Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms

The gamma subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different gamma isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the gamma 1, gamma 2 or gamma 3 isoform. When assayed at a physiological ATP concentration (5 mM), gamma 1- and gamma 2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, gamma 3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr(172) phosphorylation, when cells were incubatedwith mitochondrial inhibitors that increase cellular AMP. With gamma 1 complexes, activation and Thr(172) phosphorylation induced by the upstream kinase LKB1 [liver kinase B1; but not calmodulin-dependent kinase kinase (CaMKK beta)] in cellfree assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the gamma 2 and gamma 3 complexes were small or insignificant. Binding of AMP or ADP protected all three gamma subunit complexes against inactivation by Thr(172) dephosphorylation; with gamma 2 complexes, ADP had similar potency to AMP, but with gamma 1 and gamma 3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different gamma subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles.