Fig 2: mTOR phosphorylate GCN2atS230.A, the indicated HEK293T cell variants were cultured in AA replete, or AA deplete conditions and treated with Torin1 [1 μM] as indicated. Phospho-tag gel (12.5%) was used to evaluate the overall phosphorylation of GCN2; retarded migration of band reflects more phosphorylated protein and vice versa. B, kinase prediction ranking of the S230-GCN2 motif. C, the relative intensity of the phosphorylated S230 peptide was determined through mass spectrometry of FLAG-GCN2 immunoprecipitated from HEK293T NPRL2 KO cells that were starved from AA for 1 h and either treated with Torin1 [1 μM] or left untreated as a control. Each column represents 3 biological replicates ± SD. The p-value was calculated using a one-tailed Type II Student's t test. D, GCN2 protein alignment of Homo sapiens (human) and Saccharomyces cerevisiae (yeast), lines represent conversative sequence while gaps represent distinct sequences. S230 motif is emphasized as distinct to human. E, WT and S230A mutant of full-length FLAG-GCN2 were transfected into HEK293T GCN2 KO and GCN2 NPRL2 DKO cells. These cells were cultured in AA replete, or AA deplete conditions and treated with Torin1 [1 μM] as indicated. Shown are phospho-tag (7.5%) and regular representative immunoblots. F, the indicated domain of GCN2 was cloned and subjected to S230A mutation. Shown are phospho-tag (12.5%) and regular representative immunoblots of HEK293T NPRL2 KO cells expressing either WT or S230A of this domain and treated with Torin1 [1 μM] for 1 h or left untreated as a control. G, HEK293T GCN2 KO cells were transfected with the indicated full-length FLAG-GCN2 and cultured in the indicated conditions. Shown are representative immunoblots and relative quantification of P-GCN2-T899 and P-eIF2α-S51.

Fig 3: mTORC1 overactivity promotes GCN2 autophosphorylation regardless of its binding ability to uncharged tRNAs or ribosomal proteins.A, Wild-type NPRL2 KO RPMI8226 cells were cultured in AA deplete medium for the indicated time points. GCN2 autophosphorylation was assessed by immunoblotting against P-GCN2-T899 and total-GCN2. mTORC1 activity was assessed by immunoblotting against P-S6K1-T389 and total-S6K1. B, human GCN2 protein domain organization scheme of full length and truncated used in (C and D). C, the indicated constructs of GCN2 were transfected into the indicated cells and their autophosphorylation on T899 was assessed as described in (A). D, HEK293T GCN2 KO cells were transfected with the indicated proteins. GCN2 and mTORC1 activities were assessed as described in (A). Shown are representative immunoblots and relative quantification of P-T899-GCN2 (to time 0).

Fig 4: Growth differentiation factor 15 (GDF15)/ glial cell‐derived neurotrophic factor family receptor a‐like (GFRAL)‐mediated signaling in cisplatin‐resistant gastric cancer cells could be through general control nonderepressible 2 (GCN2). (A, B) siGDF15 and siGFRAL were transfected into (A) AGS cisplatin‐resistant (CisR) and (B) NUGC‐3CisR cells. (C) AGSCisR and (D) NUGC‐3CisR cells were treated with SPP86 (5 μΜ) for 24 h. Upstream regulators of the eukaryotic initiation factor 2α (eIF2α) and eIF2α‐activating transcription factor 4 (ATF4)‐xCT pathways were analyzed using western blotting. Graph is presented as mean ± SEM (n ≥ 3). *Significant vs. individual control (Con). PERK, PKR‐like endoplasmic reticulum kinase; PKR, protein kinase R.

Fig 5: General control nonderepressible 2 (GCN2) is responsible for growth differentiation factor 15 (GDF15)‐mediated glial cell‐derived neurotrophic factor family receptor a‐like (GFRAL)‐eukaryotic initiation factor 2α (eIF2α)‐activating transcription factor 4 (ATF4)‐xCT signaling and cisplatin resistance. (A, B, D) After GDF15 overexpression by pcDNA‐GDF15 (G, pcDNA‐GDF15; V, pcDNA alone), cells were treated with siRNAs against (A) protein kinase R (PKR), (B) heme‐regulated eIF2α kinase (HRI), and (D) GCN2 for 48 h. The effect of siRNAs against PKR, HRI, and GCN2 on GDF15‐mediated eIF2α‐ATF4‐xCT regulation was assessed using western blotting. (C) Effects of siHRI and siPKR on cisplatin resistance in cisplatin‐resistant (CisR) cells was evaluated with sulforhodamine B (SRB) assay. Graph is presented as mean ± SEM (n ≥ 3). *Significant vs. individual control. **Significant vs. siScr with GDF15 overexpression.