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

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5′-ends, we show that an MNK inhibitor, {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}}CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5′-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357–1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m⁷GTP-Sepharose reveals that both CGP and eIF4G(1357–1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5′-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.     

Adenovirus-specific translation by displacement of kinase Mnk1 from cap-initiation complex eIF4F

Translation of cellular mRNAs involves formation of a cap-binding translation initiation complex known as eIF4F, containing phosphorylated cap-binding protein eIF4E, eIF4E kinase Mnk1, eIF4A, poly(A)-binding protein and eIF4G. Adenovirus is shown to prevent cellular translation by displacing Mnk1 from eIF4F, thereby blocking phosphorylation of eIF4E. Over expression of an eIF4E mutant that cannot be phosphorylated by Mnk1 impairs translation of cellular but not viral late mRNAs. Adenovirus 100k protein is shown to bind the C-terminus of eIF4G in vivo and in vitro, the same region bound by Mnk1. In vivo, 100k protein displaces Mnk1 from eIF4G during adenovirus infection, or in transfected cells. Purified 100k protein also evicts Mnk1 from isolated eIF4F complexes in vitro. A mutant adenovirus with a temperature-sensitive 100k protein that cannot inhibit cellular protein synthesis at restrictive temperature no longer blocks Mnk1 binding to eIF4G, or phosphorylation of eIF4E. We describe a mechanism whereby adenovirus selectively inhibits the translation of cellular but not viral mRNAs by displacement of Mnk1 from eIF4G and inhibition of eIF4E phosphorylation.     

Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.     

Molecular cross-talk between MEK1/2 and mTOR signaling during recovery of 293 cells from hypertonic stress

To investigate the role for initiation factor phosphorylation in de novo translation, we have studied the recovery of human kidney cells from hypertonic stress. Previously, we have demonstrated that hypertonic shock causes a rapid inhibition of protein synthesis, the disaggregation of polysomes, the dephosphorylation of eukaryotic translation initiation factor (eIF)4E, 4E-BP1, and ribosomal protein S6, and increased association of 4E-BP1 with eIF4E. The return of cells to isotonic medium promotes a transient activation of Erk1/2 and the phosphorylation of initiation factors, promoting an increase in protein synthesis that is independent of a requirement for eIF4E phosphorylation. As de novo translation is associated with the phosphorylation of 4E-BP1, we have investigated the role of the signaling pathways required for this event by the use of cell-permeable inhibitors. Surprisingly, although rapamycin, RAD001, wortmannin, and LY294002 inhibited the phosphorylation of 4E-BP1 and its release from eIF4E, they did not prevent the recovery of translation rates. These data suggest that only a small proportion of the available eIF4F complex is required for maximal translation rates under these conditions. Similarly, prevention of Erk1/2 activity alone with low concentrations of PD184352 did not impinge upon de novo translation until later times of recovery from salt shock. However, U0126, which prevented the phosphorylation of Erk1/2, ribosomal protein S6, TSC2, and 4E-BP1, attenuated de novo protein synthesis in recovering cells. These results indicate that the phosphorylation of 4E-BP1 is mediated by both phosphatidylinositol 3-kinase-dependent rapamycin-sensitive and Erk1/2-dependent signaling pathways and that activation of either pathway in isolation is sufficient to promote de novo translation.     

Sensitivity of global translation to mTOR inhibition in REN cells depends on the equilibrium between eIF4E and 4E-BP1

Initiation is the rate-limiting phase of protein synthesis, controlled by signaling pathways regulating the phosphorylation of translation factors. Initiation has three steps, 43S, 48S and 80S formation. 43S formation is repressed by eIF2α phosphorylation. The subsequent steps, 48S and 80S formation are enabled by growth factors. 48S relies on eIF4E-mediated assembly of eIF4F complex; 4E-BPs competitively displace eIF4E from eIF4F. Two pathways control eIF4F: 1) mTORc1 phosphorylates and inactivates 4E-BPs, leading to eIF4F formation; 2) the Ras-Mnk cascade phosphorylates eIF4E. We show that REN and NCI-H28 mesothelioma cells have constitutive activation of both pathways and maximal translation rate, in the absence of exogenous growth factors. Translation is rapidly abrogated by phosphorylation of eIF2α. Surprisingly, pharmacological inhibition of mTORc1 leads to the complete dephosphorylation of downstream targets, without changes in methionine incorporation. In addition, the combined administration of mTORc1 and MAPK/Mnk inhibitors has no additive effect. The inhibition of both mTORc1 and mTORc2 does not affect the metabolic rate. In spite of this, mTORc1 inhibition reduces eIF4F complex formation, and depresses translocation of TOP mRNAs on polysomes. Downregulation of eIF4E and overexpression of 4E-BP1 induce rapamycin sensitivity, suggesting that disruption of eIF4F complex, due to eIF4E modulation, competes with its recycling to ribosomes. These data suggest the existence of a dynamic equilibrium in which eIF4F is not essential for all mRNAs and is not displaced from translated mRNAs, before recycling to the next.     

Hypoxia-inducible Factor-1α (HIF-1α) Promotes Cap-dependent Translation of Selective mRNAs through Up-regulating Initiation Factor eIF4E1 in Breast Cancer Cells under Hypoxia Conditions

Hypoxia promotes tumor evolution and metastasis, and hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxia-related cellular processes in cancer. The eIF4E translation initiation factors, eIF4E1, eIF4E2, and eIF4E3, are essential for translation initiation. However, whether and how HIF-1α affects cap-dependent translation through eIF4Es in hypoxic cancer cells has been unknown. Here, we report that HIF-1α promoted cap-dependent translation of selective mRNAs through up-regulation of eIF4E1 in hypoxic breast cancer cells. Hypoxia-promoted breast cancer tumorsphere growth was HIF-1α-dependent. We found that eIF4E1, not eIF4E2 or eIF4E3, is the dominant eIF4E family member in breast cancer cells under both normoxia and hypoxia conditions. eIF4E3 expression was largely sequestered in breast cancer cells at normoxia and hypoxia. Hypoxia up-regulated the expression of eIF4E1 and eIF4E2, but only eIF4E1 expression was HIF-1α-dependent. In hypoxic cancer cells, HIF-1α-up-regulated eIF4E1 enhanced cap-dependent translation of a subset of mRNAs encoding proteins important for breast cancer cell mammosphere growth. In searching for correlations, we discovered that human eIF4E1 promoter harbors multiple potential hypoxia response elements. Furthermore, using chromatin immunoprecipitation (ChIP) and luciferase and point mutation assays, we found that HIF-1α utilized hypoxia response elements in the human eIF4E1 proximal promoter region to activate eIF4E1 expression. Our study suggests that HIF-1α promotes cap-dependent translation of selective mRNAs through up-regulating eIF4E1, which contributes to tumorsphere growth of breast cancer cells at hypoxia. The data shown provide new insights into protein synthesis mechanisms in cancer cells at low oxygen levels.     

Phosphorylation of eukaryotic initiation factor (eIF) 4E is not required for de novo protein synthesis following recovery from hypertonic stress in human kidney cells

Previous work has suggested that increased phosphorylation of eukaryotic initiation factor (eIF) 4E at Ser-209 in the C-terminal loop of the protein often correlates with increased translation rates. However, the functional consequences of phosphorylation have remained contentious with our understanding of the role of eIF4E phosphorylation in translational control far from complete. To investigate the role for eIF4E phosphorylation in de novo translation, we studied the recovery of human kidney cells from hypertonic stress. Results show that hypertonic shock caused a rapid inhibition of protein synthesis and the disaggregation of polysomes. These changes were associated with the dephosphorylation of eIF4G, eIF4E, 4E-binding protein 1 (4E-BP1), and ribosomal protein S6. In addition, decreased levels of the eIF4F complex and increased association of 4E-BP1 with eIF4E were observed over a similar time course. The return of cells to isotonic medium rapidly promoted the phosphorylation of these initiation factors, increased levels of eIF4F complexes, promoted polysome assembly, and increased rates of translation. However, by using a cell-permeable, specific inhibitor of eIF4E kinase, Mnk1 (CGP57380), we show that de novo initiation of translation and eIF4F complex assembly during this recovery phase did not require eIF4E phosphorylation.     

eIF4E-binding protein regulation of mRNAs with differential 5'-UTR secondary structure: a polyelectrostatic model for a component of protein-mRNA interactions

Control of translation in eukaryotes is complex, depending on the binding of various factors to mRNAs. Available data for subsets of mRNAs that are translationally up- and down-regulated in yeast eIF4E-binding protein (4E-BP) deletion mutants are coupled with reported mRNA secondary structure measurements to investigate whether 5'-UTR secondary structure varies between the subsets. Genes with up-regulated translational efficiencies in the caf20Δ mutant have relatively high averaged 5'-UTR secondary structure. There is no apparent wide-scale correlation of RNA-binding protein preferences with the increased 5'-UTR secondary structure, leading us to speculate that the secondary structure itself may play a role in differential partitioning of mRNAs between eIF4E/4E-BP repression and eIF4E/eIF4G translation initiation. Both Caf20p and Eap1p contain stretches of positive charge in regions of predicted disorder. Such regions are also present in eIF4G and have been reported to associate with mRNA binding. The pattern of these segments, around the canonical eIF4E-binding motif, varies between each 4E-BP and eIF4G. Analysis of gene ontology shows that yeast proteins containing predicted disordered segments, with positive charge runs, are enriched for nucleic acid binding. We propose that the 4E-BPs act, in part, as differential, flexible, polyelectrostatic scaffolds for mRNAs.     

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.     

Role of p70S6K1-mediated Phosphorylation of eIF4B and PDCD4 Proteins in the Regulation of Protein Synthesis

Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5′-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.     

Hypermethylated-capped selenoprotein mRNAs in mammals

Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5′-end m⁷G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5′-end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo.     

Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.     

Targeting the eIF4F translation initiation complex for cancer therapy

Abstract In multiple human cancers, the function of the eukaryotic translation initiation factor 4E (eIF4E) is elevated and directly related to disease progression. Overexpression or hyperactivation of eIF4E in experimental models can drive cellular transformation and malignant progression. Elevated eIF4E function triggers enhanced assembly of the eIF4F translation initiation complex and thereby drives cap-dependent translation. Though all capped mRNAs require eIF4F for translation, a pool of mRNAs are exceptionally dependent on elevated eIF4F activity for translation and are thereby selectively and disproportionately affected by altered eIF4F activity. These mRNAs encode proteins that play significant roles in all aspects of malignancy including angiogenesis factors (VEGF, FGF-2), onco-proteins (c-myc, cyclin D1, ODC), pro-survival proteins (survivin, BCL-2) and proteins involved in tumor invasion and metastasis (MMP-9, heparanase). Recent advances in targeting the eIF4F complex have highlighted the role for this complex in tumor cell survival and angiogenesis and have illuminated the enhanced susceptibility of the tumor cells to inhibition of the eIF4F complex. These studies have demonstrated the attractiveness and plausibility of targeting eIF4E and the eIF4F translation initiation complex for cancer therapy and have prompted the advance of the first eIF4E-specific therapy to the clinic.     

Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo

Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5' cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen- and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogen-induced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.