Activation and phosphorylation of heterotrimeric AMPK complexes by AMPKK1, AMPKK2 and recombinant GST-LKB1:STRADα:MO25α complexes, and the effect of AMP. (a) Activation of α1β1γ1 and α2β1γ1 complexes separated from purified rat liver AMPK. The AMPKα1- or AMPKα2-containing complexes were purified by immunoprecipitation and activation of the resuspended immunoprecipitates by the three AMPKK preparations examined. The results are expressed as activation relative to the control without added AMPKK. (b) Quantification by western blotting of the relative amounts of LKB1, STRADα and MO25α polypeptides in the three AMPKK preparations used in (a). A small amount of degradation is detectable due in part to the heavy loadings of the GST-LKB1 and FLAG-STRADα. The identity of the polypeptide labeled '?' in the anti-LKB1 blot is not known. (c) Effect of AMP on the activation of α1β1γ1 and α2β1γ1 heterotrimers of AMPK, and of GST-AMPKα1 catalytic domain, by AMPKK1, AMPKK2 and the recombinant GST-LKB1:STRADα:MO25α complex. AMPKK activity was measured as in Figure 3 with or without 200 μM AMP. The results are expressed as ratios of the activities obtained with and without AMP.