Journal: The Journal of Biological Chemistry

Article Title: A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling

doi: 10.1016/j.jbc.2022.102629

Figure Lengend Snippet: Torins inhibit the GCN2 pathway. A , effect of sapanisertib, omipalisib, or ETP-46464 on Ddit3 ::mCherry activity. Drugs were used at 1 μM. Shown in each panel are WT Ddti3::mCherry 3T3 and GCN2-deficient cells ( Eif2ak4 -/- ) untreated (Ctrl) over time and normalized to cellular confluence. The first panel (DMSO control) indicated the control for effects of the solvent. Note that a common control was used for sapanisertib and omipalisib. B , torin-1 or torin-2 was added to Ddti3 ::mCherry 3T3 cells in Leu-free media at 0.1, 1, or 10 μM and mCherry signal recorded and normalized to confluence. C , pathway immunoblotting analysis of the effect of torins on the GCN2 ISR versus the mTORC1 pathway. 3T3 cells were starved in Leu-deficient media for 4 h in the presence of the drugs indicated (0.01–10 μM) added at time zero. Lysates were probed for activation of GCN2 (p-GCN2 T898), total GCN2, ATF4, p-4EBP1 T36/45, or total 4EBP1. GRB2 was used as a loading control. Data are representative of at least five independent experiments. DMSO, dimethyl sulfoxide.

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies in blocking buffer: PERK (C33E10) (1:2000; 3192, Cell Signaling Technology, CST), p-PERK T980 (16F8) (1:300; 3179, CST), GCN2 (1:800; 3302, CST), p-GCN2 T899 (1:1000; ab75836, Abcam), eIF2α (D7D3) XP (1:1000; 5324, CST), p-eIF2α S51 (119A11) (1:1000; 3597, CST), GRB2 (1:1000; 610112, Becton Dickinson), 4EBP1 (53H11) (1:1000; 9644, CST), p-4EBP1 T37/46 (236B4) (1:1000; 2855, CST), mCherry (1:2000; ab167453, Abcam), Akt (pan) (C67E7) (1:1000; 4691, CST), p-Akt S473 (1:1000; D9E9 XR, 4060, CST), Vinculin (1:1000; 13901, CST), ATF4 (1:1000, sc-390063, Santa Cruz), and CHOP L63F7 (1:500; 2895, CST).

Techniques: Activity Assay, Western Blot, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling

doi: 10.1016/j.jbc.2022.102629

Figure Lengend Snippet: Role of eIF2α S52 and PERK . A , pathway immunoblotting analysis in mutant eIF2α cells. 3T3 WT cells or 3T3 cells with a biallelic point mutation at serine 52 to alanine ( Eif2s1 S52A) were cultured in media with Leu (+) or lacking Leu (−) for 4 h without or with GCN2-IN-6 (10 μM), torin-1 (1 μM) rapamycin (1 μM) coincident with amino acid starvation. Data are depicted as representative one of two independent experiments. B , pathway immunoblotting analysis for GCN2- versus PERK-ISR specificity. 3T3 cells were cultured in media with Leu (Ctrl) or lacking Leu (-) for 4 h in the absence or presence of 1 μM thapsigargin. Cells were treated additionally with exogenous leucine, GCN2-IN-6 (10 μM), rapamycin (1 μM), or torin-1 (1 μM). PERK activation was detected with a polyclonal antibody to T980, required for kinase activation or an antibody to total PERK, which detects the mobility shift associated with PERK activation. Note that torin-1 does not inhibit PERK activation, controlled by the inhibition of p-4EBP1 T36/45, while GCN2-IN-6 inhibits PERK activation. Data are depicted as one of two independent experiments. ISR, integrated stress response.

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies in blocking buffer: PERK (C33E10) (1:2000; 3192, Cell Signaling Technology, CST), p-PERK T980 (16F8) (1:300; 3179, CST), GCN2 (1:800; 3302, CST), p-GCN2 T899 (1:1000; ab75836, Abcam), eIF2α (D7D3) XP (1:1000; 5324, CST), p-eIF2α S51 (119A11) (1:1000; 3597, CST), GRB2 (1:1000; 610112, Becton Dickinson), 4EBP1 (53H11) (1:1000; 9644, CST), p-4EBP1 T37/46 (236B4) (1:1000; 2855, CST), mCherry (1:2000; ab167453, Abcam), Akt (pan) (C67E7) (1:1000; 4691, CST), p-Akt S473 (1:1000; D9E9 XR, 4060, CST), Vinculin (1:1000; 13901, CST), ATF4 (1:1000, sc-390063, Santa Cruz), and CHOP L63F7 (1:500; 2895, CST).

Techniques: Western Blot, Mutagenesis, Cell Culture, Activation Assay, Mobility Shift, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling

doi: 10.1016/j.jbc.2022.102629

Figure Lengend Snippet: mTORC1 regulates GCN2 activity postactivation. A , time-dependent GCN2 pathway analysis of torin-1 on GCN2 activation. 3T3 cells were starved for Leu for 1 h or 4 h in the absence or presence of GCN2-IN-6 (10 μM), rapamycin (1 μM), everolimus, (1 μM), and torin-1 (1 μM). Lysates were probed for activation of GCN2 (p-GCN2 T898), total GCN2, ATF4 as a marker of the ISR or p-4EBP1. GRB2 was used as a loading control. Note that torin-1 does not inhibit p-GCN2 or ATF4 at 1 h. B , 3T3 cells were cultured in Leu-free media for the times indicated in the absence of presence of torin-1 (100 nM). The GRB2 loading control indicates the expected time-dependent decline in total protein amounts in the lysates over the 24 h starvation period. C , quantification of the inhibitory effect of RapaLink-1 (10 μM) in Ddti3 ::mCherry 3T3 cells upon L-leu starvation compared with rapamycin (1 μM) and sapanisertib (10 μM). Data are depicted as mean ± SEM of three technical replicates and represent one of two independent experiments. D , pathway immunoblotting analysis of the effects of RapaLink-1 compared to its parent inhibitors (rapamycin and sapanisertib) using Leu-starved 3T3 cells for 4 h. Data are representative of three independent experiments. ISR, integrated stress response.

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies in blocking buffer: PERK (C33E10) (1:2000; 3192, Cell Signaling Technology, CST), p-PERK T980 (16F8) (1:300; 3179, CST), GCN2 (1:800; 3302, CST), p-GCN2 T899 (1:1000; ab75836, Abcam), eIF2α (D7D3) XP (1:1000; 5324, CST), p-eIF2α S51 (119A11) (1:1000; 3597, CST), GRB2 (1:1000; 610112, Becton Dickinson), 4EBP1 (53H11) (1:1000; 9644, CST), p-4EBP1 T37/46 (236B4) (1:1000; 2855, CST), mCherry (1:2000; ab167453, Abcam), Akt (pan) (C67E7) (1:1000; 4691, CST), p-Akt S473 (1:1000; D9E9 XR, 4060, CST), Vinculin (1:1000; 13901, CST), ATF4 (1:1000, sc-390063, Santa Cruz), and CHOP L63F7 (1:500; 2895, CST).

Techniques: Activity Assay, Activation Assay, Marker, Cell Culture, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling

doi: 10.1016/j.jbc.2022.102629

Figure Lengend Snippet: 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 . 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.

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies in blocking buffer: PERK (C33E10) (1:2000; 3192, Cell Signaling Technology, CST), p-PERK T980 (16F8) (1:300; 3179, CST), GCN2 (1:800; 3302, CST), p-GCN2 T899 (1:1000; ab75836, Abcam), eIF2α (D7D3) XP (1:1000; 5324, CST), p-eIF2α S51 (119A11) (1:1000; 3597, CST), GRB2 (1:1000; 610112, Becton Dickinson), 4EBP1 (53H11) (1:1000; 9644, CST), p-4EBP1 T37/46 (236B4) (1:1000; 2855, CST), mCherry (1:2000; ab167453, Abcam), Akt (pan) (C67E7) (1:1000; 4691, CST), p-Akt S473 (1:1000; D9E9 XR, 4060, CST), Vinculin (1:1000; 13901, CST), ATF4 (1:1000, sc-390063, Santa Cruz), and CHOP L63F7 (1:500; 2895, CST).

Techniques: Activation Assay, Cell Culture, Marker, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Lysis

Journal: eLife

Article Title: Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace

doi: 10.7554/eLife.68799

Figure Lengend Snippet: ( A ) Inhibition of the mTORC1 kinase activity by DEPTOR. mTORC1 schematic illustrating the two known substrate-binding sites, the TOS-binding site on RAPTOR and the FRB-binding site on mTOR, for 4EBP1 and S6K1 (left panel). Phosphorylation of substrates was analyzed by western blots using anti p-4EBP1 (Thr37/46) or anti p-S6K1 (Thr389) primary antibodies (right panel). Images of western blots with long (L) and short exposures (S) are shown. The S6K1 367-404 peptide encompasses only the FRB-binding site of S6K1. The bottom panels show the quantification of the phosphorylation levels of the substrates based on the western blots from three independent experiments (mean ± SD). Band intensities were normalized to the control (0 µM DEPTOR) and data were plotted and fit by non-linear regression to determine IC 50 and y min (the residual activity at high [DEPTOR]) as described in Materials and methods. See for complementary experiment using Phos-tag SDS PAGE detection. ( B ) Inhibition of mTORC1 by PRAS40. Inhibition of 4EBP1 phosphorylation is complete under identical reaction conditions as carried out for DEPTOR. ( C ) DEPTOR has no effect on the apparent K M,4EBP1 . The phosphorylation of 4EBP1 in the absence and presence of DEPTOR (25 µM or 50 µM), normalized to the 5 µM 4EBP1 is plotted (mean ± SD, n ≥ 3) and K M values were fit as described in Materials and methods. ( D ) Inhibition of monomeric mTOR ΔN -mLST8 (left panel) by DEPTOR (mean ± SD, n ≥ 3). Similar to the wild-type mTORC1 complex, partial inhibition is observed (right panel). Figure 1—source data 1. Uncropped blots. Figure 1—source data 2. Data values for .

Article Snippet: Antibody , Anti-P-4EBP1 (T37/46) (Rabbit, polyclonal) , Cell Signalling , Cat#9459L; RRID: AB_330985 , WB (1:1000).

Techniques: Inhibition, Activity Assay, Binding Assay, Western Blot, SDS Page

Journal: eLife

Article Title: Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace

doi: 10.7554/eLife.68799

Figure Lengend Snippet: ( A ) DEPTOR deletion variants tested as inhibitors and substrates for mTORC1. ( B ) Immunoblots showing the residual phosphorylation of 4EBP1 in the presence of various DEPTOR deletion variants. Images of western blots with long (L) and short exposures (S) are shown. ( C ) and ( D ) quantification of western blots of phosphorylated 4EBP1 plotted as a fraction of the control (0 µM inhibitor) vs. inhibited (mean ± SD, n ≥ 3) and fit to a non-linear regression for all deletion variants to determine IC 50 . While all experiments were performed at 30°C, inhibition of mTORC1 by the PDZ domain was tested at 20°C for 20 min as the domain stability was low. N-terminally extended PDZ constructs that showed increased temperature stability showed no inhibition of mTORC1 . To demonstrate that the temperature difference had no effect on the inhibition, DEPTOR (WT) was tested at 20°C for mTORC1 inhibition . The data shown for DEPTOR (WT) is also part of . DEPTOR 13A inhibition of the mTORC1 A1459P mutant is shown in . Figure 2—source data 1. Uncropped blots. Figure 2—source data 2. Data values for .

Article Snippet: Antibody , Anti-P-4EBP1 (T37/46) (Rabbit, polyclonal) , Cell Signalling , Cat#9459L; RRID: AB_330985 , WB (1:1000).

Techniques: Western Blot, Inhibition, Construct, Mutagenesis

Journal: eLife

Article Title: Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace

doi: 10.7554/eLife.68799

Figure Lengend Snippet: ( A ) Increasing concentrations of RHEB-GTP lead to activated mTORC1 and at the same time result in a decreased DEPTOR IC 50 (substrate was 4EBP1). Band intensities reflecting P-4EBP1 were normalized to the control (0 µM DEPTOR) and the data (mean ± SD, n ≥ 3) was fit by a nonlinear regression to determine IC 50 . ( B ) Cancer-associated, hyperactive mutant mTORC1-A1459P also shows a decreased DEPTOR IC 50 . Band intensities reflecting P-4EBP1 were normalized to the control (0 µM DEPTOR) and the data (mean ± SD, n ≥ 3) was fit by a nonlinear regression to determine IC 50 . ( C ) Cryo-EM reconstruction of A1459P mTORC1 mutant and DEPTOR reveals the loss of an mTOR helix at the mutation site. The site of mutation lies within the hinges of mTORC1 which are involved in introducing major conformational changes in mTOR upon RHEB-induced activation. ( D ) Alignment of WT and mutant A1459 mTORC1 on the PDZ-binding site in the FAT domain (shown with green density bound to the PDZ domain illustrated with magenta spheres) reveals a shift of mTOR N-heat domain for mTORC1 A1459P/DEPTOR (yellow density) with respect to mTORC1 WT/DEPTOR (gray density). This shift increases the crevice between the FAT and the N-heat domains in mTORC1 A1459P and creates an easier access for DEPTOR PDZ to its binding site. ( E ) Comparison of PDZ binding to wild-type and mutant mTORC1 analyzed by SPR. PDZ (construct 305–409) binds to wild-type mTORC1 with slow on/off kinetics and lower affinity, whereas it binds the mutant mTORC1, with fast on/off kinetics and 10-fold greater affinity. The total binding data were fit to a model with one-site specific binding combined with a linear non-specific component. Figure 6—source data 1. Data values for .

Article Snippet: Antibody , Anti-P-4EBP1 (T37/46) (Rabbit, polyclonal) , Cell Signalling , Cat#9459L; RRID: AB_330985 , WB (1:1000).

Techniques: Mutagenesis, Cryo-EM Sample Prep, Activation Assay, Binding Assay, Construct

Journal: eLife

Article Title: Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace

doi: 10.7554/eLife.68799

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-P-4EBP1 (T37/46) (Rabbit, polyclonal) , Cell Signalling , Cat#9459L; RRID: AB_330985 , WB (1:1000).

Techniques: Recombinant, Plasmid Preparation, Variant Assay, Mutagenesis, Protease Inhibitor, Staining, Expressing, Software