The AMP-activated protein kinase (AMPK) is a metabolic stress sensing αβγ heterotrimer critical for energy homeostasis, making it a therapeutic target for metabolic diseases such as type 2 diabetes and obesity¹. An increasing number of direct AMPK activating drugs such as the thienopyridone derivative A-769662 have been identified², but little progress has been made in elucidating their mechanism of action. A-769662 preferentially activates AMPK complexes containing the β1-isoform³, and a requirement for the β-subunit carbohydrate binding module (CBM) as well as the autophosphorylation site Ser108 within the β-CBM has been demonstrated. We investigated mechanisms governing Ser108 autophosphorylation with the aim of increasing potency of AMPK direct activators.

            We show that AMPK sensitivity to A-769662 is conferred exclusively by β-Ser108 phosphorylation and is independent of activation loop (α-Thr172) phosphorylation. β-Ser108 and α-Thr172 are largely unphosphorylated in the basal state in AMPK expressed in COS7 mammalian cells, and are phosphorylated to a similar extent following induction of AMPK signalling with the Ca²⁺-ionophore ionomycin.  We were unable to detect β-Ser108 phosphorylation in a kinase dead AMPK mutant, even after ionomycin treatment, demonstrating that β-Ser108 is a cis-autophosphorylation site. Dephosphorylation of β-Ser108 in cells occurred at a significantly slower rate compared to α-Thr172, and this was reflected in reduced rate of phosphatase PP2cα-mediated dephosphorylation in vitro. Thus, basal and stimulated levels of β-Ser108 autophosphorylation are intrisically linked to α-Thr172 phosphorylation, however regulatory disconnectivity is achieved at the dephosphorylation step. Our findings highlight the potential benefits of combinatorial therapies involving AMPK-targetting drugs and currently available AMP-elevating drugs such as metformin.