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.     

Acute treatment with TNF-alpha attenuates insulin-stimulated protein synthesis in cultures of C2C12 myotubes 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.     

Phosphorylation states of translational initiation factors affect mRNA cap binding in wheat

Phosphorylation of eukaryotic translational initiation factors (eIFs) has been shown to be an important means of regulating protein synthesis. Plant initiation factors undergo phosphorylation/dephosphorylation under a variety of stress and growth conditions. We have shown that recombinant wheat cap-binding protein, eIF(iso)4E, produced from E. coli can be phosphorylated in vitro. Phosphorylation of eIF(iso)4E has effects on m(7)G cap-binding affinity similar to those of phosphorylation of mammalian eIF4E even though eIF(iso)4E lacks an amino acid that can be phosphorylated at the residue corresponding to Ser-209, the phosphorylation site in mammalian eIF4E. The cap-binding affinity was reduced 1.2-2.6-fold when eIF(iso)4E was phosphorylated. The in vitro phosphorylation site for wheat eIF(iso)4E was identified as Ser-207. Addition of eIF(iso)4G and eIF4B that had also been phosphorylated in vitro further reduced cap-binding affinity. Temperature-dependent studies showed that DeltaH(degrees) was favorable for cap binding regardless of the phosphorylation state of the initiation factors. The entropy, however, was unfavorable (negative) except when eIF(iso)4E was phosphorylated and interacting with eIF(iso)4G. Phosphorylation may modulate not only cap-binding activity, but other functions of eukaryotic initiation factors as well.     

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.     

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.     

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.     

A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E.

In the yeast Saccharomyces cerevisiae a small protein named p20 is found associated with translation initiation factor eIF4E, the mRNA cap-binding protein. We demonstrate here that p20 is a repressor of cap-dependent translation initiation. p20 shows amino acid sequence homology to a region of eIF4G, the large subunit of the cap-binding protein complex eIF4F, which carries the binding site for eIF4E. Both, eIF4G and p20 bind to eIF4E and compete with each other for binding to eIF4E. The eIF4E-p20 complex can bind to the cap structure and inhibit cap-dependent but not cap-independent translation initiation: the translation of a mRNA with the 67 nucleotide omega sequence of tobacco mosaic virus in its 5' untranslated region (which was previously shown to render translation cap-independent) is not inhibited by p20. Whereas the translation of the same mRNA lacking the omega sequence is strongly inhibited by p20. Disruption of CAF20, the gene encoding p20, stimulates the growth of yeast cells, overexpression of p20 causes slower growth of yeast cells. These results show that p20 is a regulator of eIF4E activity which represses cap-dependent initiation of translation by interfering with the interaction of eIF4E with eIF4G, e.g. the formation of the eIF4F-complex.     

Striking multiplicity of eIF4E-BP1 phosphorylated isoforms identified by 2D gel electrophoresis regulation by heat shock.

Eukaryotic initiation factor eIF4E-binding protein 1 (eIF4E-BP1), or PHAS-I, is multiply phosphorylated by insulin-stimulated protein kinase(s). Estimates for the number of phosphorylation sites range from two to greater than eight. IEF/SDS/PAGE can precisely differentiate protein isoforms based on their differences in charge (phosphorylation) and molecular mass. In this study, the diversity of eIF4E-BP1 isoforms was determined using IEF/SDS/PAGE/immunoblotting of unfractionated cell lysates. To investigate the molecular regulation of phosphorylation, alterations in eIF4E-BP1 in response to heat shock in HeLa cells were determined. In exponentially growing cells, 8-10 prominent eIF4E-BP1 isoforms were detected. Following heat shock, a rapid, temperature-dependent dephosphorylation of eIF4E-BP1 occurs roughly concurrent with protein synthesis inhibition; during recovery from heat shock rephosphorylation of eIF4E-BP1 parallels restoration of protein synthesis. However, eIF4E-BP1 and eIF4E kinases remain highly active during heat shock, as okadaic acid treatment restores phosphorylation of both factors in heat shocked cells. eIF4E-BP1 dephosphorylation is associated with eIF4E dissociation from large molecular mass complexes and increased binding to eIF4E-BP1. The amount of eIF4E-BP1 converted to the dephosphorylated state is sufficient to titrate all the eIF4E present. eIF4E-BP1 phosphorylation changes regulated by heat shock also occur in Drosophila. Of the 10 isoforms of eIF4E-BP1 resolved by IEF/SDS/PAGE, at least seven are labelled with [32P] and all 10 are recognized by (eIF4E-BP1)-specific antibodies. These results identify a complex set of eIF4E-BP1 phosphorylation isoforms; changes in the expression of these isoforms in response to stresses such as heat shock may contribute to translation repression.     

Dissociation of eIF4E-Binding Protein 2 (4E-BP2) from eIF4E Independent of Thr37/Thr46 Phosphorylation in the Ischemic Stress Response

Eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) are translational repressors that bind specifically to eIF4E and are critical in the control of protein translation. 4E-BP2 is the predominant 4E-BP expressed in the brain, but their role is not well known. Here, we characterized four forms of 4E-BP2 detected by two-dimensional gel electrophoresis (2-DGE) in brain. The form with highest electrophoretic mobility was the main form susceptible to phosphorylation at Thr³⁷/Thr⁴⁶ sites, phosphorylation that was detected in acidic spots. Cerebral ischemia and subsequent reperfusion induced dephosphorylation and phosphorylation of 4E-BP2 at Thr³⁷/Thr⁴⁶, respectively. The induced phosphorylation was in parallel with the release of 4E-BP2 from eIF4E, although two of the phosphorylated 4E-BP2 forms were bound to eIF4E. Upon long-term reperfusion, there was a decrease in the binding of 4E-BP2 to eIF4E in cerebral cortex, demonstrated by cap binding assays and 4E-BP2-immunoprecipitation experiments. The release of 4E-BP2 from eIF4E was without changes in 4E-BP2 phosphorylation or other post-translational modification recognized by 2-DGE. These findings demonstrated specific changes in 4E-BP2/eIF4E association dependent and independent of 4E-BP2 phosphorylation. The last result supports the notion that phosphorylation may not be the uniquely regulation for the binding of 4E-BP2 to eIF4E under ischemic stress.     

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 (H2O2) 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 H2O2-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 H2O2-induced eIF4F complex formation inhibition. Besides the inhibitor induces a similar decrease in 4E-BP1 phosphorylated levels to that promote by H2O2. 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 H2O2-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.     

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.     

The translation initiation factor eIF2beta is an interactor of protein phosphatase-1

It is reasonably well understood how the initiation of translation is controlled by reversible phosphorylation of the eukaryotic translation initiation factors eIF2alpha, eIF2Bepsilon and eIF4E. Other initiation factors, including eIF2beta, are also established phosphoproteins but the physiological impact of their phosphorylation is not known. Using a sequence homology search we found that the central region of eIF2beta contains a putative PP1-(protein phosphatase-1) binding RVxF-motif. The predicted eIF2beta-PP1 interaction was confirmed by PP1 binding and co-immunoprecipitation assays on cell lysates as well as with the purified components. Site-directed mutagenesis showed that eIF2beta contains, in addition to an RVxF-motif, at least one other PP1-binding site in its C-terminal half. eIF2beta functioned as an inhibitor for the dephosphorylation of glycogen phosphorylase and Ser51 of eIF2alpha by PP1, but did not affect the dephosphorylation of Ser464 of eIF2Bepsilon by this phosphatase. Strikingly, eIF2beta emerged as an activator of its own dephosphorylation (Ser2, Ser67, Ser218) by associated PP1, since the substrate quality of eIF2beta was decreased by the mere mutation of its RVxF-motif. These results make eIF2beta an attractive candidate substrate for associated PP1 in vivo. The overexpression of wild-type eIF2beta or eIF2beta with a mutated RVxF-motif did not differentially affect the rate of translation, indicating that the binding of PP1 is not rate-limiting for translation under basal conditions.     

Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA 5' cap binding protein, which plays an important role in the control of translation. The activity of eIF4E is regulated by a family of repressor proteins, the 4E-binding proteins (4E-BPs), whose binding to eIF4E is determined by their phosphorylation state. When hyperphosphorylated, 4E-BPs do not bind to eIF4E. Phosphorylation of the 4E-BPs is effected by the phosphatidylinositol (PI) 3-kinase signal transduction pathway and is inhibited by rapamycin through its binding to FRAP/mTOR (FK506 binding protein-rapamycin-associated protein or mammalian target of rapamycin). Phosphorylation of 4E-BPs can also be induced by protein synthesis inhibitors. These observations led to the proposal that FRAP/mTOR functions as a "sensor" of the translational apparatus (E. J. Brown and S. L. Schreiber, Cell 86:517-520, 1996). To test this model, we have employed the tetracycline-inducible system to increase eIF4E expression. Removal of tetracycline induced eIF4E expression up to fivefold over endogenous levels. Strikingly, upon induction of eIF4E, 4E-BP1 became dephosphorylated and the extent of dephosphorylation was proportional to the expression level of eIF4E. Dephosphorylation of p70(S6k) also occurred upon eIF4E induction. In contrast, the phosphorylation of Akt, an upstream effector of both p70(S6k) and 4E-BP phosphorylation, was not affected by eIF4E induction. We conclude that eIF4E engenders a negative feedback loop that targets a component of the PI 3-kinase signalling pathway which lies downstream of PI 3-kinase.     

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.     

IGF-I/IGFBP-3 ameliorates alterations in protein synthesis, eIF4E availability, and myostatin in alcohol-fed rats

Chronic alcohol consumption decreases the concentration of the anabolic hormone IGF-I, and this change is associated with impaired muscle protein synthesis. The present study evaluated the ability of IGF-I complexed with IGF-binding protein (IGFBP)-3 to modulate the alcohol-induced inhibition of muscle protein synthesis in gastrocnemius. After 16 wk on an alcohol-containing diet, either the IGF-I/IGFBP-3 binary complex (BC) or saline was injected two times daily for three consecutive days. After the final injection of BC (3 h), plasma IGF-I concentrations were elevated in alcohol-fed rats to values not different from those of similarly treated control animals. Alcohol feeding decreased the basal rate of muscle protein synthesis by limiting translational efficiency. BC treatment of alcohol-fed rats increased protein synthesis back to basal control values, but the rate remained lower than that of BC-injected control rats. The BC partially reversed the alcohol-induced decrease in the binding of eukaryotic initiation factor (eIF)4E with eIF4G. This change was associated with reversal of the alcohol-induced dephosphorylation of eIF4G but was independent of changes in the phosphorylation of either 4E-BP1 or eIF4E. However, BC reversed the alcohol-induced increase in IGFBP-1 and muscle myostatin, known negative regulators of IGF-I action and muscle mass. Hence, exogenous IGF-I, administered as part of a BC to increase its circulating half-life, can in part reverse the decreased protein synthesis observed in muscle from chronic alcohol-fed rats by stimulating selected components of translation initiation. The data support the role of IGF-I as a mediator of chronic alcohol myopathy in rats.     

Amino acids and insulin are both required to regulate assembly of the eIF4E. eIF4G complex in rat skeletal muscle

The respective roles of insulin and amino acids in regulation of skeletal muscle protein synthesis and degradation after feeding were examined in rats fasted for 17 h and refed over 1 h with either a 25 or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained (control groups: C(25) and C(0)) or blocked with diazoxide injections (diazoxide groups: DZ(25) and DZ(0)). Muscle protein metabolism was examined in vitro in epitrochlearis muscles. Only feeding the 25% amino acid/protein meal in the presence of increased plasma insulin concentration (C(25) group) stimulated protein synthesis and inhibited proteolysis in skeletal muscle compared with the postabsorptive state. The stimulation of protein synthesis was associated with increased phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1), reduced binding of eIF4E to 4E-BP1, and increased assembly of the active eIF4E. eIF4G complex. The p70 S6 kinase (p70(S6k)) was also hyperphosphorylated in response to the 25% amino acid/protein meal. Acute postprandial insulin deficiency induced by diazoxide injections totally abolished these effects. Feeding the 0% amino acid/protein meal with or without postprandial insulin deficiency did not stimulate muscle protein synthesis, reduce proteolysis, or regulate initiation factors and p70(S6k) compared with fasted rats. Taken together, our results suggest that both insulin and amino acids are required to stimulate protein synthesis, inhibit protein degradation, and regulate the interactions between eIF4E and 4E-BP1 or eIF4G in response to feeding.     

Acute attenuation of translation initiation and protein synthesis by glucocorticoids in skeletal muscle

Glucocorticoids are diabetogenic factors that not only antagonize the action of insulin in target tissues but also render these tissues catabolic. Therefore, in rats, we endeavored to characterize the effects in skeletal muscle of glucocorticoids on translation initiation, a regulated process that, in part, governs overall protein synthesis through the modulated activities of eukaryotic initiation factors (eIFs). Four hours after intraperitoneal administration of dexamethasone (100 microg/100 g body wt), protein synthesis in skeletal muscle was reduced to 59% of the value recorded in untreated control animals. Furthermore, translation initiation factor eIF4E preferred association with its endogenous inhibitor 4E-BP1 rather than eIF4G. Dexamethasone treatment resulted in dephosphorylation of both 4E-BP1 and the 40S ribosomal protein S6 kinase concomitant with enhanced phosphorylation of eIF4E. Moreover, the guanine nucleotide exchange activity of eIF2B was unaffected as was phosphorylation of the alpha-subunit of eIF2. Hence glucocorticoids negatively modulate the activation of a subset of the protein synthetic machinery, thereby contributing to the catabolic properties of this class of hormones in vivo.     

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.