Fig 2: p38 promotes mTORC1 activation and stress granule formation when PI3K is inactive.(A) p38 mediates mTORC1 activation when PI3K is inactive. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO) or wortmannin (100 nM, PI3K inhibitor). In addition, the cells were treated with carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor). MK2-pT334, Akt-pT308, Akt-pS473, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent five biological replicates. (B) Quantification of data shown in (A) when PI3K is active. Akt-pS473 and p70-S6K-pT389 were compared between carrier (DMSO) and LY2228820-treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The P-values for the Bonferroni multiple comparison tests are shown. ***P ≤ 0.001. (C) Quantification of data shown in (A) when PI3K is inactive. Akt-pS473 and p70-S6K-pT389 were compared between wortmannin- and wortmannin + LY2228820–treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The P-values for the Bonferroni multiple comparison tests are shown. *P ≤ 0.05; ***P ≤ 0.001. (D) p38 drives mTORC1 activity when PI3K is inactive. Quantification of data shown in Fig S14A. 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed t test across five biological replicates. Data represent the mean ± SEM. *P ≤ 0.05. (E) Prediction on the extent of mTORC1 inhibition upon LY2228820 treatment when PI3K is active or inactive. Prediction was performed with model V. The red lines depict the time points measured experimentally (Fig 5A–C). (F) When PI3K activity declines, p38 drives mTORC1 activity. Quantification of data shown in Fig S13L. MCF-7 cells were serum-starved and treated with arsenite for 60 min in the presence of different concentrations of wortmannin (as indicated, PI3K inhibitor) in carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor)-treated cells. p70-S6K-pT389 relative intensity was normalized separately for each wortmannin concentration. Significance of p70-S6K-pT389 inhibition by LY2228820 was tested using a two-tailed t test across five biological replicates. Data represent the mean ± SEM. *P ≤ 0.05. (G) p38 drives mTORC1 activity in several cell lines, as PI3K activity declines. Quantification of data shown in Fig S14D–G. MCF-7, CAL51, LN18, HEK293T, and HeLa cells were serum-starved and exposed to arsenite for 60 min in combination with wortmannin (100 nM, PI3K inhibitor) and/or LY2228820 (1 mM, p38 inhibitor). Data represent 3–4 biological replicates (see Fig S14D–G). 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed t test across three biological replicates. Data represent the mean ± SEM. *P ≤ 0.05; **P ≤ 0.01. (H) Stress granule numbers upon PI3K and p38 inhibition. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO), wortmannin (100 nM, PI3K inhibitor), LY2228820 (1 μM, p38 inhibitor), or wortmannin + LY2228820. Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent four biological replicates. White square indicates region of insert and blue arrow highlights stress granules; scale bar 10 μm. (I) Quantification of data shown in (H). The number of stress granules (SGs) per cell (normalized to the arsenite condition) across four biological replicates. Stress granule formation between carrier and LY2228820 as well as wortmannin- and wortmannin + LY2228820–treated cells was compared using a two-tailed t test across four biological replicates. Data represent the mean ± SEM. *P ≤ 0.01. ns, not significant.

Fig 3: Computational model predicts three stress inputs on PI3K, Akt, and mTORC1.(A) Scheme of model II, with only one stress input on PI3K (Fig S5). The corresponding AIC value is indicated at the top. (B) Simulated response of Akt-pT308, Akt-pS473, and p70-S6K-pT389 to arsenite stress in a system without (blue) or with (orange) wortmannin (PI3K perturbation) using model II, with one stress input on PI3K. Blue dots represent experimental data from arsenite time course (Fig S1C and D) and orange dots represent experimental data from arsenite time course + PI3K perturbation (Fig S3A and B). Lines represent computational simulation. Data represent the mean ± SEM. Simulations of all used observables are shown in Fig S6. (C) Scheme of model III, with two stress inputs on PI3K and Akt-pS473 (Fig S7). The corresponding AIC value is indicated at the top. (D) Simulated response of Akt-pT308, Akt-pS473, and p70-S6K-pT389 to arsenite stress in a system without (blue) or with (orange) wortmannin (PI3K perturbation) using model III, with two stress inputs on PI3K and Akt-pS473. Dots represent experimental data and lines represent computational simulation as described in (B). Data represent the mean ± SEM. Simulations of all used observables are shown in Fig S8. (E) Scheme of model V, with three stress inputs on PI3K, Akt-pS473, and mTORC1 (Fig S11). The corresponding AIC value is indicated at the top. (F) Simulated response of Akt-pT308, Akt-pS473, and p70-S6K-pT389 to arsenite stress in a system without (blue) or with (orange) wortmannin (PI3K perturbation) using model V, with three stress inputs on PI3K, Akt-pS473, and mTORC1. Dots represent experimental data and lines represent computational simulation as described in (B). Data represent the mean ± SEM. Simulations of all used observables are shown in Fig S12.

Fig 4: Stress activates mTORC1 to promote stress granule formation.(A) Arsenite stress enhances phosphorylation of eIF2α-S51 and mTORC1 substrates. MCF-7 cells were serum-starved and treated with arsenite. p70-S6K-pT389, 4EBP1-pT37/46, and eIF2α-pS51 were monitored by immunoblot. Data represent six biological replicates. (B) Quantification of data shown in (A). eIF2α-pS51, p70-S6K-pT389, and 4E-BP1-pT37/46 levels were compared between control and arsenite-treated cells using a two-tailed t test across six biological replicates. Data represent the mean ± SEM. **P ≤ 0.01; ***P ≤ 0.001. (C) Arsenite induces stress granules. MCF-7 cells were serum-starved and exposed to arsenite for 30 min. Stress granules were visualized by immunofluorescence staining of G3BP1. Data represent three biological replicates. Nuclei were visualized using Hoechst 33342. Scale bar: 10 μm. (D) mTOR mediates induction of p70-S6K-pT389 and 4EBP1-pT37/46 by stress. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO), everolimus (100 nM, mTORC1 inhibitor), or AZD8055 (100 nM, mTOR inhibitor). p70-S6K-pT389, 4E-BP1-pT37/46, G3BP1, and eIF2α-pS51 were monitored by immunoblot. Data represent four biological replicates. (E) Quantification of data shown in (D). p70-S6K-pT389 and 4E-BP1-pT37/46 levels were compared between the different treatments using a two-tailed t test across four biological replicates. Data represent the mean ± SEM. ns, not significant; *P ≤ 0.05; **P ≤ 0.01. (F) mTOR inhibition reduces stress granule numbers. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO), everolimus (100 nM, mTORC1 inhibitor), or AZD8055 (100 nM, mTOR inhibitor). Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent three biological replicates. White squares indicate region of insert and blue arrows highlight stress granules; scale bar 10 μm. (G) Quantification of data shown in (F): number of stress granules (SGs) per cell (normalized to the arsenite condition) across three biological replicates. Stress granule numbers were compared between the carrier and everolimus, or carrier and AZD8055-treated cells, using a two-tailed t test across three biological replicates. Data represent the mean ± SEM. *P ≤ 0.05.

Fig 5: Dynamic mTOR model.(A) Scheme summarizing the mTOR pathway. Amino acids directly activate mTORC1, which targets PRAS40-pS183, p70-S6K-pT389, and 4E-BP1-pT37/46. The insulin signal is transduced via the IR. The IR phosphorylates and recruits IRS1 which in turn recruits and activates PI3K. PI3K promotes the translocation of PDK1 to the membrane, where PDK1 phosphorylates Akt-pT308. Akt phosphorylates and inhibits the endogenous mTORC1 inhibitors PRAS40 at T246 and TSC2 at T1462. Thus, Akt activates mTORC1. p70-S6K inhibits IRS1 by direct phosphorylation at S636/639. PI3K also activates mTORC2, which targets Akt-pS473. In this study, we show that upon stress, mTORC1 is activated via the PI3K axis to promote stress granule assembly. (B) Topology of model I without stress input (Supplemental Data 1). Brown squares = species included in the model, circles = species variants (P, phosphorylation at the indicated site; cyt, cytosolic localization; mem, cell membrane localization; and *, active state), dark brown = observable species, species in ellipses = possible inputs to the model (insulin and amino acids) and inhibitory agents (MK-2206 and wortmannin), dark blue lines = mTORC2 activity, and light blue lines = mTORC1 activity. (C) Wortmannin fully inhibits PI3K (100%) in MCF-7 cells upon arsenite treatment. Akt-T308 phosphorylation (data shown in Fig S3A) was used to calculate the extent of PI3K inhibition upon wortmannin treatment. A one-way ANOVA was used to calculate P-values of Akt-T308 phosphorylation upon arsenite and arsenite + wortmannin treatment. As under wortmannin treatment, there was no significant induction of Akt-pT308 (P = 0.3226); the extent of inhibition for wortmannin was set to 100%. (D) MK-2206 partially inhibits Akt (83%) in MCF-7 cells upon arsenite treatment. TSC2-T1462 phosphorylation (data shown in Fig S3C) was used as to calculate the extent of Akt inhibition upon MK-2206 treatment. A one-way ANOVA was used to calculate P-values of TSC2-1462 phosphorylation upon arsenite treatment and arsenite + MK-2206 treatment. As under MK-2206 treatment, there was still a significant induction of TSC2-pT1462 (P = 0.0239), the extent of inhibition for MK-2206 was calculated by comparing the slopes of induction curves (83%) between carrier and MK-2206 treatment (indicated as red lines). (E) Summary of the extent of inhibition for wortmannin and MK-2206 in MCF-7 cells under arsenite treatment.