A Genetically-Encoded FRET Sensor based on AMP-Activated Protein Kinase Reports Allosteric Kinase Activation
Résumé
AMPK is a multi-protein nanomachine that is activated by multiple, complex mechanisms, allowing fine tuning of AMPK activity in different situations of metabolic stress. Binding of adenine nucleotides to the gamma subunit plays a major role in either direct allosteric activation of AMPK or modulation of AMPK phosphorylation and dephosphorylation by upstream kinases and phosphatases. These activation mechanisms require crosstalk between AMPK subunits by a nucleotide-induced conformational switch. We have engineered an AMPK complex that allows a direct, real-time readout of the AMPK conformational state by fluorescence energy transfer (FRET). This molecular sensor confirms the exquisite sensitivity of AMPK to low micromolar concentrations of AMP, shows the exclusive ability of ATP, but not MgATP, to compete with AMP, and allows insight into the role of CBS domains for allosteric AMPK activation. It has potential applications as a tool for screening of allosteric activators of AMPK, and as a reporter of cellular energy state.