Fig. 2
From: Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism
FRET changes report AMPK allosteric activation. a AMP-concentration dependence of FRET (black) compared to AMP-induced allosteric activation (red) of AMPfret 1.0. This activation was determined with pre-activated AMPK in presence of 200 µM ATP, followed by immunoblotting for phosphorylated acetyl-CoA carboxylase (ACC; insert); quantified band intensities were normalized to control at 0 µM AMP. Data and error bars represent mean ± SEM (n ≥ 3). Note: ATP in the kinase assay shifts the curve to higher AMP concentrations. b GMP-concentration dependence of FRET, showing absence of FRET change. Points represent mean ± SEM (n ≥ 3). c Phosphorylation of T172 does not interfere with AMP-induced conformational changes reported by FRET. AMPfret 1.0 wild type (WT), pre-phosphorylated (P-T172), or mutated (T172A/D) proteins were incubated in absence (blue) or in presence of 20 µM AMP (orange), or with additional 3 mM Mg2+-complexed ATP (orange dashed red). Note: FRET changes are independent of MgATP. Data and error bars represent mean ± SEM (n ≥ 3). d AMPfret response to different AMPK activators. AMPfret 1.1 (left) or 2.1 (right) were incubated in absence (blue) or in presence of AMP (20 µM, orange), A-769662 (20 µM, green), 991 (2 µM, green), metformin (500 µM, white) or staurosporine (25 µM, purple). Note: no FRET change is induced by the indirect activator metformin or the protein kinase inhibitor staurosporine. Data and error bars represent mean ± SEM (n ≥ 4). e Additive effect of allosteric activators bound to CBS (AMP) and ADaM sites (A-769662). FRET ratio of AMPfret 2.0 incubated with A-769662 (5–20 µM) in absence (green) or presence of 20 µM AMP (green dashed orange). Note: a concentration-dependent effect of A-769662 is only significant in presence of AMP. Data and error bars represent mean ± SEM (n ≥ 3). All FRET signals are normalized to control (no adenylates). After checking normality and equality of variance, statistical significance was analysed by two-way ANOVA followed by Bonferroni multiple comparison. Means sharing the same letter do not differ significantly (p ≤ 0.02)
