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Phosphorylation of eIF4E Confers Resistance to Cellular Stress and DNA-Damaging Agents through an Interaction with 4E-T: A Rationale for Novel Therapeutic Approaches.

Martínez A, Sesé M, Losa JH, Robichaud N, Sonenberg N, Aasen T, Ramón Y Cajal S - PLoS ONE (2015)

Bottom Line: Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1).We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required.Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology, Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca, VHIR, Universitat Autònoma de Barcelona, Barcelona, Spain.

ABSTRACT
Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents in vitro. Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D) form of eIF4E, but not phospho-dead (S209A) eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin), starvation (glucose+glutamine withdrawal), and oxidative stress (arsenite). De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1). We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

No MeSH data available.


Related in: MedlinePlus

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).
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pone.0123352.g007: 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).

Mentions: To validate whether phosphorylation of eIF4E mediates a selective synthesis of proteins under stress (as shown under normal conditions by other researchers [35]), we screened a number of proteins by western blot under normal conditions and after arsenite treatment. Indeed, S209D expression, but not that of S209A or GFP, caused an increase in the level of some proteins 2 hours after arsenite stress, including proteins associated with resistance to apoptosis (Mcl-1) and cell cycle progression (cyclin D1) whereas other proteins were either maintained or decreased (Fig 7A). To exclude the possibility that S209D works through transcriptional pathways, we treated cells with actinomycin D before and after arsenite stress to block de novo transcription. As seen in Fig 7B, S209D prevented the loss of HuR, Mcl-1, and cyclin D1 also in the presence of actinomycin D, strongly suggesting post-transcriptional regulation, possibly by maintenance of mRNA translation. After cycloheximide treatment, blocking protein synthesis, the levels of some proteins, like cyclin D1, Mcl1, and HuR, was significantly reduced after 2 hours of recovery from arsenite treatment (Fig 7C), indicating that phosphomimetic eIF4E indeed acts by maintaining mRNA translation rather than stabilizing these proteins through an alternative mechanism.


Phosphorylation of eIF4E Confers Resistance to Cellular Stress and DNA-Damaging Agents through an Interaction with 4E-T: A Rationale for Novel Therapeutic Approaches.

Martínez A, Sesé M, Losa JH, Robichaud N, Sonenberg N, Aasen T, Ramón Y Cajal S - PLoS ONE (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4414544&req=5

pone.0123352.g007: 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).
Mentions: To validate whether phosphorylation of eIF4E mediates a selective synthesis of proteins under stress (as shown under normal conditions by other researchers [35]), we screened a number of proteins by western blot under normal conditions and after arsenite treatment. Indeed, S209D expression, but not that of S209A or GFP, caused an increase in the level of some proteins 2 hours after arsenite stress, including proteins associated with resistance to apoptosis (Mcl-1) and cell cycle progression (cyclin D1) whereas other proteins were either maintained or decreased (Fig 7A). To exclude the possibility that S209D works through transcriptional pathways, we treated cells with actinomycin D before and after arsenite stress to block de novo transcription. As seen in Fig 7B, S209D prevented the loss of HuR, Mcl-1, and cyclin D1 also in the presence of actinomycin D, strongly suggesting post-transcriptional regulation, possibly by maintenance of mRNA translation. After cycloheximide treatment, blocking protein synthesis, the levels of some proteins, like cyclin D1, Mcl1, and HuR, was significantly reduced after 2 hours of recovery from arsenite treatment (Fig 7C), indicating that phosphomimetic eIF4E indeed acts by maintaining mRNA translation rather than stabilizing these proteins through an alternative mechanism.

Bottom Line: Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1).We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required.Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology, Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca, VHIR, Universitat Autònoma de Barcelona, Barcelona, Spain.

ABSTRACT
Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents in vitro. Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D) form of eIF4E, but not phospho-dead (S209A) eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin), starvation (glucose+glutamine withdrawal), and oxidative stress (arsenite). De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1). We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

No MeSH data available.


Related in: MedlinePlus