New Hierarchical Phosphorylation Pathway of the Translational Repressor eIF4E-binding Protein 1 (4E-BP1) in Ischemia-Reperfusion Stress

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr⁶⁹-phosphorylated alone, Thr⁶⁹- and Thr³⁶/Thr⁴⁵-phosphorylated, all these plus Ser⁶⁴ phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr³⁶/Thr⁴⁵ phosphorylation alone was detected without Thr⁶⁹ phosphorylation, and neither was Ser⁶⁴ phosphorylation without Thr³⁶/Thr⁴⁵/Thr⁶⁹ phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr⁶⁹, Thr³⁶/Thr⁴⁵, and Ser⁶⁴ residues, with 4E-BP1 remaining phosphorylated at Thr⁶⁹ alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr³⁶/Thr⁴⁵ and Ser⁶⁴, in addition to Thr⁶⁹. Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr⁶⁹ is phosphorylated first followed by Thr³⁶/Thr⁴⁵ phosphorylation, and Ser⁶⁴ is phosphorylated last. Thr⁶⁹ phosphorylation alone allows binding to eIF4E, and subsequent Thr³⁶/Thr⁴⁵ phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.     

eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo

The mRNA's cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.     

Norovirus Translation Requires an Interaction between the C Terminus of the Genome-linked Viral Protein VPg and Eukaryotic Translation Initiation Factor 4G

Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5′ end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation.     

High-dose rapamycin induces apoptosis in human cancer cells by dissociating mTOR complex 1 and suppressing phosphorylation of 4E-BP1

mTOR, the mammalian target of rapamycin, has been widely implicated in signals that promote cell cycle progression and survival in cancer cells. Rapamycin, which inhibits mTOR with high specificity, has consequently attracted much attention as an anti-cancer therapeutic. Rapamycin suppresses phosphorylation of S6 kinase at nano-molar concentrations, however at higher micro-molar doses, rapamycin induces apoptosis in several human cancer cell lines. While much is known about the effect of low dose rapamycin treatment, the mechanistic basis for the apoptotic effects of high-dose rapamycin treatment is not understood. We report here that the apoptotic effects of high-dose rapamycin treatment correlate with suppressing phosphorylation of the mTOR complex 1 substrate, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). Consistent with this observation, ablation of eIF4E also resulted in apoptorsis in MDA-MB 231 breast cancer cells. We also provide evidence that the differential dose effects of rapamycin are correlated with partial and complete dissociation of Raptor from mTORC1 at low and high doses, respectively. In contrast with MDA-MB-231 cells, MCF-7 breast cancer cells survived rapamycin-induced suppression of 4E-BP1 phosphorylation. We show that survival correlated with a hyper-phosphorylation of Akt at S473 at high rapamycin doses, the suppression of which conferred rapamycin sensitivity. This study reveals that the apoptotic effect of rapamycin requires doses that completely dissociate Raptor from mTORC1 and suppress that phosphorylation of 4E-BP1 and inhibit eIF4E.     

Activation of p53 stimulates proteasome-dependent truncation of eIF4E-binding protein 1 (4E-BP1)

Background information. The translational inhibitor protein 4E‐BP1 [eIF4E (eukaryotic initiation factor 4E)‐binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5′ cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E‐BP1. Phosphorylation of 4E‐BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E‐BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. Results. We now report that activation of p53 also results in modification of 4E‐BP1 to a truncated form. Unlike full‐length 4E‐BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full‐length 4E‐BP1. Inhibitor studies indicate that the p53‐induced cleavage of 4E‐BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full‐length 4E‐BP1. Measurements of the turnover of 4E‐BP1 indicate that the truncated form is much more stable than the full‐length protein. Conclusions. The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E‐BP1, which may exert a long‐term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth‐inhibitory and pro‐apoptotic effects of p53.     

4E-BP1、4E-BP1-4A 重组慢病毒载体的构建及对胃癌细胞 HGC27生长的影响

目的:构建4E-BP1及其 T37A、T46A、S65A、T70A 突变体4E-BP1-4A 基表达的重组慢病毒载体,研究其对胃癌 HGC27细胞生长的影响.方法:PCR 扩增4E-BP1基及其突变体4E-BP1-4A 基并克隆到 pCDH 载体,构建成 pCDH-4E-BP1、pCDH-4E-BP1-4A,将其与包装载体共转染293T 细胞,包装成 Lenti-4E-BP1及 Lenti-4E-BP1-4A重组慢病毒载体,将此慢病毒感染胃癌 HGC27细胞,Western 印迹鉴定病毒载体介导的4E-BP1、4E-BP1-4A 蛋白的表达,MTT、克隆形成和软琼脂方法研究过量表达4E-BP1、4E-BP1-4A 对胃癌 HGC27细胞生长的影响.结果:包装成 Lenti-4E-BP1及 Lenti-4E-BP1-4A 重组慢病毒载体,并将此慢病毒载体感染胃癌 HGC27细胞;MTT、克隆形成、软琼脂实验表明过量表达4E-BP1可抑制胃癌 HGC27细胞的生长,过量表达4E-BP1-4A 时抑制效果更明显.结论:构建了4E-BP1、4E-BP1-4A 的重组慢病毒表达载体,在胃癌 HGC27细胞中过量表达4E-BP1可抑制细胞生长,过量表达4E-BP1-4A 的抑制效果更明显.     

mTOR／4E—BP1参与诺考达唑诱导的Dami细胞多倍体化调控

目的：多倍性是物种形成的重要机制,决定一些重要器官细胞产生的数量和功能,而且与某些病理过程（如恶性肿瘤）的发生有密切关系。我们通过建立相对同步化的多倍体细胞模型,已经证实mTOR／S6K1参与多倍体细胞周期的调控。本课题主要研究roTOR下游的另一个重要信号分子4E-BP1是否也参与细胞的倍体化调控。方法：诺考达唑诱导Dami细胞建立相对同步化的多倍体细胞模型,Western-blot分析多倍体细胞模型中mTOR／4E—BP1通路信号分子表达和磷酸化修饰位点的变化,流式细胞仪双荧光分析4E—BP1不同结构域磷酸化位点修饰与细胞周期各时相的关系。结果：诺考达唑诱导的Dami细胞可作为相对同步化的多倍体细胞周期模型,在二倍体和多倍体细胞周期中,mTOR表达增加及第2448位丝氨酸位点磷酸化发生在G1期进入S期,4E—BP1的第37,46位苏氨酸和第65位丝氨酸位点磷酸化发生在G2／M期。结论：mTOR／4E-BP1通路参与多倍体细胞周期的调控。     

Truncated initiation factor eIF4G lacking an eIF4E binding site can support capped mRNA translation

Picornavirus proteases cleave translation initiation factor eIF4G into a C-terminal two-thirds fragment (hereafter named p100) and an N-terminal one-third fragment, which interacts with the cap-binding factor eIF4E. As the timing of this cleavage correlates broadly with the shut-off of host cell protein synthesis in infected cells, a very widespread presumption has been that p100 cannot support capped mRNA translation. Through the use of an eIF4G-depleted reticulocyte lysate system, we show that this presumption is incorrect. Moreover, recombinant p100 can also reverse the inhibition of capped mRNA translation caused either by m7GpppG cap analogue, by 4E-BP1, which sequesters eIF4E and thus blocks its association with eIF4G, or by cleavage of endogenous eIF4G by picornavirus proteases. The concentration of p100 required for maximum translation of capped mRNAs is approximately 4-fold higher than the endogenous eIF4G concentration in reticulocyte lysates. Our results imply that picornavirus-induced shut-off is not due to an intrinsic inability of p100 to support capped mRNA translation, but to the viral RNA outcompeting host cell mRNA for the limiting concentration of p100.     

Hyperglycemia-induced O-GlcNAcylation and truncation of 4E-BP1 protein in liver of a mouse model of type 1 diabetes

4E-BP1 is a protein that, in its hypophosphorylated state, binds the mRNA cap-binding protein eIF4E and represses cap-dependent mRNA translation. By doing so, it plays a major role in the regulation of gene expression by controlling the overall rate of mRNA translation as well as the selection of mRNAs for translation. Phosphorylation of 4E-BP1 causes it to release eIF4E to function in mRNA translation. 4E-BP1 is also subject to covalent addition of N-acetylglucosamine to Ser or Thr residues (O-GlcNAcylation) as well as to truncation. In the truncated form, it is both resistant to phosphorylation and able to bind eIF4E with high affinity. In the present study, Ins2(Akita/+) diabetic mice were used to test the hypothesis that hyperglycemia and elevated flux of glucose through the hexosamine biosynthetic pathway lead to increased O-GlcNAcylation and truncation of 4E-BP1 and consequently decreased eIF4E function in the liver. The amounts of both full-length and truncated 4E-BP1 bound to eIF4E were significantly elevated in the liver of diabetic as compared with non-diabetic mice. In addition, O-GlcNAcylation of both the full-length and truncated proteins was elevated by 2.5- and 5-fold, respectively. Phlorizin treatment of diabetic mice lowered blood glucose concentrations and reduced the expression and O-GlcNAcylation of 4E-BP1. Additionally, when livers were perfused in the absence of insulin, 4E-BP1 phosphorylation in the livers of diabetic mice was normalized to the control value, yet O-GlcNAcylation and the association of 4E-BP1 with eIF4E remained elevated in the liver of diabetic mice. These findings provide insight into the pathogenesis of metabolic abnormalities associated with diabetes.     

Phosphorylation of Eukaryotic Translation Initiation Factor 4E and Eukaryotic Translation Initiation Factor 4E-binding Protein (4EBP) and Their Upstream Signaling Components Undergo Diurnal Oscillation in the Mouse Hippocampus: IMPLICATIONS FOR MEMORY PERSISTENCE*

Translation of mRNA plays a critical role in consolidation of long-term memory. Here, we report that markers of initiation of mRNA translation are activated during training for contextual memory and that they undergo diurnal oscillation in the mouse hippocampus with maximal activity observed during the daytime (zeitgeber time 4–8 h). Phosphorylation and activation of eukaryotic translation initiation factor 4E (eIF4E), eIF4E-binding protein 1 (4EBP1), ribosomal protein S6, and eIF4F cap-complex formation, all of which are markers for translation initiation, were higher in the hippocampus during the daytime compared with night. The circadian oscillation in markers of mRNA translation was lost in memory-deficient transgenic mice lacking calmodulin-stimulated adenylyl cyclases. Moreover, disruption of the circadian rhythm blocked diurnal oscillations in eIF4E, 4EBP1, rpS6, Akt, and ERK1/2 phosphorylation and impaired memory consolidation. Furthermore, repeated inhibition of translation in the hippocampus 48 h after contextual training with the protein synthesis inhibitor anisomycin impaired memory persistence. We conclude that repeated activation of markers of translation initiation in hippocampus during the circadian cycle might be critical for memory persistence.     

Coordinated and selective recruitment of eIF4E and eIF4G factors for potyvirus infection in Arabidopsis thaliana

The translation initiation factors eIF4E and eIF(iso)4E play a key role during virus infection in plants. During mRNA translation, eIF4E provides the cap-binding function and is associated with the protein eIF4G to form the eIF4F complex. Susceptibility analyses of Arabidopsis mutants knocked-out for At-eIF4G genes showed that eIF4G factors are indispensable for potyvirus infection. The colonization pattern by a viral recombinant carrying GFP indicated that eIF4G is involved at a very early infection step. Like eIF4E, eIF4G isoforms are selectively recruited for infection. Moreover, the eIF4G selective involvement parallels eIF4E recruitment. This is the first report of a coordinated and selective recruitment of eIF4E and eIF4G factors, suggesting the whole eIF4F recruitment.     

Mechanistic Target of Rapamycin Complex 1 (mTORC1)-mediated Phosphorylation Is Governed by Competition between Substrates for Interaction with Raptor

In this study, the interaction of mTORC1 with its downstream targets p70S6K1 and 4E-BP1 was evaluated in both mouse liver and mouse embryonic fibroblasts following combined disruption of the genes encoding 4E-BP1 and 4E-BP2. Phosphorylation of p70S6K1 was dramatically elevated in the livers of mice lacking 4E-BP1 and 4E-BP2 following feeding-induced activation of mTORC1. Immunoprecipitation of mTORC1 suggested that elevated phosphorylation was the result of enhanced interaction of p70S6K1 with raptor. These findings were extended to a cell culture system wherein loss of 4E-BP1 and 4E-BP2 resulted in elevated interaction of p70S6K1 with IGF1-induced activation of mTORC1 in conjunction with an enhanced rate of p70S6K1 phosphorylation at Thr-389. Furthermore, cotransfecting HA-p70S6K1 with 4E-BP1, but not 4E-BP1(F114A), reduced recovery of mTORC1 in HA-p70S6K1 immunoprecipitates. Together, these findings support the conclusion that, in the absence of 4E-BP proteins, mTORC1-mediated phosphorylation of p70S6K1 is elevated by a reduction in competition between the two substrates for interaction with raptor.     

Human Eukaryotic Initiation Factor 4G (eIF4G) Protein Binds to eIF3c, -d,and -e to Promote mRNA Recruitment to the Ribosome

Recruitment of mRNA to the 40S ribosomal subunit requires the coordinated interaction of a large number of translation initiation factors. In mammals, the direct interaction between eukaryotic initiation factor 4G (eIF4G) and eIF3 is thought to act as the molecular bridge between the mRNA cap-binding complex and the 40S subunit. A discrete ∼90 amino acid domain in eIF4G is responsible for binding to eIF3, but the identity of the eIF3 subunit(s) involved is less clear. The eIF3e subunit has been shown to directly bind eIF4G, but the potential role of other eIF3 subunits in stabilizing this interaction has not been investigated. It is also not clear if the eIF4A helicase plays a role in stabilizing the interaction between eIF4G and eIF3. Here, we have used a fluorescence anisotropy assay to demonstrate that eIF4G binds to eIF3 independently of eIF4A binding to the middle region of eIF4G. By using a site-specific cross-linking approach, we unexpectedly show that the eIF4G-binding surface in eIF3 is comprised of the -c, -d and -e subunits. Screening multiple cross-linker positions reveals that eIF4G contains two distinct eIF3-binding subdomains within the previously identified eIF3-binding domain. Finally, by employing an eIF4G-dependent translation assay, we establish that both of these subdomains are required for efficient mRNA recruitment to the ribosome and stimulate translation. Our study reveals unexpected complexity to the eIF3-eIF4G interaction that provides new insight into the regulation of mRNA recruitment to the human ribosome.     

4E-BP1 degradation and eIF4E truncation occur spatially distinctly in the porcine uterine epithelia and are features of noninvasive implantation in the pig

The implantation of the blastocyst into the endometrium is an indispensable premise for successful embryonic development. This process is regulated by maternal and embryonic signals that influence gene expression at the translational level, among other processes. Recently, we have shown that proteolytical cleavage of the prototypical 25‐kDa, mRNA cap‐binding protein eIF4E produces a stable variant with a molecular mass of approximately 23 kDa exclusively in the porcine endometrium during implantation. This is accompanied by dephosphorylation and reduction of the abundant repressor 4E‐BP1. Here, we investigate the distribution of the truncated eIF4E and of 4E‐BP1 in the porcine uterine tissue, their binding in native samples, and we analyzed eIF4E‐, eIF4G‐, and 4E‐BP1‐specific proteolytic activities. Our results show that in pigs, the truncated eIF4E is located in the endometrial luminal epithelium during implantation. Neither glandulary tissue nor stroma expressed any truncated eIF4E. The reduced abundance of 4E‐BP1 during implantation is mainly the result of decay in the glandular epithelia. Moreover, steroid replacements, in vitro protease assays, and cell lysate fractionation showed that eIF4E cleavage and 4E‐BP1 decay both depended on the ovarian steroid hormones estradiol and progestrone, but these effects are the result of different proteolytic activities. Although eIF4G cleavage also depends on calcium, stimulation by these steroids could not be established. We propose that the translation initiation process in the endometrium is differently regulated by the truncated eIF4E, utilizing different abundances of 4E‐BP1 and binding dynamic of eIF4E/4E‐BP1 in distinct forms of implantation. Mol. Reprod. Dev. 78:895–905, 2011. © 2011 Wiley Periodicals, Inc.     

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: A reappraisal of translation initiation during mitosis

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G₁/S boundary) or the Cdk1 inhibitor, RO3306 (G₂/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.     

Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection

Protein synthesis is a tightly controlled process responding to several stimuli, including viral infection. As obligate intracellular parasites, viruses depend on the translation machinery of the host and can manipulate it by affecting the availability and function of specific eukaryotic initiation factors (eIFs). Human norovirus is a member of the Caliciviridae family and is responsible for gastroenteritis outbreaks. Previous studies on feline calicivirus and murine norovirus 1 (MNV1) demonstrated that the viral protein, genome-linked (VPg), acts to direct translation by hijacking the host protein synthesis machinery. Here we report that MNV1 infection modulates the MAPK pathway to activate eIF4E phosphorylation. Our results show that the activation of p38 and Mnk during MNV1 infection is important for MNV1 replication. Furthermore, phosphorylated eIF4E relocates to the polysomes, and this contributes to changes in the translational state of specific host mRNAs. We propose that global translational control of the host by eIF4E phosphorylation is a key component of the host-pathogen interaction.     

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.