covalent modification of Thr172 and allosteric activation and
give the most sensitive indication of the degree of AMPK
activation within the cell. An example of this is provided by
the ADaM site ligand A769662, which usually causes only a
modest increase in AMPK activity as measured in IP-kinase
assays or pT172 blots yet causes a large allosteric activation
that is reflected in a much larger increase in pACC
phosphorylation.4 Notes
- PBS for washing cells must be ice-cold to preserve the activa-
tion and phosphorylation status of AMPK and of downstream
targets. - Lysis buffer can be made in advance and stored at 20 C.
Lysis buffer must be ice-cold when used, to preserve the acti-
vation and phosphorylation status of AMPK and of down-
stream targets. - Such DNAs may encode either the WTor R299G mutant ofγ 1
or the wild type or R531G mutant ofγ2 (human numbering).
Although it is also possible to mutate the equivalent arginine in
AMPK-γ3 (R454), AMPK complexes containing WT γ 3
appear to respond rather differently in that they are not signifi-
cantly allosterically activated by AMP [13]. - To detect changes in AMPK activity or phosphorylation of
Thr172 in response to energy stress, the cells need to express
LKB1 and its accessory subunits, STRAD-αor STRAD-βand
MO25-α or MO25-β. Some tumor cell lines, do not
express LKB1. - The S108A mutation can also be made in AMPK-β2, although
β2-containing complexes are usually much less sensitive to
ADAM site activators thanβ1-containing complexes. - Addition of hydroxyethyl cellulose decreases electro-osmotic
flow; to dissolve it, boil in a microwave with gentle agitation. - Some AMPK activators, such as phenformin and berberine,
inhibit mitochondrial ATP synthesis and thus activate AMPK
by increasing cellular levels of AMP and ADP. However many
cell lines, especially when maintained in medium containing
25 mM glucose (as in Dulbecco’s Modified Eagle’s Medium,
DMEM), are highly glycolytic, and mitochondrial inhibitors
may fail to elicit much of a response. In such cases, transferring
to a more physiological glucose concentration (5 mM) 16 h
prior to treatment with mitochondrial inhibitors can lead to
greater AMPK activation.
250 Simon A. Hawley et al.