Dephosphorylation Of eIF4E Research Articles

Decreased NOTCH1 Activation Correlates with Response to Ibrutinib in Chronic Lymphocytic Leukemia.

Ibrutinib, a Bruton tyrosine kinase inhibitor (BTKi), has improved the outcomes of chronic lymphocytic leukemia (CLL), but primary resistance or relapse are issues of increasing significance. While the predominant mechanism of action of BTKi is the B-cell receptor (BCR) blockade, many off-target effects are unknown. We investigated potential interactions between BCR pathway and NOTCH1 activity in ibrutinib-treated CLL to identify new mechanisms of therapy resistance and markers to monitor disease response. NOTCH activations was evaluated either in vitro and ex vivo in CLL samples after ibrutinib treatment by Western blotting. Confocal proximity ligation assay (PLA) experiments and analyses of down-targets of NOTCH1 by qRT-PCR were used to investigate the cross-talk between BTK and NOTCH1. In vitro ibrutinib treatment of CLL significantly reduced activated NOTCH1/2 and induced dephosphorylation of eIF4E, a NOTCH target in CLL. BCR stimulation increased the expression of activated NOTCH1 that accumulated in the nucleus leading to HES1, DTX1, and c-MYC transcription. Results of in situ PLA experiments revealed the presence of NOTCH1-ICD/BTK complexes, whose number was reduced after ibrutinib treatment. In ibrutinib-treated CLL patients, leukemic cells showed NOTCH1 activity downregulation that deepened over time. The NOTCH1 signaling was restored at relapse and remained activated in ibrutinib-resistant CLL cells. We demonstrated a strong clinical activity of ibrutinib in a real-life context. The ibrutinib clinical efficacy was associated with NOTCH1 activity downregulation that deepened over time. Our data point to NOTCH1 as a new molecular partner in BCR signaling with potential to further improve CLL-targeted treatments.

4EGI-1 induces apoptosis and enhances radiotherapy sensitivity in nasopharyngeal carcinoma cells via DR5 induction on 4E-BP1 dephosphorylation.

The eIF4F complex regulated by a various group of eIF4E-binding proteins (4E-BPs) can initial the protein synthesis. Small molecule compound 4EGI-1, an inhibitor of the cap-dependent translation initiation through disturbing the interaction between eIF4E and eIF4G which are main elements of the eIF4E complex, has been reported to suppress cell proliferation by inducing apoptosis in many types of cancer. And death receptor 5 (DR5) is a major component in the extrinsic apoptotic pathway. However, the correlation among 4EGI-1, DR5 and 4E-BPs have not been discovered in NPC now. Therefore, we intend to find out the effect of 4EGI-1 on the apoptosis process of NPC and the relationship among 4EGI-1, DR5 and 4E-BPs. Our results revealed a significant down regulation of DR5 expression in NPC tissues, which inversely correlated with lymph node metastasis status and clinical stages. Depressed DR5 expression was an independent biomarker for poor prognosis in NPC, and elevated DR5 expression showed longer overall survival time in 174 NPC patients. Besides, 4EGI-1 induced apoptosis in NPC cells through the DR5-caspase-8 axis on 4E-BP1 and eIF4E dephosphorylation exerting positive influence on their anti-tumor activities. The induction of DR5 also sensitized NPC cells to radiotherapy, and the SER was 1.195. These results establish the death receptor pathway as a novel anticancer mechanism of eIF4E/eIF4G interaction inhibitor in NPC.

Dual modulation of Ras-Mnk and PI3K-AKT-mTOR pathways: A Novel c-FLIP inhibitory mechanism of 3-AWA mediated translational attenuation through dephosphorylation of eIF4E.

The eukaryotic translation initiation factor 4E (eIF4E) is considered as a key survival protein involved in cell cycle progression, transformation and apoptosis resistance. Herein, we demonstrate that medicinal plant derivative 3-AWA (from Withaferin A) suppressed the proliferation and metastasis of CaP cells through abrogation of eIF4E activation and expression via c-FLIP dependent mechanism. This translational attenuation prevents the de novo synthesis of major players of metastatic cascades viz. c-FLIP, c-Myc and cyclin D1. Moreover, the suppression of c-FLIP due to inhibition of translation initiation complex by 3-AWA enhanced FAS trafficking, BID and caspase 8 cleavage. Further ectopically restored c-Myc and GFP-HRas mediated activation of eIF4E was reduced by 3-AWA in transformed NIH3T3 cells. Detailed underlying mechanisms revealed that 3-AWA inhibited Ras-Mnk and PI3-AKT-mTOR, two major pathways through which eIF4E converges upon eIF4F hub. In addition to in vitro studies, we confirmed that 3-AWA efficiently suppressed tumor growth and metastasis in different mouse models. Given that 3-AWA inhibits c-FLIP through abrogation of translation initiation by co-targeting mTOR and Mnk-eIF4E, it (3-AWA) can be exploited as a lead pharmacophore for promising anti-cancer therapeutic development.

Involvement of p38 in signal switching from autophagy to apoptosis via the PERK/eIF2α/ATF4 axis in selenite-treated NB4 cells.

Selenite has emerged as an optional chemotherapeutic agent for hematological malignancies. Autophagy and apoptosis are both engaged in selenite-induced cell death. In a previous report, we have identified heat shock protein 90 (Hsp90) as a critical modulator of the balance between autophagy and apoptosis in selenite-treated leukemia cells. However, the mechanisms by which selenite mediates the crosstalk between autophagy and apoptosis remain largely unknown. Herein, we demonstrate that the endoplasmic reticulum (ER) stress-related PERK/eIF2α/ATF4 pathway and p38 are core modules for the selenite-induced switch to apoptosis from autophagy. We found that selenite activated PERK and eIF2α/ATF4 downstream to promote apoptosis. During this progression, p38 was dissociated from PERK-inhibiting Hsp90 and became autophosphorylated. Then, activated p38 further enhanced the docking of activating transcription factor 4 (ATF4) onto the CHOP (CCAAT/enhancer-binding protein homologous protein) promoter via eIF2α to enhance apoptosis. We also found that activated p38 suppressed the phosphorylation of eIF4E that directed ATF4 to bind to the MAP1LC3B (microtubule-associated protein 1 light chain 3B) promoter. Because of the deactivation of eIF4E, the association of ATF4 with the MAP1LC3B promoter was inhibited, and autophagy was compromised. Intriguingly, p53 played important roles in mediating the p38-mediated regulation of eIF2α and eIF4E. When activated by p38, p53 induced the phosphorylation of eIF2α and the dephosphorylation of eIF4E, particularly in the nucleus where the ATF4 transcription factor was modulated, ultimately resulting in differential expression of CHOP and LC3. Moreover, selenite exhibited potent antitumor effects in vivo. In an NB4 cell xenograft model, selenite induced apoptosis and hampered autophagy. In addition, related signaling proteins demonstrated similar changes to those observed in vitro. These data suggest that selenite may be a candidate drug for leukemia therapy.

Proteasome Inhibition Leads to Dephosphorylation and Downregulation of Protein Expression of Members of the Akt/mTOR Pathway In MCL

AbstractAbstract 4449 Background:Mantle cell Lymphoma (MCL) is a distinct B-cell subtype characterized by the chromosomal translocation t(11;14)(q13;q32), an especially poor clinical outcome and low response to chemotherapy. The proteasome inhibitor bortezomib is approved for treatment of relapsed and refractory MCL and achieves a response rate of 30–40%. However, little is known which molecules represent the critical targets of proteasome inhibition and how different regulators of cell cycle and apoptosis are affected. Bortezomib has been shown to inactivate the NFkB pathway in MCL, but recent findings indicate Bortezomib is also active in a proteasome –independent manner suggesting Bortezomib targets multiple pathways. Method:Four MCL cell lines (HBL2, Granta 519, Jeko-1, NCEB-1), two CLL cell lines (Mec1, Mec2) and two hematological control cell lines (Jurkat, Karpas 422) were exposed to Bortezomib at a previously defined cytotoxic concentration (25nmol). Western blot and mRNA analysis were performed for various members of the PI3K/Akt/mTOR and the MEK/ERK pathway after 24h Bortezomib exposure. Results were compared to cell proliferation (WST1, trypan blue staining), induction of apoptosis (Annexin V PE/7-AAD staining) and cell cycle data (FACS). Result:Western blot analysis revealed reduced phosphorylation of Akt at Ser473 in all cell lines while autophosphorylation of mTOR at Ser2481 was completely downregulated only in the susceptible cell lines. In addition further members of the mTOR pathway were affected by bortezomib treatment. Dephosphorylation of the 4EBP1, downregulation of p70S6 protein expression was detected in all cell lines whereas EIF4E dephosphorylation was only observed in the sensitive MCL cell lines. In contrast Bortezomib did not affect members of the MEK/ERK pathway (MEK1/2, p42/44MAPK). Interestingly the mRNA and protein expression profile of CCND1 were differently altered suggesting an involvement of bortezomib in the regulation of translation initiation. This data were also confirmed by microarray analysis. Conclusion:In this study Bortezomib treatment was shown to target the Akt/mTOR pathway especially by dephosphorylation of the translation initiation factor EIF4E and other molecules of this signal pathway. This knowledge will play a crucial role in the development of future combinations of biologicals to target the molecular pathogenesis of MCL. Disclosures:Dreyling:Johnson & Johnson: support of investigators initial trials.

Overexpression and Phosphorylation of eIF4E Is Required for Beta-Catenin Activation in Blast Crisis Chronic Myelogenous Leukemia.

Abstract 41▪▪This icon denotes an abstract that is clinically relevant.Despite the success of tyrosine kinase inhibitors (TKI) in chronic phase (CP) CML, blast crisis (BC) CML remains difficult to control, with most patients eventually developing either primary or secondary TKI-resistance (TKI-R). In addition to TKI-R, myeloid BC GMPs acquire the ability to self-renew, a feature that is mediated by b-catenin activation, and which may contribute to disease persistence. The mechanisms underlying b-catenin activation remain obscure, including its dependence on Bcr-Abl activity. Long-term control of BC CML will require overcoming the mechanisms mediating TKI-R, as well as reversing b-catenin-mediated self-renewal. In this study, we investigated the role of the mRNA translation machinery in mediating b-catenin-mediated self-renewal, since our prior work had implicated aberrant mRNA translation in TKI-R and BC pathophysiology (Ly et al. Cancer Research 2003; Prabhu et al. Oncogene, 2007; Zhang et al. MCB, 2008). First, we showed that primary CD34+ BC cells overexpressed eIF4E, a rate-limiting translation initiation factor, compared to CP cells. We also found that both eIF4E overexpression and phosphorylation at serine 209 (S209) occurred in a Bcr-Abl-independent manner. Next, by using a combination of genetic and biochemical approaches, we showed that forced overexpression of eIF4E was sufficient to activate b-catenin in the K562 BC cell line, as measured by a b-catenin reporter (a 2-fold increase in TOP/FOP ratio) and by an increase in nuclear b-catenin (a 2.5-fold increase in mean fluorescence intensity using immunofluorescence). Importantly, overexpression of an eIF4E S209A mutant did not show increased b-catenin activity, whereas overexpression of a phospho-mimetic S209D mutant did, indicating b-catenin activation was dependent on S209 phosphorylation. Next, we found that siRNA knock-down or small molecule inhibition (CGP57380) of the Mnk 1/2 kinases (which are responsible for in vivo phosphorylation of eIF4E) prevented the increased b-catenin activity induced by eIF4E overexpression. Also, Mnk1/2 inactivation had no effect on b-catenin activity in the S209D mutants. Because recent work has implicated GSK3b inactivation as a contributor to b-catenin activity in BC GMPs (Abrahamsson et al. PNAS, 2009), we repeated these experiments in the presence of the GSK3b inhibitor, BIO. Here, we found that both eIF4E overexpression and BIO treatment cooperated to further augment b-catenin activity (a 3 to 4-fold increase in the TOP/FOP ratio, and 3-fold increase in nuclear b-catenin), and that this was an effect that required eIF4E phosphorylation. Based on these data, we performed biochemical and functional analyses of primary CP and BC CML cells, as well as normal cord blood (CB) progenitors. As reported by others, we found increased nuclear b-catenin in CD34+CD38+ BC cells compared CP or CB cells. Inhibition of Mnk 1/2 was also able to prevent accumulation of nuclear b-catenin in CD34+CD38+ BC cells, as was a known b-catenin inhibitor (CGP049090), but not imatinib. Functionally, Mnk kinase inhibition by CGP57380 (2.5-10 microM) also significantly decreased the ability of BC CFCs to serially replate (>8 weeks) in methylcellulose (by >90%) compared to CB CFCs (25-50%). Finally, since recent work has demonstrated that activation of the serine/threonine phosphatase, PP2A, can impair b-catenin activity and self-renewal in CML stem cells (Neviani et al, ASH Meeting Abstracts, 2008), we tested the ability of the PP2A activator, FTY720, to inhibit b-catenin activity in the context of eIF4E overexpression. We found that while FTY720 was able to inhibit b-catenin activity in cells overexpressing wild-type eIF4E, it could not do so in the presence of the S209D mutants. In summary, our results indicate that: (i) increased expression and phosphorylation of eIF4E at S209 is sufficient to activate b-catenin in CML cells; (ii) GSK3b inactivation and eIF4E phosphorylation cooperate to augment b-catenin activity; (iii) the serial-replating ability of BC CML progenitors is dependent on eIF4E phosphorylation; (iv) dephosphorylation of eIF4E at S209 is important in enabling the PP2A activator, FTY720, to inhibit b-catenin activity. Taken together, our data show that targeting eIF4E function, by modulating the activity of the kinases or phosphatases which regulate S209 phosphorylation, is a novel approach to overcome b-catenin-mediated self-renewal in BC CML cells, but not normal progenitors. Disclosures:No relevant conflicts of interest to declare.

Regulation of cap‐dependent translation initiation in the early stage porcine parthenotes

The binding of mRNAs to ribosomes is mediated by the protein complex eIF4F in conjunction with eIF4B (eukaryotic initiation factor 4F and 4B). EIF4F is a three subunit complex consisting of eIF4A (RNA helicase), eIF4E (mRNA cap binding protein), and eIF4G (bridging protein). The crucial role is played by eIF4E, which directly binds the 5'-cap structure of the mRNA and facilitates the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. EIF4E binding to mRNA and to other initiation factors is regulated on several levels, including its phosphorylation on Ser-209, and association with its regulatory protein 4E-binding protein (4E-BP1). In this study we document that both the translation initiation factor eIF4E and its regulator 4E-BP1 become dephosphorylated in the early stage porcine zygotes already 8 hr post-activation. Similarly, the activities of ERK1/2 MAP and Mnk1 kinases, which are both involved in eIF4E phosphorylation, gradually decrease during this period with the timing similar to that of eIF4E dephosphorylation. The formation of an active eIF4F complex is also diminished after 9-15 hr post-activation, although substantial amounts of this complex have been detected also 24 hr post-activation (2-cell stage). The overall protein synthesis in the parthenotes decreases gradually from 12 hr post-activation reaching a minimum after 48 hr (4-cell stage). Although the translation is gradually decreasing during early preimplantation development, the eIF4F complex, which is temporarily formed, might be a premise for the translation of a small subset of mRNAs at this period of development.

Ras Transformation of RIE-1 Cells Activates Cap-Independent Translation of Ornithine Decarboxylase: Regulation by the Raf/MEK/ERK and Phosphatidylinositol 3-Kinase Pathways

Ornithine decarboxylase (ODC) is the first and generally rate-limiting enzyme in polyamine biosynthesis. Deregulation of ODC is critical for oncogenic growth, and ODC is a target of Ras. These experiments examine translational regulation of ODC in RIE-1 cells, comparing untransformed cells with those transformed by an activated Ras12V mutant. Analysis of the ODC 5' untranslated region (5'UTR) revealed four splice variants with the presence or absence of two intronic sequences. All four 5'UTR species were found in both cell lines; however, variants containing intronic sequences were more abundant in Ras-transformed cells. All splice variants support internal ribosome entry site (IRES)-mediated translation, and IRES activity is markedly elevated in cells transformed by Ras. Inhibition of Ras effector targets indicated that the ODC IRES element is regulated by the phosphorylation status of the translation factor eIF4E. Dephosphorylation of eIF4E by inhibition of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) or the eIF4E kinase Mnk1/2 increases ODC IRES activity in both cell lines. When both the Raf/MEK/ERK and phosphatidylinositol 3-kinase/mammalian target of rapamycin pathways are inhibited in normal cells, ODC IRES activity is very low and cells arrest in G(1). When these pathways are inhibited in Ras-transformed cells, cell cycle arrest does not occur and ODC IRES activity increases, helping to maintain high ODC activity.

N-acetyl-cysteine abolishes hydrogen peroxide-induced modification of eukaryotic initiation factor 4F activity via distinct signalling pathways

During the oxidative stress generated by hydrogen peroxide (H 2O 2) in nerve growth factor (NGF)-differentiated PC12 cells, eIF4E binding protein (4E-BP1) and initiation factor 4E (eIF4E) phosphorylated levels decrease significantly, and an enhancement of the association of 4E-BP1 to eIF4E, which in turn decreases eIF4F formation is observed. The treatment with N-acetyl-cysteine (NAC) completely abolishes the H 2O 2-induced decrease in eIF4E phosphorylated levels, whereas the decrease in 4E-BP1 phosphorylated levels and eIF4F activity inhibition are significantly but not fully reversed. Rapamycin, the mammalian target of rapamycin (FRAP/mTOR) inhibitor, prevents the effect of NAC on H 2O 2-induced eIF4F complex formation inhibition. Besides the inhibitor induces a similar decrease in 4E-BP1 phosphorylated levels to that promote by H 2O 2. However, rapamycin has no effect on the NAC-induced recovery in phosphorylated eIF4E levels. Neither the MAP kinase inhibitors, PD98056 and SB203580, or the protein phosphatase 2A inhibitor, okadaic acid, mimic NAC effect on the H 2O 2-induced eIF4E dephosphorylation. Altogether our findings suggest that the effects caused by oxidative stress on eIF4s factors depends on two MAP kinase-independent signal transduction pathways, being at least one of them rapamycin-dependent.

Acute treatment with TNF-α attenuates insulin-stimulated protein synthesis in cultures of C2C12myotubes through a MEK1-sensitive mechanism

Insulin and TNF-alpha exert opposing effects on skeletal muscle protein synthesis that are mediated in part by the rapamycin-sensitive mammalian target of rapamycin (mTOR) pathway and the PD-98059-sensitive, extracellular signal-regulated kinase (ERK)1/2 pathway. The present study examined the separate and combined effects of insulin (INS), TNF, PD-98059, or dnMEK1 adenovirus on the translational control of protein synthesis in C(2)C(12) myotubes. Cultures were treated with INS, TNF, PD-98059, dnMEK1, or a combination of INS + TNF with PD-98059 or dnMEK1. INS stimulated protein synthesis, enhanced eIF4E.eIF4G association, and eIF4G phosphorylation and repressed eIF4E.4E-BP1 association vs. control. INS also promoted phosphorylation of ERK1/2, S6K1, and 4E-BP1 and dephosphorylation of eIF4E. TNF alone did not have an effect on protein synthesis (vs. control), eIF4E.eIF4G association, or the phosphorylation of eIF4G, S6K1, or 4E-BP1, although it transiently increased ERK1/2 and eIF4E phosphorylation. When myotubes were treated with TNF + INS, the cytokine blocked the insulin-induced stimulation of protein synthesis. This appeared to be due to an attenuation of insulin-stimulated eIF4E.eIF4G association, because other stimulatory effects of INS, e.g., phosphorylation of ERK1/2, 4E-BP1, S6K1, eIF4G, and eIF4E and eIF4E.4E-BP1 association, were unaffected. Finally, treatment of myotubes with PD-98059 or dnMEK1 adenovirus before TNF + INS addition resulted in a derepression of protein synthesis and the association of eIF4G with eIF4E. These findings suggest that TNF abrogates insulin-induced stimulation of protein synthesis in myotubes through a decrease in eIF4F complex assembly independently of S6K1 and 4E-BP1 signaling and dependently on a MEK1-sensitive signaling pathway.

Structural basis for competitive inhibition of eIF4G-Mnk1 interaction by the adenovirus 100-kilodalton protein.

Translation of most cellular mRNAs involves cap binding by the translation initiation complex. Among this complex of proteins are cap-binding protein eIF4E and the eIF4E kinase Mnk1. Cap-dependent mRNA translation generally correlates with Mnk1 phosphorylation of eIF4E when both are bound to eIF4G. During the late phase of adenovirus (Ad) infection translation of cellular mRNA is inhibited, which correlates with displacement of Mnk1 from eIF4G by the viral 100-kDa (100K) protein and dephosphorylation of eIF4E. Here we describe the molecular mechanism for 100K protein displacement of Mnk1 from eIF4G and elucidate a structural basis for eIF4G interaction with Mnk1 and 100K proteins and Ad inhibition of cellular protein synthesis. The eIF4G-binding site is located in an N-terminal 66-amino-acid peptide of 100K which is sufficient to bind eIF4G, displace Mnk1, block eIF4E phosphorylation, and inhibit eIF4F (cap)-dependent cellular mRNA translation. Ad 100K and Mnk1 proteins possess a common eIF4G-binding motif, but 100K protein binds more strongly to eIF4G than does Mnk1. Unlike Mnk1, for which binding to eIF4G is RNA dependent, competitive binding by 100K protein is RNA independent. These data support a model whereby 100K protein blocks cellular protein synthesis by coopting eIF4G and cap-initiation complexes regardless of their association with mRNA and displacing or blocking binding by Mnk1, which occurs only on preassembled complexes, resulting in dephosphorylation of eIF4E.

An activity-dependent switch to cap-independent translation triggered by eIF4E dephosphorylation.

The rate of translation of egg-laying hormone (ELH) in Aplysia californica bag cell neurons rises after an afterdischarge (AD), the physiological trigger for egg-laying1. We found that the 5′ untranslated region (5′ UTR) of ELH possessed an internal ribosomal entry site (IRES), and that an AD was accompanied by a switch to cap-independent, IRES-mediated translation, indicating that the use of a cellular IRES can be regulated by physiological activity. Furthermore, the AD caused dephosphorylation of the initiation factor eIF4E, which was sufficient to increase use of the ELH IRES, showing that dephosphorylation of eIF4E can trigger a switch to IRES-mediated translation.

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1.

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.

Regulation of Cap-Dependent Translation by Insulin-like Growth Factor-1 in Neuronal Cells

Insulin-like growth factor-1 (IGF-1) both promotes survival and activates protein synthesis in neurons. In the present paper, we investigate the effect of IGF-1 treatment on cap-dependent translation in primary cultured neuronal cells. IGF-1 treatment increased the phosphorylation of eukaryotic initiation factor (eIF)-4E-binding protein 1 (4E-BP1), exclusively at Thr-36 and Thr-45 residues, and eIF-4G phosphorylation at Ser-1108. In contrast, a significant eIF-4E dephosphorylation was found. In parallel, increased eIF-4E/4G assembly and protein synthesis activation in response to IGF-1 treatment were observed. The phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the mammalian target of rapamycin (mTOR) inhibitor rapamycin, but not the mitogen-activated protein kinase (MAPK)-activating kinase (MEK) inhibitor PD98059, reversed the IGF-1-induced effects observed on eIF-4E/4G assembly and phosphorylation status of 4E-BP1, eIF-4E, and eIF-4G. Therefore, our findings show that the IGF-1-induced regulation of cap-dependent translation is largely dependent on the PI-3K and mTOR pathway in neuronal cells.

Rapid dephosphorylation of eIF4E by dietary protein in the skeletal muscle and liver of food-deprived rats.

The effect of dietary protein on eIF4E phosphorylation was examined in rats starved for 18 h and then fed on either a 20% casein diet (20C) or a protein-free diet (0C). Refeeding with the 20C diet, but not the 0C diet, resulted in partial dephosphorylation of eIF4E in both the skeletal muscle and liver. The results suggest that the dephosphorylation of eIF4E in response to food intake was regulated by the increase in plasma amino acid concentration that occurred after feeding with the 20C diet.

Ischaemia induces changes in the association of the binding protein 4E-BP1 and eukaryotic initiation factor (eIF) 4G to eIF4E in differentiated PC12 cells

Ischaemia was obtained in vitro by subjecting nerve-growth-factor-differentiated PC12 cells to glucose deprivation plus anoxia. During ischaemia the rate of protein synthesis was significantly inhibited, and eIF4E-binding protein (4E-BP1) and eukaryotic initiation factor 4E (eIF4E) were significantly dephosphorylated in parallel. In addition, ischaemia induced an enhancement of the association of 4E-BP1 to eIF4E, which in turn decreased eIF4F formation, whereas no degradation of initiation factor 4G was observed. The treatment of PC12 cells with the specific p38 mitogen-activated protein kinase inhibitor SB203580 induced eIF4E dephosphorylation but did not cause any effect on protein synthesis rate. Rapamycin, the inhibitor of mammalian target of rapamycin ('mTOR'), but not PD98059, the inhibitor of extracellular signal-regulated protein kinases ('ERK1/2'), induced similar effects on 4E-BP1 phosphorylation to ischaemia; nevertheless, 4E-BP1-eIF4E complex levels were higher in ischaemia than in rapamycin-treated cells. In addition, both protein synthesis rate and eIF4F formation were lower in ischaemic cells than in rapamycin-treated cells.

Phosphorylation of eIF-4E on Ser 209 in Response to Mitogenic and Inflammatory Stimuli Is Faithfully Detected by Specific Antibodies

Phosphorylation of Ser 209 is thought to modulate the activity of the cap-binding factor eIF-4E which is a crucial component in the initiation complex for cap-dependent translation of mRNA. We report here the full reconstitution of the p38 Map kinase cascade leading to phosphorylation of eIF-4E in vitro and the generation of antibodies specific for phospho-serine 209 in eIF-4E. These antibodies were used to probe the phosphorylation of eIF-4E in mammalian cells stimulated with mitogens and pro-inflammatory cytokines. Treatment of human dermal fibroblasts with FCS led to a transient hyperphosphorylation, followed by hypophosphorylation and return to normal state phosphorylation at 16 h after the initial stimulation. By using a potent small molecular weight inhibitor of Mnk1, the upstream kinase for eIF-4E, we observed a rapid dephosphorylation of eIF-4E within 45 min after addition of the inhibitor, suggesting a high turnover of phosphate on eIF-4E mediated by Mnk1 and a yet unidentified phosphatase.

Effect of dietary protein on translation initiation in rat skeletal muscle and liver

The effect of dietary protein on the initiation of mRNA translation was examined in rats starved for 18 h and then fed isocaloric diets containing either 20% protein (20P) or no added protein (0P). Feeding the 20P diet, but not the 0P diet, stimulated protein synthesis in skeletal muscle and liver by 38 and 41%, respectively. The stimulation was associated with reduced binding of eukaryotic initiation factor (eIF) 4E to the translational repressor 4E-BP1, increased formation of the active eIF4E-eIF4G complex, and increased phosphorylation of 4E-BP1. In contrast, feeding a 0P diet had no effect on any of these parameters. Feeding a 20P diet resulted in partial dephosphorylation of eIF4E in both tissues. In liver, refeeding a 0P diet also resulted in partial eIF4E dephosphorylation, suggesting that the phosphorylation state of eIF4E is not important in the stimulation of protein synthesis under these conditions. Finally, plasma insulin concentrations were the same in rats fed either diet (14.8 +/- 4.9 vs. 15.5 +/- 4.5 microU/ml for 20P and 0P groups, respectively), suggesting that feeding-induced changes in plasma insulin are not sufficient to stimulate protein synthesis. Instead, a combination of dietary protein and insulin may be required to stimulate translation initiation.

Signal transduction pathways involved in the regulation of protein synthesis by insulin in L6 myoblasts.

The phosphorylation states of three proteins implicated in the action of insulin on translation were investigated, i.e., 70-kDa ribosomal protein S6 kinase (p70S6k), eukaryotic initiation factor (eIF) 4E, and the eIF-4E binding protein 4E-BP1. Addition of insulin caused a stimulation of protein synthesis in L6 myoblasts in culture, an effect that was blocked by inhibitors of phosphatidylinositide-3-OH kinase (wortmannin), p70S6k (rapamycin), and mitogen-activated protein kinase (MAP kinase) kinase (PD-98059). The stimulation of protein synthesis was accompanied by increased phosphorylation of p70S6k, an effect that was blocked by rapamycin and wortmannin but not PD-98059. Insulin caused dephosphorylation of eIF-4E, an effect that appeared to be mediated by the p70S6k pathway. Insulin also stimulated phosphorylation of 4E-BP1 as well as dissociation of the 4E-BP1.eIF-4E complex. Both rapamycin and wortmannin completely blocked the insulin-induced changes in 4E-BP1 phosphorylation and association of 4E-BP1 and eIF-4E; PD-98059 had no effect on either parameter. Finally, insulin stimulated formation of the active eIF-4G.eIF-4E complex, an effect that was not prevented by any of the inhibitors. Overall, the results suggest that insulin stimulates protein synthesis in L6 myoblasts in part through utilization of both the p70S6k and MAP kinase signal transduction pathways.

Cap-Binding Protein (Eukaryotic Initiation Factor 4E) and 4E-Inactivating Protein BP-1 Independently Regulate Cap-Dependent Translation

Cap-dependent protein synthesis in animal cells is inhibited by heat shock, serum deprivation, metaphase arrest, and infection with certain viruses such as adenovirus (Ad). At a mechanistic level, translation of capped mRNAs is inhibited by dephosphorylation of eukaryotic initiation factor 4E (eIF-4E) (cap-binding protein) and its physical sequestration with the translation repressor protein BP-1 (PHAS-I). Dephosphorylation of BP-I blocks cap-dependent translation by promoting sequestration of eIF-4E. Here we show that heat shock inhibits translation of capped mRNAs by simultaneously inducing dephosphorylation of eIF-4E and BP-1, suggesting that cells might coordinately regulate translation of capped mRNAs by impairing both the activity and the availability of eIF-4E. Like heat shock, late Ad infection is shown to induce dephosphorylation of eIF-4E. However, in contrast to heat shock, Ad also induces phosphorylation of BP-1 and release of eIF-4E. BP-1 and eIF-4E can therefore act on cap-dependent translation in either a mutually antagonistic or cooperative manner. Three sets of experiments further underscore this point: (i) rapamycin is shown to block phosphorylation of BP-1 without inhibiting dephosphorylation of eIF-4E induced by heat shock or Ad infection, (ii) eIF-4E is efficiently dephosphorylated during heat shock or Ad infection regardless of whether it is in a complex with BP-1, and (iii) BP-1 is associated with eIF-4E in vivo regardless of the state of eIF-4E phosphorylation. These and other studies establish that inhibition of cap-dependent translation does not obligatorily involve sequestration of eIF-4E by BP-1. Rather, translation is independently regulated by the phosphorylation states of eIF-4E and the 4E-binding protein, BP-1. In addition, these results demonstrate that BP-1 and eIF-4E can act either in concert or in opposition to independently regulate cap-dependent translation. We suggest that independent regulation of eIF-4E and BP-1 might finely regulate the efficiency of translation initiation or possibly control cap-dependent translation for fundamentally different purposes.