Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cellsMore about Open Access at the Crick
Authors listRobin Willows Matthew J Sanders Bing Xiao Bhakti R Patel Stephen R Martin Jon Read Jon Wilson Julia Hubbard Steve Gamblin David Carling
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 (Thr). 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 Thr AMPK is activated by AMP and A769662. We present here the crystal structure of non-phosphorylated Thr AMPK in complex with AMP and 991. This structure reveals that the activation loop, as well as the complex overall, is similar to the Thr phosphorylated complex. We find that in the presence of AMP and 991 non-phosphorylated Thr, AMPK is much less active than the Thr phosphorylated enzyme. In human cells, the basal level of Thr phosphorylation is very low (∼1%), but is increased 10-fold by treatment with 2-deoxyglucose. In cells lacking the major Thr kinases, LKB1 and CaMKKβ, Thr phosphorylation is almost completely abolished, and AMPK activity is virtually undetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr AMPK can induce an ordered, active-like, conformation of the activation loop explaining how AMPK activity can be measured without Thr phosphorylation. However, in a cellular context, phosphorylation of Thr is critical for significant activation of AMPK.
Journal Biochemical Journal
Issue number 17