Figure 2.

Activator-3 and AMP share common activation mode for AMPK activation and Activator-3 enhances AMPK phosphorylation by upstream kinase LKB1 and protects AMPK complex from PP2C mediated dephosphorylation. (A–D) Recombinant human AMPK α1β1γ1 (A), AMPK α2β1γ1 (B), AMPK α2β2γ2 (C) or AMPK α2β2γ3 (D) were assayed in the presence of AMP (100 µM) alone and AMP (100 µM) and increasing concentration of Activator-3 (0–10 µM). Results are expressed as percentage increase of AMPK activity relative to DMSO control and represent the mean ±SD for three independent experiments. (E) Recombinant human AMPK α1β1γ1 was assayed in the presence of Activator-3 (30 nM; ~EC₅₀ concentration in vitro kinase assay) alone and Activator-3 (30 nM) and increasing concentrations of AMP (0–300 µM). Results are expressed as percentage increase of AMPK activity relative to DMSO control and represent the mean ± SD for three independent experiments. (F) Recombinant human AMPK α1β1γ1 was assayed in the presence of low concentration of ATP (20 µM) and high and physiological concentration of ATP (2 mM) and increasing concentration of Activator-3 (0–1 µM). Results are expressed as percentage change of AMPK activity relative to DMSO control (set as 100%). Results represent the mean ± SD for three independent experiments. (G) Recombinant AMPK complex was purified from HEK-293T cells by transient overexpression of the indicated construct as indicated in methods and assayed in presence of LKB1 alone or in presence of LKB1 and 100 µM AMP or increasing concentration of Activator-3 (10–100 nM) and the representative blot was quantified. (H) The effects of Activator-3 and AMP on dephosphorylation and inactivation of human recombinant AMPK α2β1γ1 by PP2C. Assays were performed either using vehicle (pAMPK alone) or with PP2C or vehicle with PP2C + Mg²⁺ or increasing concentration of Activator-3 (10–100 nM) or 100 µM AMP. The blots were probed with indicated antibodies.