Inna A Nikonorova, Emily T Mirek, Christina C Signore, Michael P Goudie, Ronald C Wek, Tracy G Anthony

Amino acid availability is sensed by general control nonderepressible 2 (GCN2) 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 ofGcn2prevented hepatic phosphorylation of eukaryotic initiation factor 2 alpha (eIF2) to asparaginase and instead unleashed mTORC1 activity. This change in intracellular signaling occurred within minutes and resulted in increased 5' terminal oligopyrimidine (TOP) mRNA translation instead of activating transcription factor 4 (ATF4) synthesis.  Asparaginase also promoted hepatic mRNA levels of several mTORC1 inhibitors between 3 and 18 h and these were blunted or blocked in the absence ofGcn2, but their timing could not explain the early discordant effects in mTORC1 signaling. Pre-conditioning mice with a chemical endoplasmic reticulum (ER) stress agent before amino acid stress rescued normal mTORC1 repression in the liver ofGcn2-/-mice but not in livers with bothGcn2and the ER stress kinase,Perk, deleted. Furthermore, treating wild type andGcn2-/-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 ATF4 is not required but eIF2 phosphorylation is necessary to prevent mTORC1 activation during amino acid stress.