The eukaryotic translation initiation factor 4E is not modified during the course of vaccinia virus replication.

The ability of vaccinia virus to inhibit processes of cap-dependent translational initiation by inactivating the eukaryotic translation initiation factor 4E (eIF-4E) has been examined. Analyses of the quantities of eIF-4E present in either uninfected mouse L929 cells or vaccinia virus-infected cells showed that during the first 12 hr of virus replication, when there is a marked decrease in host gene expression in infected cells, there is no change in the total amount of eIF-4E present. Analyses of eIF-4E that was metabolically labeled with [32P] and then purified by affinity chromatography using m7GTP-Sepharose 4B, indicated that neither the incorporation of radiolabel into eIF-4E nor the amounts of eIF-4E capable of binding to cap structures changed significantly during virus replication. Immunodetection of phosphorylated and unphosphorylated eIF-4E in cell lysates fractionated by two-dimensional gel electrophoresis showed that the steady-state levels of phosphorylated and unphosphorylated forms of eIF-4E were similar in uninfected and virus-infected cells. These results suggest that vaccinia virus does not gain preferential translation of viral mRNAs over other mRNAs in the cell by reducing either eIF-4E phosphorylation or its ability to bind to the cap structure.     

Vaccinia virus-mediated inhibition of host protein synthesis involves neither degradation nor underphosphorylation of components of the cap-binding eukaryotic translation initiation factor complex eIF-4F.

Recent reports indicated that vaccinia virus late mRNAs contain a unique 5' poly(A) leader sequence and that the in vitro translation of these mRNAs may be relatively cap-independent. These observations led us to examine the possibility that the mechanism of inhibition of host protein synthesis by vaccinia virus, like that of certain other viruses, involves specific modifications of the cap-binding translation initiation factor complex eIF-4F. The eIF-4F complex was affinity-purified from human cells infected with vaccinia virus and analyzed by one- and two-dimensional electrophoresis and immunoblotting. No evidence of vaccinia virus-induced degradation of p220, as occurs during poliovirus infection, or alteration of phosphorylation of eIF-4E (p24), as occurs during adenovirus infection, was detected at the time of severe inhibition of host protein synthesis.     

Translational control and metastatic progression: enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs

To form metastases, tumors must break from the primary tumor site, invade surrounding tissues, enter and survive within the circulation and ultimately colonize a distal tissue. Each of these steps requires the cooperative function of numerous proteins – proteins that facilitate angiogenesis (e.g., VEGF), cell survival (e.g., Bcl-2), invasion (e.g., MMPs), and autocrine growth stimulation (e.g., c-myc, cyclin D1). Although expression of these proteins is regulated at many levels by disparate stimuli, translation of these key malignancy-related proteins is regulated primarily by the activity of the mRNA cap-binding protein eIF-4E, the rate-limiting member of the eIF-4F translation initiation complex. By binding the cap structure at the 5′ terminus of cellular mRNAs, eIF-4E recruits mRNAs to the eIF-4F complex, which then scans from the 5′ cap through the untranslated region (5′UTR), unwinding secondary structure to reveal the translation initiation codon and to enable ribosome loading. Messenger RNAs with short unstructured 5′ UTRs are more easily translated than mRNAs harboring lengthy, highly structured 5′ UTRs, as these prohibit efficient scanning and start codon recognition. As such, the translation of these mRNAs, which typically encode proteins involved in angiogenesis (e.g., VEGF), tumor growth (cyclin D1) and survival (Bcl-2), is suppressed except when eIF-4E is engaged with the eIF-4F complex – a common event in many human and experimental cancers. This review focuses on the hypothesis that enhanced eIF-4E function contributes to metastatic progression by selectively upregulating the translation of key malignancy-related proteins that together conspire to drive the metastatic process. This revised version was published online in July 2006 with corrections to the Cover Date.     

阻抑eIF-4E对大肠腺癌LS-174T细胞乙酰肝素酶mRNA及其翻译表达水平的影响

目的:确定阻抑结肠癌LS-174T细胞中过量表达的真核细胞起始因子-4E(eukaryoticinitiationfactor-4E,eIF-4E)是否促进乙酰肝素酶(heparanase)mRNA的降解,并改变其翻译表达水平。方法:应用脂质体包裹与eIF-4EmRNA翻泽起始点互补的asODN,转染处理人大肠腺癌细胞LS-174T。使用Westernblot和RT-PCR方法分别检测eIF-4E被阻抑后其转录和翻译水平的改变。乙酰肝素酶mRNA在细胞内水平采用Northernblot定量检测,其蛋白表达水平改变采用Westernblot检测。结果:asODN经脂质体转染LS-174T细胞后,eIF-4E基因表达明显受到抑制,其蛋白表达产物也显著下降。伴随eIF-4E被阻抑表达,Northernblot结果显示乙酰肝素酶mRNA水平下降,且其蛋白翻译表达量也降低。结论:阻抑eIF-4E影响LS-174T细胞乙酰肝素酶mRNA稳定、促使其降解,并降低乙酰肝素酶表达。     

Dissecting eIF4E action in tumorigenesis

Genetically engineered mouse models are powerful tools for studying cancer genes and validating targets for cancer therapy. We previously used a mouse lymphoma model to demonstrate that the translation initiation factor eIF4E is a potent oncogene in vivo. Using the same model, we now show that the oncogenic activity of eIF4E correlates with its ability to activate translation and become phosphorylated on Ser 209. Furthermore, constitutively activated MNK1, an eIF4E Ser 209 kinase, promotes tumorigenesis in a manner similar to eIF4E, and a dominant-negative MNK mutant inhibits the in vivo proliferation of tumor cells driven by mutations that deregulate translation. Phosphorylated eIF4E promotes tumorigenesis primarily by suppressing apoptosis and, accordingly, the anti-apoptotic protein Mcl-1 is one target of both phospho-eIF4E and MNK1 that contributes to tumor formation. Our results provide insight into how eIF4E contributes to tumorigenesis and pinpoint a level of translational control that may be suitable for therapeutic intervention.     

GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation in Saccharomyces cerevisiae

We have isolated and characterized two suppressor genes, SUI4 and SUI5, that can initiate translation in the absence of an AUG start codon at the HIS4 locus in Saccharomyces cerevisiae. Both suppressor genes are dominant in diploid cells and lethal in haploid cells. The SUI4 suppressor gene is identical to the GCD11 gene, which encodes the γ subunit of the eIF-2 complex and contains a mutation in the G₂ motif, one of the four signature motifs that characterizes this subunit to be a G-protein. The SUI5 suppressor gene is identical to the TIF5 gene that encodes eIF-5, a translation initiation factor known to stimulate the hydrolysis of GTP bound to eIF-2 as part of the 43S preinitiation complex. Purified mutant eIF-5 is more active in stimulating GTP hydrolysis in vitro than wild-type eIF-5, suggesting that an alteration of the hydrolysis rate of GTP bound to the 43S preinitiation complex during ribosomal scanning allows translation initiation at a non-AUG codon. Purified mutant eIF-2γ complex is defective in ternary complex formation and this defect correlates with a higher rate of dissociation from charged initiator-tRNA in the absence of GTP hydrolysis. Biochemical characterization of SUI3 suppressor alleles that encode mutant forms of the β subunit of eIF-2 revealed that these mutant eIF-2 complexes have a higher intrinsic rate of GTP hydrolysis, which is eIF-5 independent. All of these biochemical defects result in initiation at a UUG codon at the his4 gene in yeast. These studies in light of other analyses indicate that GTP hydrolysis that leads to dissociation of eIF-2GDP from the initiator-tRNA in the 43S preinitiation complex serves as a checkpoint for a 3-bp codon/anticodon interaction between the AUG start codon and the initiator-tRNA during the ribosomal scanning process.     

Identification of a coelomic mitogenic factor in Eisenia foetida earthworm

Coelomic fluid of earthworms Eisenia foetida (Oligochaeta, Annelida) exerts a mitogenic activity on murine splenocytes. Total coelomic fluid was subjected to size-exclusion chromatography and a semi-purified mitogenic fraction (fraction 5) was isolated and further characterized. Both coelomic fluid and the semi-purified fraction 5 block concanavalin A (ConA)-induced spleen cell proliferation but exert a synergistic effect on LPS-triggered spleen cell proliferation. Using a polyclonal antiserum neutralizing the mitogenic activity of the semi-purified fraction 5, a 60-kDa component was identified and named CMF (coelomic mitogenic factor). CMF was found to bind ConA which could account for its ability to inhibit ConA-induced spleen cell proliferation. CMF is present in the coelomic fluid as a trimer of a 20-kDa protein. N-terminal amino acid sequence of monomeric CMF reveals partial sequence homology with phospholipase A2 (PLA2). Moreover, CMF-enriched coelomic fluid fraction 5 exerts phospholipase activity comparable with that of bovine pancreatic PLA2. Our results suggest that coelomic fluid of E. foetida contains a ubiquitous PLA2-like enzyme which might be involved in immune reactions in earthworms such as anti-bacterial mechanisms.     

Phosphorylation of eIF4E by Mnk-1 enhances HSV-1 translation and replication in quiescent cells

Although the activity of the translation initiation factor eIF4F is regulated in part by translational repressors (4E-BPs) that prevent incorporation of eIF4E, the cap-binding protein, into the initiation complex, the contribution of eIF4E phosphorylation to translational control remains controversial. Here, we demonstrate that the herpes simplex virus-1 (HSV-1) ICP0 gene product, a multifunctional transactivator of viral gene expression with ubiquitin E3 ligase activity that is important for vegetative replication and reactivation of latent infections, is required to stimulate phosphorylation of eIF4E as well as 4E-BP1, and promote assembly of eIF4F complexes in infected cells. Furthermore, 4E-BP1 is degraded by the proteasome in an ICP0-dependent manner, establishing that the proteasome can control 4E-BP1 steady-state levels. Preventing eIF4E phosphorylation by inhibiting the eIF4E kinase mnk-1 dramatically reduced viral replication and the translation of viral polypeptides in quiescent cells, providing the first evidence that phosphorylation of eIF4E by mnk-1 is critical for viral protein synthesis and replication. Thus, in marked contrast to many viruses that inactivate eIF4F, HSV-1 stimulates eIF4F complex assembly in quiescent, differentiated cells; moreover, this is important for viral replication, and may be crucial for HSV-1 to initiate its productive growth cycle in resting cells, such as latently infected neurons.     

Reversal of the CD4(+)/CD8(+) T-cell ratio in lymph node cells upon in vitro mitogenic stimulation by highly purified, water-soluble S3-S4 dimer of pertussis toxin

Pertussis toxin (PT), a holomer consisting of a catalytic S1 subunit and a B oligomer composed of S2-S4 and S3-S4 dimers, held together by the S5 subunit, exerts profound effects on immune cells, including T-cell mitogenicity. While the mitogenic activity of PT was shown to reside fully within the B oligomer, it could not be assigned to any particular B-oligomer component. In this study, we purified the S3-S4 dimer to homogeneity under conditions propitious to maintenance of the native conformation. In contrast to previous reports which suggested that both S3-S4 and S2-S4 dimers are necessary for mitogenic activity, our preparation of the highly purified S3-S4 dimer was as strongly mitogenic as the B oligomer, suggesting that the S3-S4 dimer accounts for the mitogenic activity of the B oligomer. Moreover, in vitro stimulation of naive lymphocytes by the S3-S4 dimer resulted in reversal of the normal CD4(+)/CD8(+) T-cell ratio from approximately 2:1 to 1:2. The reversal of the CD4(+)/CD8(+) T-cell ratio is unlikely to be due to preferential apoptosis-necrosis of CD4(+) T cells, as indicated by fluorescence-activated cell sorter analysis of annexin-stained T-cell subsets, or to preferential stimulation of CD8(+) T cells. The mechanism underlying the reversal requires further investigation. Nevertheless, the data presented indicate that the S3-S4 dimer may have potential use in the context of diseases amenable to immunological modulation.     

Oxidative stress induces arachidonate release from human lung cells through the epithelial growth factor receptor pathway

Oxidative stress is thought to be a factor influencing many inflammatory responses, including arachidonic acid (AA) release. We have studied the effect of hydrogen peroxide on AA and prostaglandin E(2) release, cytosolic phospholipase (cPLA(2)) steady-state mRNA, cPLA(2) protein levels, cPLA(2) enzyme activity, and cPLA(2) phosphorylation in a human lung epithelial cell line: A549 cells. Hydrogen peroxide caused a dose-dependent increase of A23187-stimulated AA and prostaglandin E(2) release, with a maximum effect at 1 h. This effect is associated with a maximum specific cPLA(2) activity at 1 h, and with a significant increase in cPLA(2) Serine 505 phosphorylation. All these effects were abolished, in a dose-related manner, by the epithelial growth factor receptor kinase inhibitor, AG 1478. To further investigate the pathway leading to the increase cPLA(2) phosphorylation, we used cells transfected with a Ras dominant negative vector and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and p38 kinase inhibitors. Cells transfected with the Ras dominant negative vector exhibited diminished hydrogen peroxide-induced AA release and cPLA(2) phosphorylation as compared with cells transfected with the Ras expression vector. Both MEK and p38 kinase inhibitors inhibited the hydrogen peroxide effect on AA release and specific cPLA(2) activity. Finally, cells stably transfected with an antisense cPLA(2) vector exhibited diminished A23187-stimulated AA release in response to hydrogen peroxide as compared with cells stably transfected with empty expression vector. Collectively, these data show that hydrogen peroxide increases cPLA(2) activity through its phosphorylation utilizing an epithelial growth factor/Ras/extracellular signal-regulated kinase and p38 pathway.     

Translation initiation factor eIF-4gamma is encoded by an amplified gene and induces an immune response in squamous cell lung carcinoma

Amplification of cellular oncogenes is an important mechanism of altered gene expression in human cancers. Using comparative genomic hybridization we recently identified an amplification at 3q26.1-q26.3 in 30% of squamous cell carcinomas of the lung. A variety of methods including microdissection-mediated procedures permit cloning of genes encoded within amplified domains but do not directly lead to the identification of biologically relevant genes. In this study, we have circumvented this problem by combining an immunological and molecular genetic approach to analyze squamous cell lung carcinoma. To identify both amplified and tumor relevant genes, we generated a cDNA expression library from a tumor with the 3q amplification and hybridized the expressed recombinant polypeptides with the autologous serum. Of 400000 cDNA clones we identified 17 antigens which induce an immune response in a patient with squamous cell lung carcinoma. While most clones represent individual genes sequence analysis revealed that four of the 17 cDNAs are nearly identical with the eukaryotic translation initiation factor (eIF)-4gamma recently assigned on 3q. We demonstrated that the gene for eIF-4gamma was amplified within 3q26-q27 in independent squamous cell lung carcinomas. In this study, we report the identification of several antigens which elicit an immune response in a squamous cell lung carcinoma patient including eIF-4gamma. eIF-4gamma is encoded by an amplified gene and possibly plays a crucial part in the development of squamous cell lung carcinoma.      

Sequences mediating the translation of mouse S16 ribosomal protein mRNA during myoblast differentiation and in vitro and possible control points for the in vitro translation

The translation of ribosomal protein (r-protein) mRNAs is generally inefficient and regulated during the differentiation of mouse myoblasts into fibers. In this discussion we show that the first 31 nucleotides of the S16 r-protein mRNA, when located at the 5' end of the mRNA, are sufficient to impart the translational properties of an r-protein mRNA to the SV-GALK mRNA, which is normally translated efficiently in both myoblasts and fibers. If the same S16 sequences are located within the interior of the 5'-untranslated region of the SV-GALK mRNA, however, they do not impart the translational properties of an r-protein mRNA to the SV-GALK mRNA. The translation of mouse r-protein mRNAs was examined in vitro to help elucidate the mechanisms controlling their translation. Mouse r-protein mRNAs are inefficiently translated in rabbit reticulocyte extracts, and the same sequences that mediate their inefficient and regulated translation during myoblast differentiation also mediate their inefficient translation in a position-dependent manner in reticulocyte extracts. To determine whether the subpolysomal r-protein mRNAs that are not actively translated in vivo are capable of translation, subpolysomal RNA was translated in reticulocyte extracts. The subpolysomal r-protein mRNAs are just as capable of translation as are polysomal mRNAs. To help identify the initiation factors and/or the steps in the initiation pathway that mediate the inefficient translation of r-protein mRNAs, reticulocyte extracts were supplemented with purified initiation factors. Only eIF-4F, the cap-binding complex, and eIF-3, which is involved in subunit dissociation and interacts with eIF-4F during initiation, stimulated the translation of r-protein mRNA. These experiments, along with m7GDP inhibition studies, suggest that eIF-4F and/or eIF-3, or the steps mediated by these factors, mediate the inefficient translation in reticulocyte extracts and raise the possibility that these steps also control the regulated translation of r-protein mRNAs during myoblast differentiation.     

Point mutations modify the response of poliovirus RNA to a translation initiation factor: a comparison of neurovirulent and attenuated strains

Upon translation of poliovirus RNA in reticulocyte lysates, initiation occurs largely "incorrectly," that is, at sites in the middle of the viral genome rather than at the beginning of the polyprotein reading frame; the anomaly appears to be due to an initiation factor deficiency. Here, a fraction which stimulated initiation at the correct site, provisionally called "initiation correcting factor" (ICF), was partially purified from Krebs-2 cells. The ICF activity appeared to copurify with a complex of initiation factors eIF-2 and eIF-2B. The ability of ICF to stimulate, in reticulocyte lysates, the correct initiation of translation on the RNAs from neurovirulent and attenuated type 1 and type 3 poliovirus strains was investigated. Like crude initiation factor preparations, ICF appeared to be relatively less active with the RNAs from attenuated strains, the difference being especially pronounced for the type 3 strains. For the latter strains, the data suggested an important role of the nucleotide at, and perhaps around, position 472 in determining a response to the addition of ICF. It is proposed that interaction of a specific segment of the viral RNA with one or more of initiation factors plays an important part in the mechanism of translation of the picornavirus genomes, poliovirus attenuation, and, possibly, pathogenesis of poliomyelitis.     

Reversal of the interferon-induced block of protein synthesis by purified preparations of eucaryotic initiation factor 2.

The translation of Mengo virus RNA in extracts of interferon-treated mouse L cells in the presence of double-stranded RNA is inhibited about 80% when compared to translation in control cell extracts. Addition of eucaryotic initiation factor 2 (eIF-2) purified from rabbit reticulocytes leads to effective reversal of the interferon-induced block. This finding suggests that in interferon-treated cell extracts, inactivation of eIF-2 is a major mechanism of translational control.     

Hierarchical phosphorylation of the translation inhibitor 4E-BP1

In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5' end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.