Time-resolved analysis of amino acid stress identifies eIF2 phosphorylation as necessary to inhibit mTORC1 activity...

Amino acid availability is sensed by GCN2 (general control nonderepressible 2) and mechanistic target of rapamycin complex 1 (mTORC1), but how these two sensors coordinate their respective signal transduction events remains mysterious. In this study we utilized mouse genetic models to investigate the role of GCN2 in hepatic mTORC1 regulation upon amino acid stress induced by a single injection of asparaginase. We found that deletion of Gcn2 prevented hepatic phosphorylation of eukaryotic initiation factor 2α to asparaginase and instead unleashed mTORC1 activity. This change in intracellular signaling occurred within minutes and resulted in increased 5'-terminal oligopyrimidine mRNA translation instead of activating transcription factor 4 synthesis. Asparaginase also promoted hepatic mRNA levels of several genes which function as mTORC1 inhibitors, and these genes were blunted or blocked in the absence of Gcn2, but their timing could not explain the early discordant effects in mTORC1 signaling. Preconditioning mice with a chemical endoplasmic reticulum stress agent before amino acid stress rescued normal mTORC1 repression in the liver of Gcn2^-/- mice but not in livers with both Gcn2 and the endoplasmic reticulum stress kinase, Perk, deleted. Furthermore, treating wildtype and Gcn2^-/- mice with ISRIB, an inhibitor of PERK signaling, also failed to alter hepatic mTORC1 responses to asparaginase, although administration of ISRIB alone had an inhibitory GCN2-independent effect on mTORC1 activity. Taken together, the data show that activating transcription factor 4 is not required, but eukaryotic initiation factor 2α phosphorylation is necessary to prevent mTORC1 activation during amino acid stress.