Regulation of AMPK inhibition by metabolism through 14-3-3 proteins – AMPKill

Phosphorylation governs signal propagation and is involved in cell growth, division and viability. 14-3-3 proteins are important phospho-transducers which bind phospho-substrates and regulate their activities by various means, being widely considered as decoders of phosphorylation events. This project aims at extending the functions of 14-3-3 proteins as regulators of phosphorylation. More specifically, we will decipher how 14-3-3s work in concert with a phosphatase (PP1) in response to nutrient cues to inhibit the conserved kinase AMPK (5'-AMP-activated kinase) using yeast as a model.
Glucose regulates many aspects of the cell’s physiology and in yeast, PP1 is in charge of the glucose-mediated dephosphorylation (inhibition) of AMPK. Yet, how glucose triggers PP1 activity towards AMPK is a long-standing question. By genetic screening, we identified mutations in conserved residues of a 14-3-3 protein leading to aberrant AMPK signaling, as well as mutations in the regulatory subunit of PP1 Reg1. 14-3-3 interacts with Reg1, but this interaction no longer occurs when 14-3-3 are mutated, suggesting a role for 14-3-3 in in the regulation of PP1 by metabolism. Interestingly, intricate connections between 14-3-3s and PP1 were previously noted in metazoans. To understand the mechanistic basis of this regulation, we will pursue our ongoing characterization of the identified 14-3-3 and Reg1 mutants and decipher the network of 14-3-3/PP1/AMPK interactions and its regulation by metabolism. To achieve these goals, we will use a variety of approaches, ranging from the characterization of mutants (phenotypes, phosphosubstrate localization/phosphorylation, signaling pathway analysis) to the analysis of protein-protein interactions in vitro or in vivo. In particular, we will measure protein interaction dynamics in real-time at a high temporal resolution using a NanoLuc-based bimolecular complementation assay. This should unravel new aspects of 14-3-3 functions and kinase regulation.