Mechanism of Action of A-769662, a Valuable Tool for Activation of AMP-activated Protein Kinase

Benoit Viollet,Marc Foretz,Andrew McBride,Natalia Shpiro,Olga Göransson,Fiona A. Ross,D. Grahame Hardie,Kei Sakamoto,Simon A. Hawley

doi:10.1074/jbc.m706536200

Abstract

We have studied the mechanism of A-769662, a new activator of AMP-activated protein kinase (AMPK). Unlike other pharmacological activators, it directly activates native rat AMPK by mimicking both effects of AMP, i.e. allosteric activation and inhibition of dephosphorylation. We found that it has no effect on the isolated alpha subunit kinase domain, with or without the associated autoinhibitory domain, or on interaction of glycogen with the beta subunit glycogen-binding domain. Although it mimics actions of AMP, it has no effect on binding of AMP to the isolated Bateman domains of the gamma subunit. The addition of A-769662 to mouse embryonic fibroblasts or primary mouse hepatocytes stimulates phosphorylation of acetyl-CoA carboxylase (ACC), effects that are completely abolished in AMPK-alpha1(-/-)alpha2(-/-) cells but not in TAK1(-/-) mouse embryonic fibroblasts. Phosphorylation of AMPK and ACC in response to A-769662 is also abolished in isolated mouse skeletal muscle lacking LKB1, a major upstream kinase for AMPK in this tissue. However, in HeLa cells, which lack LKB1 but express the alternate upstream kinase calmodulin-dependent protein kinase kinase-beta, phosphorylation of AMPK and ACC in response to A-769662 still occurs. These results show that in intact cells, the effects of A-769662 are independent of the upstream kinase utilized. We propose that this direct and specific AMPK activator will be a valuable experimental tool to understand the physiological roles of AMPK.

Highlights

Phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in Response to A-769662 Requires an Upstream Kinase but Is Independent of the Upstream Kinase Utilized—To determine whether the phosphorylation and activation of AMPK and the phosphorylation of ACC in intact cells in response to A-769662 requires the upstream kinase LKB1, we examined the effects of A-769662 in muscle tissue isolated from wild-type mice or from mice with a muscle-specific deletion of LKB1 [43]
This is even lower than the EC50 of 800 nM reported for A-769662 by Cool et al [39], this may be due to differences in the preparation of AMPK used and/or the assay conditions, because our estimated EC50 for the natural activator, AMP, was lower than that reported by Cool et al [39] (8 versus 56 ␮M)
The ability of A-769662 to directly activate AMPK both in cell-free assays and in intact cells makes it unique among currently known cell-permeable activators. Other activators, such as aminoimidazole-4-carboxamide riboside (AICAR), metformin, and the thiazolidinediones, do not activate AMPK directly in cellfree assays, and either are pro-drugs that are converted to active components inside the cell (e.g. AICAR, which is converted to the AMP analogue ZMP [21]) or work even more indirectly, e.g. by inhibiting the respiratory chain or by triggering release of adiponectin from adipocytes [3]

Summary

Introduction

The compound had no effect on the activity of a T172D mutant of the ␣1 kinase domain that had been expressed in bacteria as a GST fusion (data not shown); because of the replacement of Thr-172 by an aspartate residue, this construct is constitutively active and does not require prior phosphorylation [42]. Effects of AMP and A-769662 on Dephosphorylation by Protein Phosphatase-2C␣—To test whether A-769662, like AMP [7], inhibited dephosphorylation of Thr-172, AMPK purified from rat liver was incubated with recombinant protein phosphatase-2C␣ in the presence and absence of Mg2ϩ, with or without AMP and A-769662 (Fig. 3B).

Results

Conclusion