Fig 1.
Overexpression of phosphomimetic eIF4E-S209D does not affect proliferation but increases clonogenic cell survival.
A, Western blot analysis showed similar, moderate levels of Myc-tagged exogenous eIF4E-S209D or-S209A and endogenous eIF4E expression in MDA-MB-231 and HaCaT cell lines. B, MTT cell proliferation assays revealed no significant difference in cell growth between GFP—or eIF4E-mutant–expressing cells under normal conditions. C, Crystal violet staining of clonogenic colony formation assays clearly indicate an increase in both colony number and size upon eIF4E-S209D expression compared with GFP control cells, which is reflected by a statistically significant increase in the total number of stained cells as measured by overall crystal violet absorbance. A moderate but not statistically significant increase in eIF4E-S209A–expressing cells was noted. Graphs depict the overall cell growth as measured in a crystal violet absorption assay. The survival and growth advantage after expression of eIF4E-S209D is highly significant in both HaCaT and MDA-MB-231 cells treated with arsenite for 90 minutes to induce oxidative stress before being plated. D, Graphs representing the percentage of the number of colonies after arsenite treatment compared to normal conditions in each case. Arsenite treatment clearly decreases the number of colonies in both cell lines. * = P<0.05, ** = P<0.01 and *** = P<0.001, compared to control, n = 3. (AU: Absorbance Units)
Fig 2.
MDA-MB-231 and HaCaT cells show increased resistance to stress after overexpression of phosphomimetic eIF4E.
A, MDA-MB-231 and HaCaT cells were subjected to either arsenite (NaAsO2), nutrient starvation, or cisplatin (CDDP) treatment, and cell viability was measured by an MTT assay after 24, 48, and 72 hours. In all cases, eIF4E-S209D significantly increased cell viability. B, Apoptotic activity measured by a caspase-3/-7 luminescence assay. Significant activation of caspase-3/-7 activity was observed following arsenite treatment in eIF4E-S209A– and GFP–expressing cells, which was completely prevented by eIF4E-S209D. * = P<0.05, ** P<0.01 and *** = P<0.001 compared to control, n = 3.
Fig 3.
Phosphorylation of endogenous eIF4E increases cellular resistance to stress.
A, Expression of phospho-eIF4E with and without CGP57380 treatment and with arsenite or cisplatin treatment in MDA-MB-231 cells. B, MDA-MB-231 cells treated with CGP57380 to inhibit Mnk1/2-mediated phosphorylation of eIF4E displayed slightly decreased cell proliferation under normal conditions, whereas cell recovery after oxidative stress by arsenite (and to less extent cisplatin) was dramatically reduced. C, Western blotting showed a lack of phospho-eIF4E in MEFs from Mnk1/2 null mice and significantly reduced phospho-eIF4E in wild-type MEFs treated with CGP57380. D, MTT growth assays demonstrated a slight decrease in the proliferation of MEFs from Mnk1/2 null mice compared to wild-type MEFs and a significant reduction in cell recovery following either arsenite or cisplatin treatment. E, Inhibiting Mnk1/2 in wild-type MEFs with CGP 57380 significantly reduced cellular recovery from arsenite or cisplatin treatment. * = P<0.05, ** = P<0.01 and *** P = <0.001 compared to control, n = 3.
Fig 4.
Phospho-eIF4E interacts with 4E-T and induces de novo cytoplasmic bodies.
A, Confocal immunofluorescence analysis of MDA-MB-231 cells demonstrated de novo formation of cytoplasmic 4E-T bodies after overexpression of eIF4E-S209D, with partial colocalization. Formation of cytoplasmic 4E-T bodies was not observed upon overexpression of eIF4E-S209A. B, The 4E-T bodies in the eIF4E-S209D–expressing cells colocalized with a marker of P-bodies (DCP1A) in normal conditions. C, Immunoprecipitation assays with anti-Myc-tag antibodies against eIF4E-S209A or-S209D demonstrated a specific interaction between phosphomimetic eIF4E-S209D and 4E-T. D, Arsenite treatment increased the phosphorylation of endogenous eIF4E and dramatically increased the interaction between eIF4E and 4E-T, whereas treatment with CGP57380 reduced phospho-eIF4E and significantly diminished the amount of 4E-T interacting with eIF4E. 10% inputs used in the immunoprecipitations is shown on the side.
Fig 5.
Phospho-eIF4E/4E-T binding is necessary for recovery after stress.
A, Cells stably expressing eIF4E-S209D or-S209A were transfected with scramble or specific short hairpins to knockdown 4E-T expression. eIF4E-S209D significantly enhanced cell recovery from arsenite stress in scramble-transfected cells, whereas knockdown of 4E-T completely abolished any protective effect of eIF4E-S209D. B, Expression of the eIF4E mutants W73A, W73A/S209D, both unable to bind 4E-T, and S209D; W73A and W73A/S209D did not confer any resistance to arsenite treatment compared with S209D. * = P<0.05 and ** = P<0.01 compared to control, n = 3.
Fig 6.
eIF4E-S209D rapidly modulates protein translation after arsenite treatment.
Polysome analysis of MDA-MB-231 cells showed that cells expressing eIF4E-S209D, but not-S209A or GFP, induced a peak in the 80S fraction 2 hours after arsenite treatment, indicating translational stalling. Twelve hours after recovery from arsenite treatment, the 80S peak in S209D-expressing cells was more moderate compared with that of S209A- or GFP-expressing cells, indicating a slightly higher level of protein synthesis. B, m7-GTP pull-down assays in normal conditions and 2 hours after arsenite treatment indicate that eIF4E-S209D strongly associates with the mRNA cap in a complex with eIF4G in the recovery period after arsenite treatment, which may allow re-initiation of protein synthesis.
Fig 7.
Selective increase in protein synthesis in cells expressing eIF4E-S209D.
A, Western blotting analysis showed that eIF4E-S209D was able to maintain the expression levels of some proteins, such as cyclin D, after stress caused by arsenite treatment (compared to S209A- or GFP-expressing cells). Other proteins showed reduced levels or no changes. B, Treatment with actinomycin D for 6 hours in normal conditions and 2 hours after stress indicated that the selective advantage of eIF4E-S209D in maintaining protein expression was due to post-transcriptional events. C, Treatment with cycloheximide after arsenite treatment showed that cyclin D and Mcl-1 are subjected to translational regulation by eIF4E-S209D, rather than modulation of protein stability. D, Quantitative PCR analysis of polysomal RNA indicated that eIF4E-S209D after stress permits a dramatic increase in actively translated cyclin D1 (expressed as a fraction of total mRNA).