(2) promotion of phosphorylation at Thr172 by upstream kinase
(s) via binding of either AMP or ADP, and (3) inhibition of
dephosphorylation of Thr172 by protein phosphatases via binding
of either AMP or ADP; all three effects are antagonized by binding
of ATP [8–10]. AMPK can also be activated by synthetic activators
such as A-769662 [11] or 991 [3] and natural products such as
salicylate [12], which bind in the so-calledallostericdrug and
metabolite (ADaM) site [13] located between the carbohydrate-
binding module (CBM) on theβsubunit and the small lobe of the
kinase domain on theαsubunit [3, 14, 15]. Most activators bind-
ing this site cause a large allosteric activation of AMPK, even with
enzyme that is not phosphorylated on Thr172, although this allo-
steric effect may require prior phosphorylation at Ser108 on theβ
subunit, a site subject to autophosphorylation [16]. However,
compounds that bind at the ADaM site also protect Thr172 from
dephosphorylation [17, 18].
When assaying AMPK, the choice of method is dependent on
the starting material to be used. AMPK heterotrimers can be
expressed in an inactive form in bacteria using a polycistronic
expression vector [19], and these can be used in cell-free in-solu-
tion kinase assays either in their unphosphorylated “naı ̈ve” state or
after phosphorylation and activation by an upstream kinase. Using
bacterially expressed AMPK, one can use solution-phase assays to
examine effects of ligands that bind at the nucleotide and ADaM
sites, particularly their effects on allosteric activation, promotion of
Thr172 phosphorylation, and protection against Thr172 dephos-
phorylation. However, if the AMPK complexes are derived from
cultured mammalian cells or tissues without prior purification,
in-solution assays are not recommended. While the synthetic pep-
tide(s) used in AMPK assays are relatively specific, other kinases
present in cell lysates may be able to phosphorylate them. Also,
different subunit isoforms of AMPK phosphorylate these peptides
equally well and therefore cannot be distinguished by in-solution
kinase assays. To circumvent both problems, AMPK is first immu-
noprecipitated using isoform-specific antibodies and then assayed
in resuspended immunoprecipitates (referred to below as IP kinase
assays). There are a number of commercially available immunopre-
cipitating antibodies that recognize AMPK subunits and do not
affect the kinase activity (although carrying out large numbers of IP
kinase assays using commercial antibodies can be expensive, in
which case it may be cheaper to get them custom-made).
Whether using bacterially expressed AMPK in in-solution
assays, or IP kinase assays using AMPK derived from cell lysates, it
is essential to ensure that the appropriate amount of enzyme is used
so that the assay operates within the linear range. Thus, pilot
experiments titrating the quantity of AMPK, or the quantity of
lysate immunoprecipitated, are essential.70 Fiona A. Fyffe et al.