AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and
has attracted significant attention as a therapeutic target for treating metabolic disorders.
AMPK activity is stimulated more than 100-fold by phosphorylation of threonine 172
(Thr172). Binding of AMP to the γ subunit allosterically activates the kinase. Additionally,
many small molecules, e.g. 991, have been identified that bind between the kinase
domain and the carbohydrate-binding module of the β subunit, stabilising their interaction
and leading to activation. It was reported recently that non-phosphorylated Thr172 AMPK
is activated by AMP and A769662. We present here the crystal structure of non-phosphorylated
Thr172 AMPK in complex with AMP and 991. This structure reveals that the
activation loop, as well as the complex overall, is similar to the Thr172 phosphorylated
complex. We find that in the presence of AMP and 991 non-phosphorylated Thr172,
AMPK is much less active than the Thr172 phosphorylated enzyme. In human cells, the
basal level of Thr172 phosphorylation is very low (∼1%), but is increased 10-fold by treatment
with 2-deoxyglucose. In cells lacking the major Thr172 kinases, LKB1 and CaMKKβ,
Thr172 phosphorylation is almost completely abolished, and AMPK activity is virtually
undetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr172
AMPK can induce an ordered, active-like, conformation of the activation loop explaining
how AMPK activity can be measured in vitro without Thr172 phosphorylation. However, in
a cellular context, phosphorylation of Thr172 is critical for significant activation of AMPK.