potentially permit formation of 12 distinct AMPK complexes
(α 1 β 1 γ1 through α 2 β 2 γ3). Much of the pioneering work on
AMPK regulation was conducted on enzyme extracted from rat
liver (predominantlyα1,α2,β1, andγ1 isoforms), purified using
extensive multistep protocols. These preparations provided break-
throughs in identifying AMP as a stimulatory signal for AMPK [3]
and phosphorylation of the activation loop residueα-Thr-172 as a
key mediator of AMPK activity [4]. Development of a bacterial
system for expression of milligram quantities of recombinant, het-
erotrimeric AMPK complexes [5] heralded a new frontier in AMPK
research, contributing to the discovery of LKB1 and CaMKK2 as
upstream kinases for Thr-172, characterization of regulatory
autophosphorylation sites (e.g.,β1-Ser108 andα-Ser485), and, of
course, providing the opportunity to conduct extensive structural
and biophysical studies that now underpin drug development
efforts. Expression of AMPK heterotrimers, and in particular spe-
cific isoform combinations, has since been described with insect
cell- and mammalian cell-based systems [6, 7].
Relevant to the majority of recombinant proteins, selection of
the most appropriate expression system is largely determined by
final application. The benefit afforded by large-scale expression of
AMPK in bacterial hosts is countered to some extent by lack of
physiological modifications such as AMPKβ-subunit myristoyla-
tion, important for full regulation by AMP/ADP, or basal Thr-172
phosphorylation. Conclusions drawn from performing analyses
with bacterial-expressed AMPK in solitudeshould therefore be
made with caution. Conversely, recombinant AMPK generated in
mammalian cells closely resembles the enzyme form found in vivo
(low basal Thr-172 and Ser108 phosphorylation, stoichiometric
myristoylation) but suffers from inferior yield (Table1). Addition-
ally, consideration must also be given to the presence of endoge-
nous AMPK isoforms contaminating purified preparations,
particularly if the intention is to analyze specific AMPK heterotri-
mer complexes.
Here, we describe detailed protocols, optimized in our labora-
tory, to individually produce all 12 AMPK heterotrimers in
cultured mammalian cells. The method describes non-liposomal
transfection of mammalian cells and harvesting conditions. The
use of subunit-specific affinity tags (GST, flag, myc, or HA) enables
extraction of specific isoforms for biochemical assay, and posthar-
vest treatments with CaMKK2 or lambda (λ) phosphatase are con-
venient methods to increase or decrease AMPK phosphorylation
and activity, respectively.160 Jonathan S. Oakhill et al.