The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis (Tunicamycin)

In response to a variety of upstream growth and oncogenic signals, the mechanistic target of rapamycin complex 1 (mTORC1) promotes anabolic metabolism, in part, through activation of downstream transcription factors. The transcription factor activating transcription factor 4 (ATF4) has been previously shown to function downstream of mTORC1 signaling to promote de novo purine synthesis and this activation of ATF4 can occur independently of the canonical activation of ATF4 through the integrated stress response (ISR). Here, we show that ATF4 activation through mTORC1 signaling drives a specific transcriptional program through ATF4 which is comprised of only a subset of genes involved in the broader cellular stress response. We find genes involved in amino acid uptake, biosynthesis, and charging by tRNA synthetases display transcriptional changes downstream of both mTORC1 and ATF4. We discovered that ATF4 activation contributes to the mTORC1-stimulated increase in protein synthesis, highlighting the importance of these transcriptional changes. Additionally, we observe regulation of the cystine transporter SLC7A11 by ATF4. We show that SLC7A11 is important for cell survival and is one mechanism by which cells with active mTORC1 signaling produce glutathione. Thus, ATF4 downstream of mTORC1 signaling regulates protein and glutathione synthesis. Atf4 WT and KO MEFs were grown to 70% confluence in 6 cm plates and were serum-starved in the presence of 2-mercaptoethanol and 1X MEM NEAA mixture and treated with vehicle (DMSO) or 20 nM rapamycin (LC Laboratories, R5000) for 30 min prior to additional treatment with vehicle (water) or 500 nM insulin (Alpha Diagnostic, INSL 16-N-5 ) for 16 h, or treated with vehicle (DMSO) or 2 mg/mL tunicamycin (Sigma-Aldrich, T7765) for 4, 8 or 16 h