Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism

Martin Pelosse, Cécile Cottet-Rousselle, Cécile M. Bidan, Aurélie Dupont, Kapil Gupta, Imre Berger () and Uwe Schlattner ()
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Martin Pelosse: Rue de la Piscine, Domaine Universitaire
Cécile Cottet-Rousselle: Rue de la Piscine, Domaine Universitaire
Cécile M. Bidan: University of Grenoble Alpes, CNRS, Laboratoire Interdisciplinaire de Physique (LIPhy)
Aurélie Dupont: University of Grenoble Alpes, CNRS, Laboratoire Interdisciplinaire de Physique (LIPhy)
Kapil Gupta: University of Bristol
Imre Berger: University of Bristol
Uwe Schlattner: Rue de la Piscine, Domaine Universitaire

Nature Communications, 2019, vol. 10, issue 1, 1-13

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-08938-z

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