Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism

Martin Pelosse,Cécile Cottet-Rousselle,Aurélie Dupont,Uwe Schlattner,Imre Berger,Cécile M Bidan,Kapil Gupta

doi:10.1038/s41467-019-08938-z

Abstract

AMP-activated protein kinase AMPK senses and regulates cellular energy state. AMPK activation by increasing AMP and ADP concentrations involves a conformational switch within the heterotrimeric complex. This is exploited here for the construction of a synthetic sensor of cellular energetics and allosteric AMPK activation, AMPfret. Based on engineered AMPK fused to fluorescent proteins, the sensor allows direct, real-time readout of the AMPK conformational state by fluorescence resonance energy transfer (FRET). AMPfret faithfully and dynamically reports the binding of AMP and ADP to AMPK γ-CBS sites, competed by Mg2+-free ATP. FRET signals correlate with activation of AMPK by allosteric mechanisms and protection from dephosphorylation, attributed here to specific CBS sites, but does not require activation loop phosphorylation. Moreover, AMPfret detects binding of pharmacological compounds to the AMPK α/β-ADaM site enabling activator screening. Cellular assays demonstrate that AMPfret is applicable in vivo for spatiotemporal analysis of energy state and allosteric AMPK activation.

Highlights

IntroductionAMPK activation by increasing AMP and ADP concentrations involves a conformational switch within the heterotrimeric complex
AMP-activated protein kinase AMPK senses and regulates cellular energy state
These two AMPK variants shared a cyan FP (CFP)-tag at the C-terminus of α2, while the yellow FP (YFP)-tag was either at the C-terminus of γ1 (AMPfret 1.0) or β2 (AMPfret pre2.0) (Supplementary Fig. 3a, b)

Summary

Introduction

AMPK activation by increasing AMP and ADP concentrations involves a conformational switch within the heterotrimeric complex. This is exploited here for the construction of a synthetic sensor of cellular energetics and allosteric AMPK activation, AMPfret. ATP is depleted due to imbalanced production and consumption, AMP and ADP levels increase and competitively replace ATP at up to two of the four cystathionine beta synthase (CBS) sites, CBS1 and CBS311–13. Allosteric activation by the ADaM, CBS1, and CBS3 sites appears to be additive[17] and, at least in vitro, sufficient for AMPK activation even in absence of α-T172 phosphorylation[16] Each of these activation mechanisms requires cross-talk between the catalytic α and the regulatory β- and/or γ-subunits. Solution studies using hydrogen/deuterium exchange mass spectroscopy (HDX-MS)[22,23] or luminescence energy transfer[24] provided insight into CBS site contributions to AMP-

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Conclusion