Differential regulation of CHOP translation by phosphorylated eIF4E under stress conditions

Abstract

Cells respond to environmental stress by inducing translation of a subset of mRNAs important for survival or apoptosis. CHOP, a downstream transcriptional target of stress-induced ATF4, is also regulated translationally in a uORF-dependent manner under stress. Low concentration of anisomycin induces CHOP expression at both transcriptional and translational levels. To study specifically the translational aspect of CHOP expression, and further clarify the regulatory mechanisms underlying stress-induced translation initiation, we developed a CMV promoter-regulated, uORFchop-driven reporter platform. Here we show that anisomycin-induced CHOP expression depends on phosphorylated eIF4E/S209 and eIF2α/S51. Contrary to phospho-eIF2α/S51, phospho-eIF4E/S209 is not involved in thapsigargin-induced CHOP expression. Studies using various kinase inhibitors and mutants uncovered that both the p38MAPK-Mnk and mTOR signaling pathways contribute to stress-responsive reporter and CHOP expression. We also demonstrated that anisomycin-induced translation is tightly regulated by partner binding preference of eIF4E. Furthermore, mutating the uORF sequence abolished the anisomycin-induced association of chop mRNA with phospho-eIF4E and polysomes, thus demonstrating the significance of this cis-regulatory element in conferring on the transcript a stress-responsive translational inducibility. Strikingly, although insulin treatment activated ERK-Mnk and mTOR pathways, and consequently eIF4E/S209 phosphorylation, it failed to induce phospho-eIF2α/S51 and reporter translation, thus pinpointing a crucial determinant in stress-responsive translation.

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

“…One mechanism by which MNK alters cell fate is by altering the translation of specific mRNAs. To date, there have been numerous mRNAs identified as translationally regulated targets of MNK, including those encoding cytokines IL-17 (32) and pro-inflammatory TNF␣ (79), the anti-apoptotic protein Mcl-1 (28), ribosomal proteins S19 and L32 (19), proteins involved in cell proliferation (CDK2, CDK8, CDK9, HIF-1␣, KAP1, proliferating cell nuclear antigen, PIAS1, and RASSF1 (19)), proteins involved in cell cycle progression (cyclins A, B, D1, and D3 (19,78)), cancer-related proteins (HER2 (21), VEGF (35), and IRF-1 (84)), transcription factors (RFLAT-1 (46), CHOP (85), and ␤-catenin (25)), and neuronal proteins (Arc, ␣CaMKII, PKM-, calmodulin, BDNF (86), and PEM1 (20)). Translation of many of these mRNAs is also responsive to eIF4E levels, indicating a cap-and eIF4E-dependent mechanism of translation: HIF-1␣ (19), HER2 (21), Mcl-1 (28), cyclin D1 (87,78), VEGF (88), RFLAT-1 (46), ␤-catenin (25), and Arc (86).…”

The effect of p-4E-BP1 and p-eIF4E on cell proliferation in a breast cancer model

“…Previously it was reported that p-eIF4E binds more efficiently to capped mRNAs (17), suggesting that the affinity of phosphorylated eIF4E for 4E-BP1 is decreased. Moreover, Chen et al (19) reported that p-eIF4E binds less well to 4E-BP1 than the unphosphorylated form. In our coimmunoprecipitation and Western blot studies, p-eIF4E seemed to show a weaker affinity for 4E-BP1 than total eIF4E in MDA-MB 468 cells, but not in MDA-MB 231 cells.…”

“…Although eIF4E phosphorylation on Ser209 was discovered some time ago, it remains unclear whether phosphorylation increases or decreases eIF4E affinity for the cap-structure. Early data indicated that eIF4E phosphorylation increased the affinity for capped mRNAs, whereas other studies have shown that eIF4E phosphorylation actually decreases affinity for the cap-structure and capped oligoribonucleotides (15,(17)(18)(19)(20).…”

Endoplasmic Reticulum Stress-activated C/EBP Homologous Protein Enhances Nuclear Factor-κB Signals via Repression of Peroxisome Proliferator-activated Receptor γ

“…is a C/EBP homologous protein that plays crucial roles in cellular translational stress conditions, making epithelial cells sensitive to xenobiotic-induced injuries (33). In the present study, chemical ER stresses were assessed for their effects on stress-inducible CHOP expression and its association with inflammatory cytokine production.…”