Regulation of the muscle-specific AMP-activated protein kinase α2β2γ3 complexes by AMP and implications of the mutations in the γ3-subunit for the AMP dependence of the enzyme

Abstract

The AMP-activated protein kinase is an evolutionarily conserved heterotrimer that is important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory γ-subunit of the kinase, AMP-activated protein kinase γ3, has a key role in glucose and fat metabolism in skeletal muscle, as suggested by metabolic characterization of humans, pigs and mice harboring substitutions in the AMP-binding Bateman domains of γ3. We demonstrate that AMP-activated protein kinase α2β2γ3 trimers are allosterically activated approximately three-fold by AMP with a half-maximal stimulation (A0.5) at 1.9 ± 0.5 or 2.6 ± 0.3 μm, as measured for complexes expressed in Escherichia coli or mammalian cells, respectively. We show that mutations in the N-terminal Bateman domain of γ3 (R225Q, H306R and R307G) increased the A0.5 values for AMP, whereas the fold activation of the enzyme by 200 μm AMP remained unchanged in comparison to the wild-type complex. The mutations in the C-terminal Bateman domain of γ3 (H453R and R454G), on the other hand, substantially reduced the fold stimulation of the complex by 200 μm AMP, and resulted in AMP dependence curves similar to those of the double mutant, R225Q/R454G. A V224I mutation in γ3, known to result in a reduced glycogen content in pigs, did not affect the fold activation or the A0.5 values for AMP. Importantly, we did not detect any increase in phosphorylation of Thr172 of α2 by the upstream kinases in the presence of increasing concentrations of AMP. Taken together, the data show that different mutations in γ3 exert different effects on the allosteric regulation of the α2β2γ3 complex by AMP, whereas we find no evidence for their role in regulating the level of phosphorylation of α2 by upstream kinases. © 2007 Biovitrum AB.