Figure 6.

Translation inhibitors reverse GCN2 activation.A, 3T3 cells were cultured in normal media (single left lane) or media lacking Leu for 4 h to induce GCN2 activation. At time zero, torin-2, cycloheximide (CHX), lactimidomycin (Lactim), puromycin (Puro), or leucine was added in the concentrations detailed in the Experimental procedures. Lysates were probed for activation of GCN2 (p-GCN2 T898), ATF4 as a marker of the ISR or p-4EBP1 T36/45 as an indicator of mTORC1 activity. GRB2 was used as a loading control. Note that translation inhibitors do not affect mTORC1 activity and have a differential effect of ATF4 (which is newly transcribed and translated after GCN2 activation). Data are representative of three independent experiments. B and C, quantification of intracellular Leu by LC-MS. 3T3 cells were cultured as indicated and washed with PBS prior to lysis in acetonitrile/methanol. Experiments were performed using cell samples cultured and processed independently (n = 4). Pairwise comparisons between experimental samples were made by t test in Prism. D, effect of torin-2 or sapansertib on halofuginone-mediated GCN2 activation. 3T3 cells were cultured in normal DMEM media (which lacks proline) in the presence of halofuginone with or without torin-2 or sapanisertib (100 nM each). Lysates were probed for activation of GCN2 (p-GCN2 T898) or mTORC1 activity using p-4EBP1 T36/45. GRB2 was used as a loading control. Data are presentative of two independent experiments.