Pharmacological eEF 2K activation promotes cell death and inhibits cancer progression
2016; Springer Nature; Volume: 17; Issue: 10 Linguagem: Inglês
10.15252/embr.201642194
ISSN1469-3178
AutoresAude De Gassart, Olivier Demaria, Rébecca Panès, Léa Zaffalon, Alexey G. Ryazanov, Michel Gilliet, Fabio Martinon,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoArticle29 August 2016free access Source DataTransparent process Pharmacological eEF2K activation promotes cell death and inhibits cancer progression Aude De Gassart Aude De Gassart Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Olivier Demaria Olivier Demaria Department of Dermatology, CHUV, Lausanne, Switzerland Search for more papers by this author Rébecca Panes Rébecca Panes Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Léa Zaffalon Léa Zaffalon Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Alexey G Ryazanov Alexey G Ryazanov Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Michel Gilliet Michel Gilliet Department of Dermatology, CHUV, Lausanne, Switzerland Search for more papers by this author Fabio Martinon Corresponding Author Fabio Martinon [email protected] orcid.org/0000-0002-6969-822X Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Aude De Gassart Aude De Gassart Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Olivier Demaria Olivier Demaria Department of Dermatology, CHUV, Lausanne, Switzerland Search for more papers by this author Rébecca Panes Rébecca Panes Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Léa Zaffalon Léa Zaffalon Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Alexey G Ryazanov Alexey G Ryazanov Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Michel Gilliet Michel Gilliet Department of Dermatology, CHUV, Lausanne, Switzerland Search for more papers by this author Fabio Martinon Corresponding Author Fabio Martinon [email protected] orcid.org/0000-0002-6969-822X Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Author Information Aude De Gassart1, Olivier Demaria2, Rébecca Panes1, Léa Zaffalon1, Alexey G Ryazanov3, Michel Gilliet2 and Fabio Martinon *,1 1Department of Biochemistry, University of Lausanne, Epalinges, Switzerland 2Department of Dermatology, CHUV, Lausanne, Switzerland 3Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers The State University of New Jersey, Piscataway, NJ, USA *Corresponding author. Tel: +41 21 692 5695; E-mail: [email protected] EMBO Reports (2016)17:1471-1484https://doi.org/10.15252/embr.201642194 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Activation of the elongation factor 2 kinase (eEF2K) leads to the phosphorylation and inhibition of the elongation factor eEF2, reducing mRNA translation rates. Emerging evidence indicates that the regulation of factors involved in protein synthesis may be critical for controlling diverse biological processes including cancer progression. Here we show that inhibitors of the HIV aspartyl protease (HIV-PIs), nelfinavir in particular, trigger a robust activation of eEF2K leading to the phosphorylation of eEF2. Beyond its anti-viral effects, nelfinavir has antitumoral activity and promotes cell death. We show that nelfinavir-resistant cells specifically evade eEF2 inhibition. Decreased cell viability induced by nelfinavir is impaired in cells lacking eEF2K. Moreover, nelfinavir-mediated anti-tumoral activity is severely compromised in eEF2K-deficient engrafted tumors in vivo. Our findings imply that exacerbated activation of eEF2K is detrimental for tumor survival and describe a mechanism explaining the anti-tumoral properties of HIV-PIs. Synopsis Elongation factor 2 kinase (eEF2K) is part of an adaptation pathway that enables cells to cope with nutrient deprivation by regulating translation rates. Pharmacological activation of the eEF2K pathway via the anti-viral drug nelfinavir rewires this stress adaptation program into a response that is detrimental for tumor growth. The anti-viral and anti-tumoral compound nelfinavir is a potent eEF2K activator. eEF2K activation contributes to NFR-mediated cell death. eEF2K deficiency impairs nelfinavir-mediated antitumoral activity. Introduction Drug repositioning is emerging as a successful strategy that accounts for a significant share of newly US Food and Drug Administration (FDA)-approved drugs in recent years 12. The study of unanticipated drug effects in patients can uncover new pathways and mechanisms of biological interest that can contribute to the development of new therapeutics. Early studies in HIV patients treated with the HIV aspartyl protease inhibitors (HIV-PIs) have suggested interesting off-target actions of these molecules in cancer 345. The HIV-PIs target the viral protease and are widely used to treat HIV. In 1997, nelfinavir (NFR) became one of the first HIV-PI to be approved by the FDA for HIV treatment. In addition to its antiretroviral effects, this safe and orally available drug shows promising anti-tumoral activity in mice and humans 678910. Several phase I and phase II clinical trials are investigating the efficacy of NFR repositioning in cancer with encouraging initial results 1112131415. NFR has been shown to affect multiple pathways regulating cellular homeostasis including the proteasome, the kinase AKT, and the unfolded protein response (UPR) 16. Recently, we found that NFR is a robust inducer of the integrated stress response (ISR), an adaptation response that promotes an ATF4-dependant transcriptional program 17. While some of these pathways may contribute to its anti-tumoral activity, none has been demonstrated to be essential and the molecular basis of NFR-mediated anti-tumoral effects remains unknown 161819. In this study, we generated clonal populations of cells with increased resistance to NFR-mediated toxicity. These clones showed an unaltered activation of most NFR-mediated responses, including those related to the ISR. However, among possible stress and survival pathways, we observed the downregulation of the eukaryotic translation elongation factor 2 kinase (eEF2K), suggesting that it could be a major player driving nelfinavir cytotoxic effects. Activation of eEF2K is one of the pathways that participate to the restoration of cellular homeostasis upon conditions of nutrient or energy depletion by decreasing translation rates at the stage of elongation 20212223. Indeed, the eEF2K activity inhibits the translation elongation factor eEF2, which mediates GTP-dependent translocation of the ribosome, thereby promoting peptide chain formation. In the tumoral context, increased activation of eEF2K downstream of mTORC1 inhibition by rapamycin has been linked to APC-deficient adenoma growth arrest, suggesting that enhancing eEF2K activity can be beneficial in patients with colorectal cancer 24. Here, we report that the anticancer molecule NFR triggered a robust eEF2K-dependent eEF2 phosphorylation leading to decreased rates of translation elongation. We found that NFR-mediated eEF2K activation decreased cell proliferation and promoted cell death. Consistent with these observations, we demonstrated in an in vivo model of engrafted tumors that NFR-mediated anti-tumoral activity is eEF2K dependent. Taken together, these data indicate that the eEF2K pathway can be therapeutically manipulated to drive a response that is detrimental for tumor survival. Results Nelfinavir resistance correlates with decreased eEF2K expression Long-term treatment of immortalized cells with NFR is toxic and triggers cell death 10. To get insight into the mechanisms involved, we treated HeLa cells with 10 μM of NFR and selected and characterized clones that survived and proliferated in the presence of the drug (Fig 1A). NFR concentration can reach up to 17 μM in the plasma of treated patients 2526 and around 10 μM in liver tissues of mice that receive a dose of NFR reproducing the plasma concentration measured in patients 17. Up to these concentrations of NFR, we can observe a significant increased viability in the resistant clones compared to the parental population (Fig 1B). We compared by RNA sequencing (RNA-seq), parental cells treated with NFR and four clones maintained in the presence of the drug (Dataset EV1). We observed that many genes involved in ribonucleoprotein complex biogenesis and mRNA translation regulation were downregulated in the resistant clones compared to NFR-treated parental cells. Among the translation regulating pathways significantly downregulated in the resistant clones, we noticed the decrease of the eukaryotic elongation factor 2 kinase (eEF2K) mRNA. This finding was confirmed by real-time PCR (Fig 1C) and at the protein level by Western blot (Fig 1D). Then, we interrogated eEF2K expression in the clones following NFR withdraw. We found that in two out of three representative clones, eEF2K downregulation was stable and maintained for more than 3 weeks in the absence of NFR (Fig EV1). This indicated that decreased eEF2K expression was not a direct consequence of prolonged treatment with NFR, but could be the result of a selective advantage in a few cells that bypassed NFR-mediated cell death. This hypothesis implied that eEF2K could be engaged by NFR to decrease viability. Figure 1. Resistance to nelfinavir triggers eEF2K downregulation Molecular structure of the HIV-PI nelfinavir (NFR) and schematic representation of the protocol used to generate NFR-resistant clones. HeLa cells were maintained with 10 μM NFR. After 15 days, clones proliferating in the constant presence of 10 μM NFR in the culture medium were selected and expanded for further analysis. Dose–response curves for viability of parental cells (in red) or selected clones (in black) upon treatment with NFR for 48 h. The green dashed box highlights the concentration in the physiologically relevant range. Curves are mean ± s.e.m. of three independent experiments performed in triplicate. P-values were calculated using two-way ANOVA between parental cells and each individual clone. Bar graph represents the EC50 (half-maximal effective concentration) of the dose responses. Data are mean ± s.e.m. of three independent experiments performed in triplicate. P-values were calculated using one-way ANOVA (P-value in red) followed by Dunnett's multiple comparison tests. *P-value ≤ 0.05, **P-value ≤ 0.01. The parental population and the NFR-resistant clones were analyzed for eEF2K mRNA expression by real-time PCR relative to β-actin (mean and s.e.m. from three independent batches of cells are shown). P-values were calculated using one-way ANOVA followed by Dunnett's multiple comparison tests. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001. The parental population and representative NFR-resistant clones were analyzed by Western blot for eEF2K total protein levels. Tubulin is used as a loading control. Histogram represents mean and s.e.m. of relative eEF2K levels quantified from Western blots of four different batches of cells collected on different dates. P-values were calculated using one-way ANOVA followed by Dunnett's multiple comparison tests. ***P-value ≤ 0.001. Source data are available online for this figure. Source Data for Figure 1 [embr201642194-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. eEF2K decreased level in NFR-resistant HeLa clonesNFR was washed out from resistant clones' culture medium for the indicated time, and cells were harvested and analyzed for eEF2K protein level. Numbers below represent the relative eEF2K expression compared to untreated parental cell line and were obtained by Western blot quantification normalizing eEF2K on tubulin levels. Download figure Download PowerPoint Nelfinavir triggers eEF2K to promote eEF2 phosphorylation EEF2K controls the rate of translation elongation through the phosphorylation of the eukaryotic elongation factor 2 (eEF2) at threonine 56 (Thr56). We therefore tested whether NFR can activate eEF2K by monitoring eEF2 phosphorylation. Short-term treatment with increasing concentrations of NFR triggered the phosphorylation of eEF2 in HeLa cells (Fig 2A). As expected, eEF2 phosphorylation was absent in eEF2K-deficient mouse embryonic fibroblasts, demonstrating that NFR engages eEF2K to regulate eEF2 (Figs 2B and EV2A). EEF2K deficiency did not affect other pathways modulated by NFR 17, including the phosphorylation of the translation initiation factor eIF2α or the expression of ATF4 (Fig EV2A). Similarly impairing eIF2α phosphorylation and therefore the integrated stress response (ISR) did not affect NFR-mediated eEF2 regulation (Fig EV2B), suggesting that these two pathways are engaged independently from one another. Then, we characterized these responses in the NFR-resistant clones. NFR was withdrawn from the culture media for a few hours, and the NFR response was analyzed at 6 h of treatment. As expected, the clones showed a diminished expression of eEF2K protein that correlated with a reduced eEF2 phosphorylation in the presence of low doses of NFR (Figs 2C and EV3A). However, other pathways triggered by NFR such as the induction of ATF4 (Fig EV3B) were not affected in NFR-resistant clones, which even expressed high level of NFR-response markers such as CHOP or DNAJB9 (Fig EV3C). Figure 2. HIV-PIs induce eEF2K-dependent eEF2 phosphorylation Immunoblot analysis of eEF2 Thr56 phosphorylation (P-eEF2) in HeLa cells treated for 6 h with increasing doses of NFR, and compared with 10 μg/ml tunicamycin (TM), 200 nM rapamycin (Rapa.), or 1-h starvation (Starv.). Tubulin is used as a loading control. eEF2K WT and KO MEFs treated for 6 h with indicated concentrations of NFR, 200 nM rapamycin (Rapa.), 1-h starvation (Starv.), or 1 mM of the AMPK activator AICAR, were analyzed by immunoblot with antibodies directed against total or phosphorylated eEF2 (Thr56). Three representative NFR-resistant HeLa clones were analyzed by immunoblot for eEF2K expression level and eEF2 phosphorylation upon NFR treatment and compared to parental HeLa cells. For treatments, medium was replaced for 6 h with medium containing DMSO (Mock), increasing doses of NFR or 10 μg/ml tunicamycin (TM), or for 1 h with PBS for starvation (Starv.). Tubulin is used as a loading control. Immunoblot analysis of eEF2 Thr56 phosphorylation in HeLa cells treated for 6 h with increasing doses of different HIV-PIs as indicated or the NFR metabolite M8 (hydroxy-tert-butylamide). Molecular structures of the different HIV-PIs used are indicated. Data information: Each panel is representative of at least three independent experiments. Source data are available online for this figure. Source Data for Figure 2 [embr201642194-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. eEF2K deficiency does not impair NFR-mediated ISR induction and vice versa Activation of the integrated stress response by NFR was not affected by eEF2K deficiency. ISR activation was measured in eEF2K−/− and eEF2K+/+ MEFs by immunoblotting of the translation factor ATF4 and the phosphorylation of the initiation factor eIF2α. NFR response (6 h of treatment) was compared with treatments using 10 μg/ml tunicamycin (TM), an inducer of the integrated stress response, 200 nM rapamycin (Rapa.), or 1 h of starvation in PBS (Starv.). Tubulin is used as a loading control. NFR mediates eEF2 phosphorylation independently from eIF2α phosphorylation, the effector of the ISR. NFR response was analyzed in eIF2α WT MEFs and cells unable to carry eIF2α phosphorylation on Ser51 (eIF2αS51A). Immunoblot analysis was performed to assess the phosphorylation of eEF2 and eIF2α as well as ATF4 expression after 6 h of treatment with indicated dose of NFR, 10 μg/ml tunicamycin (TM), 200 nM rapamycin (Rapa.), and after 1 h of starvation (Starv.). Tubulin is used as a loading control. Data information: Each panel is representative of at least three independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. ISR is not affected in NFR-resistant clones and NFR triggers eEF2 phosphorylation independently of Akt Three representative NFR-resistant HeLa clones were analyzed by immunoblot for eEF2 phosphorylation upon NFR and compared to parental HeLa cells (see Fig 2). Histogram shows the mean and s.e.m. of p-eEF2/-eEF2 ratio obtained from Western blot quantification of three independent experiments. Data were normalized using 40 μM NFR-treated parental cell line as the maximum (100%) p-eEF2 signal for each individual experiment. Immunoblot analysis of NFR-mediated activation of ER-stress markers in three NFR-resistant HeLa clones compared to parental HeLa cells. EIF2α phosphorylation and expression of ATF4 are shown. For treatments, medium was replaced for 6 h with medium containing increasing doses of NFR or 10 mg/ml tunicamycin (TM), or for 1 h with PBS for starvation (Starv.). These data are representative of three experiments. NFR-resistant clones and parental HeLa cells were analyzed for expression of the stress factors CHOP and DNAjB9 by real-time PCR relative to β-actin (mean and s.e.m. from three independent batches of cells are shown). P-values were calculated using one-way ANOVA followed by Dunnett's multiple comparison test; **P-value ≤ 0.01, ***P-value ≤ 0.001. eEF2K phosphorylation was measured using phos-tag SDS–PAGE and specific antibodies in HeLa cells subjected to the indicated treatments. These data are representative of three experiments. MK-2206-mediated Akt inhibition does not affect the ability of NFR to induce eEF2 phosphorylation. MEFs were treated with the indicated concentrations of the potent Akt inhibitor MK-2206 or left untreated. After 30 min, NFR was added for an additional 6 h. Immunoblot analysis was performed for phosphorylated and total Akt and eEF2 as indicated. Panel is representative of three independent experiments. Tubulin is used as a loading control. Download figure Download PowerPoint Investigation of a panel of HIV-PIs used in the clinic as well as hydroxy-t-butylamidenelfinavir (M8), the active NFR metabolite, showed that most HIV-PIs trigger eEF2 phosphorylation (Fig 2D). Yet, we consistently found that among the HIV-PIs, NFR is the most robust inducer of this pathway at relevant concentrations. Nelfinavir does not inhibit the mTORC1 pathway to promote eEF2K activation EEF2K activity is regulated by phosphorylation, which occurs at several sites downstream of specific signaling pathways. In particular, the mTORC1 downstream p70 S6 kinase negatively regulates eEFK by promoting its phosphorylation at inhibitory sites, thereby allowing translation to proceed 20, and AMP-activated protein kinase (AMPK), a sensor of low energy status, has been shown to promote activator phosphorylation of eEF2K 27 (Fig 3A). Inhibition of mTORC1 with chemical inhibitors such as rapamycin or the starvation-induced activation of AMPK impairs eEF2K phosphorylation leading to its activation and inhibition of eEF2 2829. Because many phosphorylation sites have been shown to regulate eEF2K 30, we separated cell extracts on a phos-tag SDS–PAGE to get a comprehensive analysis of its phosphorylation status. No significant changes on the eEF2K phosphorylation pattern were detected in the presence of NFR, while rapamycin or starvation considerably affected the overall eEF2K phosphorylation (Fig EV3D). In line with these observations, the dephosphorylation of the mTORC1 effectors S6 ribosomal protein or 4EBP1, a hallmark of mTORC1 inhibition and treatment with rapamycin, was only minimally affected in the presence of NFR (Fig 3B, compare lanes 2–4 with 6–7). Importantly, this effect was not observed in eEF2K-deficient cells (Fig 3B, compare lane 2–4 with lanes 14–16), suggesting that NFR only slightly affects the mTORC1 pathway downstream of eEF2K activation. Independence from mTORC1 was also confirmed by the observation that rapamycin, which does not trigger a strong eEF2 phosphorylation per se, did not affect NFR-mediated eEF2 phosphorylation (Fig 3C). AMPK activation has been shown to trigger eEF2K activation both indirectly through mTORC1 inhibition and directly by phosphorylating eEF2K 27. Indeed, the AMPK agonist AICAR is a potent inducer of eEF2K-dependent eEF2 phosphorylation (Figs 2B and 3B). Interestingly, we observed that concentrations above 20 μM of NFR triggered AMPK phosphorylation (Fig 3B, lane 3,4 and 15,16), suggesting that this pathway could be involved in mediating NFR responses. Yet, deletion of both AMPK isoforms α1 and α2 did not affect NFR-induced eEF2 phosphorylation whereas it impaired AICAR response (Fig 3D). AKT has been shown to be a target of NFR 103132; we therefore monitored AKT phosphorylation. Upon treatment with NFR, we did not observe decreased basal AKT phosphorylation (Fig 3B). Moreover, inhibition of AKT with the selective inhibitor MK-2206 did not affect NFR-mediated eEF2 phosphorylation (Fig EV3C). Similarly, inhibition of AKT phosphorylation upon treatment with the phosphoinositide 3-kinase (PI3K) inhibitors 3-methyladenine (3-MA) and wortmannin did not affect NFR-induced eEF2 phosphorylation (Appendix Fig S1). Altogether, these data demonstrate that NFR signals eEF2K activation independently of the eEF2K activating pathways such as mTORC1 inhibition, AMPK, or the ISR. Figure 3. NFR-mediated eEF2 phosphorylation is AMPK and mTOR independent Schematic representation of signaling pathways targeted by the inhibitors used in this study. eEF2K WT and KO MEFs treated for 6 h with the indicated concentrations of NFR, rapamycin (Rapa), AICAR, or starved for the indicated time in PBS (Starv.), were analyzed by immunoblot using the indicated antibodies. Tubulin is used as a loading control. Potent mTOR inhibition does not impair NFR-mediated eEF2 phosphorylation. WT MEFs were treated with the indicated concentrations of rapamycin or with vehicle (Mock). After 30 min, indicated doses of NFR were added and cells were incubated for an additional 6 h and analyzed for phosphorylated S6R and eEF2. Tubulin is used as a loading control. NFR-mediated eEF2 phosphorylation is not affected in AMPKα1α2 dKO. AMPKα1α2 WT and dKO MEFs treated for 6 h with indicated concentration of NFR, rapamycin (Rapa), AICAR, or starved for 1 h in PBS (Starv.), were analyzed by immunoblot with the indicated antibodies. Anti-total AMPKα antibody gives an unspecific band (*) with a slightly higher molecular weight in dKO cells. Tubulin is used as a loading control. Data information: Each panel is representative of at least three independent experiments. Source data are available online for this figure. Source Data for Figure 3 [embr201642194-sup-0006-SDataFig3.pdf] Download figure Download PowerPoint eEF2K activation contributes to decreased translation rates To quantify the role of eEF2K in NFR-mediated translation inhibition, we measured global translation rates using 35S-labeled methionine incorporation. We found that eEF2K deficiency partially rescued NFR-mediated decreased translation rates without impacting on tunicamycin-mediated regulation of translation (Fig 4A and B). In these cells, rapamycin triggered a relatively weak eEF2 phosphorylation (Fig 2B); thus, eEF2K did not significantly contribute to rapamycin-mediated translation decrease (Fig 4B). Similar to eEF2K deficiency, impairing eIF2α phosphorylation partially restored methionine incorporation, indicating that both pathways contribute to NFR-mediated reprogramming of mRNA translation (Fig 4B). We also examined the role of eEF2K in NFR-mediated mRNA translation control by measuring polysomal distribution. Treatment with NFR resulted in a decrease of mRNA associated with polysomes and an increase of free ribosomes, overall reflecting a decrease of protein synthesis, whereas eEF2K deficiency reversed this effect (Fig 4C). On the contrary, treatment with the ER-stress inducer tunicamycin triggered a decrease of the polysomal fraction that was unaffected by eEF2K deficiency but was rescued in the presence of the ISR inhibitor ISRIB or in cells unable to phosphorylate eIF2α (Fig EV4A and B). Inhibiting the ISR did not significantly affect the NFR-mediated decrease in polysomes (Fig EV4A and B). Interestingly, while NFR clearly affected the polysomal profile in an eEF2K-dependent manner, we did not observe the expected increase of the polysomal fraction that is predicted to accumulate upon elongation defects as observed in the presence of cycloheximide, a molecule that exerts its effect by interfering with the translocation step in protein synthesis (Fig EV4C). Instead, we observed a decreased polysomal fraction and increased free ribosomes signal. This indicates that specific eEF2K activation and eEF2 phosphorylation may affect additional steps in the translation program beyond its function during elongation. Previous reports have suggested that eEF2 could be involved in the splitting of the 80S ribosomes into subunits, a process required for the initiation steps of translation 33. Figure 4. NFR regulates translation rates by phosphorylating eEF2 A, B. EEF2K contributes to the decreased translation observed with NFR without impacting translation in the presence of TM. (A) Quantification of newly synthesized proteins at 0, 2, 4, and 6 h after 20 μM NFR (left panels) or 10 μg/ml TM (right panels) treatment in eEF2K+/+ and eEF2K−/− MEFs. Treated cells were labeled for 15 min with 35S-methionine and visualized by SDS–PAGE and subsequent autoradiography. Autoradiographs from four different experiments (see also source data) were quantified, and results show percentage of translation compared to untreated cells. The mean and s.e.m. of four independent metabolic labeling experiments are shown. (B) eEF2K+/+, eEF2K−/−, eIF2αWT, and eIF2αS51A MEFs were treated for 6 h with indicated doses of NFR, 200 nM rapamycin, or 10 μg/ml TM, or for 30 min with 10 μg/ml CHX. 35S-methionine incorporation was measured by liquid scintillation counting. Data are shown as the percentage of translation compared to untreated cells. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001 obtained using two-way ANOVA (in red) followed by Bonferroni post-test (in black). C. Representative polysome profiles of eEF2K WT and KO MEFs treated 6 h with NFR or TM. Area under the curve for sub-polysomes (S) and polysomes (P) used to calculate the P/S ratio was indicated (see also source data). Bar graph represents the ratio normalized to untreated cells. OD254 nm is optical density at 254 nm. Data are mean ± s.e.m. of P/S ratio calculated from three independent experiments. P-values were calculated using one-way ANOVA followed by Bonferroni post-test; *P-value ≤ 0.05, **P-value ≤ 0.01. D. The ribosome half-transit time in eEF2K+/+ and eEF2K−/− MEFs was determined as described in Materials and Methods. Incorporation of 35S-methionine into total protein within the PMS and PRS was obtained by linear regression analysis. Presented graphs are representative of two (CHX 1 μg/ml for 30 min and 30-min starvation in PBS in eEF2K+/+ cells) to four (NFR 20 μM for 6 h in eEF2K+/+ and eEF2K−/− cells) independent experiments. Indicated values represent the x displacement measurement (in time) between the PMS line at 300 s and the PRS line (see also source data). Histogram represents mean and s.e.m. of the ribosome half-transit time from four independent experiments. P-values were calculated using two-tailed unpaired Student's t-test; *P-value ≤ 0.05. Source data are available online for this figure. Source Data for Figure 4 [embr201642194-sup-0007-SDataFig4.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV4. NFR-mediated changes in polysomal profile are not dependent on eIF2α phosphorylation A. Representative polysome profiles of WT MEFs treated 6 h with NFR or TM with or without 500 nM ISRIB. Bar graph represents the ratio of sub-polysomes compared with polysomes (P/S). OD254 nm is optical density at 254 nm. Data are mean ± s.e.m. of P/S ratio calculated from two independent experiments. P-values were calculated using two-tailed unpaired Student's t-tests comparing ratio +/− ISRIB; *P-value ≤ 0.05. ISRIB efficiency at inhibiting NFR- or TM-mediated ATF4 induction was tested by immunoblot. B, C. Representative polysome profiles of eIF2αWT and eIF2αS51A MEFs treated 6 h with NFR or TM (B) or WT MEFs treated for 6 h with 20 μM NFR or 200 nM rapamycin (C, upper panel) or 30 min with 1 μg/ml CHX (C, lower panel). Data are mean ± s.e.m. of P/S ratio calculated from 2 to 3 independent experiments. P-values were calculated using one-way ANOVA followed by Bonferroni post-test (B and C, upper panel) or two-tailed unpaired Student's t-test (C, lower panel); *P-value ≤ 0.05, **P-value ≤ 0.01. Download figure Download PowerPoint The observation that eEF2
Referência(s)