmunoprecipitation, AMPK activity, Skeletal muscle, Isoform-specific, Radioactive

tracer^33 P, AMARA peptide

1 Introduction

A key element of an enzyme is its activity, i.e., for a kinase the ability

to phosphorylate its target proteins. The 5^0 -AMP-activated protein

kinase (AMPK) is activated both allosterically and by covalent

modifications such as phosphorylation. The allosteric activation

by AMP and to a lesser degree ADP increases the kinase activity,

but more importantly, it increases the susceptibility for phosphory-

lation by upstream kinases and reduces the dephosphorylation by

phosphatases [1]. The canonical phosphorylation site Thr-172 on

the activation loop of the catalyticαsubunit is extremely important

for the activity of AMPK. Phosphorylation of Thr-172 increases the

activity of AMPK with two to three orders of magnitude [2].

The initial measurements of AMPK activity were performed

before antibodies were produced against AMPK, and hence no

immunoprecipitation allowed AMPK purification from tissue

lysates. Using crude tissue extracts and ammonium sulfate precipi-

tation of proteins, the specificity of the kinase activity had to be

insured by a substrate peptide constructed to mimic the phosphor-

ylation site on the known AMPK target acetyl-CoA carboxylase

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_14,©Springer Science+Business Media, LLC 2018

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