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The Axin-dependent phosphorylation of beta-catenin catalysed by glycogen synthase kinase-3 (GSK3) is inhibited during embryogenesis. This protects beta-catenin against ubiquitin-dependent proteolysis, leading to its accumulation in the nucleus, where it controls the expression of genes important for development. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) is a mammalian homologue of a GSK3-binding protein (GBP), which appears to play a key role in the correct establishment of the dorsal-ventral axis in Xenopus laevis. Here, we demonstrate that FRATtide (a peptide corresponding to residues 188-226 of FRAT1) binds to GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3-catalysed phosphorylation of Axin and beta-catenin, suggesting a potential mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not suppress GSK3 activity towards other substrates, such as glycogen synthase and eIF2B, whose phosphorylation is independent of Axin but dependent on a 'priming' phosphorylation. This may explain how the essential cellular functions of GSK3 can continue, despite the suppression of beta-catenin phosphorylation.

In the initiation phase of eukaryotic translation, eIF5 stimulates the hydrolysis of GTP bound to eIF2 in the 40S ribosomal pre-initiation complex, and the resultant GDP on eIF2 is replaced with GTP by the complex nucleotide exchange factor, eIF2B. Bipartite motifs rich in aromatic and acidic residues are conserved at the C-termini of eIF5 and the catalytic (epsilon) subunit of eIF2B. Here we show that these bipartite motifs are important for the binding of these factors, both in vitro and in vivo, to the beta subunit of their common substrate eIF2. We also find that three lysine-rich boxes in the N-terminal segment of eIF2beta mediate the binding of eIF2 to both eIF5 and eIF2B. Thus, eIF5 and eIF2Bepsilon employ the same sequence motif to facilitate interaction with the same segment of their common substrate. In agreement with this, archaea appear to lack eIF5, eIF2B and the lysine-rich binding domain for these factors in their eIF2beta homolog. The eIF5 bipartite motif is also important for its interaction with the eIF3 complex through the NIP1-encoded subunit of eIF3. Thus, the bipartite motif in eIF5 appears to be multifunctional, stimulating its recruitment to the 40S pre-initiation complex through interaction with eIF3 in addition to binding of its substrate eIF2.

The present study was designed to investigate the mechanism through which leucine and histidine regulate translation initiation in L6 myoblasts. The results show that both amino acids stimulate initiation and coordinately regulate the activity of eukaryotic initiation factor eIF2B. The changes in eIF2B activity could be explained in part by modulation of the phosphorylation state of the alpha-subunit of eIF2. The activity changes might also be a result of modulation of the phosphorylation state of the eIF2B epsilon-subunit, because deprivation of either amino acid caused a decrease in eIF2Bepsilon kinase activity. Leucine, but not histidine, additionally caused a redistribution of eIF4E from the inactive eIF4E.4E-BP1 complex to the active eIF4E.eIF4G complex. The redistribution was a result of increased phosphorylation of 4E-BP1. The changes in 4E-BP1 phosphorylation and eIF4E redistribution associated with leucine deprivation were not observed in the presence of insulin. However, the leucine- and histidine-induced alterations in global protein synthesis and eIF2B activity were maintained in the presence of the hormone. Overall, the results suggest that both leucine and histidine regulate global protein synthesis through modulation of eIF2B activity. Furthermore, under the conditions employed herein, alterations in eIF4E availability are not rate-controlling for global protein synthesis but might be necessary for regulation of translation of specific mRNAs.

Protein synthesis in rat H35 Reuber hepatoma cells is rapidly inhibited on heat shock. At mild heat-shock temperatures the main cause for inhibition is the inactivation of the guanine nucleotide exchange factor eukaryotic initiation factor 2B (eIF2B); under more severe heat-shock conditions the activity of several initiation factors is compromised. eIF2B is required for GDP/GTP exchange on eIF2, which delivers methionyl-tRNA to the 40 S ribosomal subunit. We have tried to elucidate the mechanism underlying the inactivation of eIF2B by assays in vitro. Incubation of cell extracts at 41 °C or higher led to the inactivation of eIF2B. In agreement with observations in cells exposed to mild heat shocks, the thermal inactivation of eIF2B could be ascribed to neither eIF2α phosphorylation nor the induction of another inhibitor. With the use of glycerol gradients we show that eIF2B forms aggregates in heat-treated extracts. Furthermore eIF2B activity is protected against heat shock in thermotolerant cells. Taken together, these results suggest a role for chaperones in the control of eIF2B activity.

Eukaryotic initiation factor eIF2B plays a key role in the regulation of protein synthesis through its ability to catalyze the exchange of GDP bound to a second initiation factor, eIF2, for free GTP. In contrast to other GDP-GTP exchange factors (GEFs), which are often single subunit proteins, eIF2B consists of five dissimilar subunits. In the studies reported here the baculovirus expression vector system (BEVS) was used to express FLAG epitope tagged alleles for the alpha, beta, gamma, delta, and epsilon subunits of rat eIF2B in Sf21 cells. The eIF2B holoprotein was reconstituted in vivo by coexpression of all five subunits in Sf21 cells and was subsequently purified to greater than 98% homogeneity using a two-step procedure involving an anti-FLAG immunoaffinity column followed by gel filtration chromatography. The purified five-subunit eIF2B complex had high GEF activity as assayed by using [3H]GDP-bound to eIF2 as a substrate. Alternatively, eIF2B with high GEF activity was reconstituted in vitro by mixing crude cell lysates containing different eIF2B subunits. The latter results suggest that eIF2B activity in vivo could involve alterations in the concentration and/or the availability of individual subunits for holoprotein assembly. Overall, the results show the utility of the baculovirus-insect cell system for the expression, assembly, and purification of active recombinant multisubunit factors.

Eukaryotic initiation factor 2B (eIF2B) is a guanine nucleotide-exchange factor which mediates the exchange of GDP (bound to initiation factor eIF2) for GTP, thus regenerating the active [eIF2.GTP] complex that is required for peptide-chain initiation. The activity of eIF2B is a key control point for eukaryotic protein synthesis and is altered in response to viral infection, hormones, nutrients, growth factors and certain stresses. It may be regulated directly by its own phosphorylation, phosphorylation of its substrate, eIF2 and by allosteric effectors. Understanding its regulation will lead to a better knowledge of insulin action, regulation of the immune system and of insulin production in the pancreas.

Recently, we synthesized and characterized vanadyl saccharides to evaluate the effects of various vanadate and vanadyl complexes, which differ in their oxidation states on various biomacromolecules and cellular activities (1, 2). Here, we report that both vanadate (+V oxidation state) and different vanadyl species (+IV oxidation state) such as vanadyl D-glucose, vanadyl diascorbate, and vanadyl sulfate, impair the formation of polysomes and inhibit the initiation of protein synthesis in hemin-supplemented rabbit reticulocyte lysates. Vanadate inhibits protein synthesis more severely than vanadyl species and is consistent with the idea that vanadate is reduced to vanadyl state intracellularly. The inhibition of protein synthesis caused by low concentrations (10-20 mu M) of vanadate and vanadyl species is effectively mitigated by reducing agents such as dithiothreitol, reduced glutathione (GSH), or reduced pyridine dinucleotide, A significant decrease in the protein synthesis inhibition in vanadate-treated lysates by GSH suggests that the mechanism of protein synthesis inhibition by vanadate is different than the action of other oxidants such as heavy metal ions and oxidized glutathione, This suggestion is also consistent with the findings that vanadium compounds do not stimulate phosphorylation of the alpha (alpha) subunit of initiation factor 2 (eIF2) or decrease the guanine nucleotide exchange activity of eIF2B, which is required to exchange GDP for GTP in eIF2.GDP binary complex. The reduction of vanadate to vanadyl state and the subsequent complex formation of vanadyl species with the endogenous reducing compounds or with the -SH groups of certain proteins may be the cause for protein synthesis inhibition in lysates. (C) 1998

The process of protein synthesis (mRNA translation) is essential for the growth and survival of all living organisms. Protein synthesis places heavy demands upon the cell—it requires a supply of amino acids as precursors and a great deal of energy. For these reasons, overall protein synthesis is under tight control. Furthermore, the translation of specific mRNAs is also subject to sophisticated control mechanisms, allowing the cell to modulate the production of certain proteins. Regulating translation rather than earlier stages in gene expression (transcription, splicing) confers important advantages—especially rapidity (by avoiding the requirement for de novo transcription and mRNA processing) and spatial control, e.g., in early development. Great strides have now been made in understanding how protein synthesis works and the functions and structures of the components involved. The chapters in this volume broadly concern two different areas of translational control. The first four primarily concern the regulation of the protein factors involved in the process of translation. This regulation generally involves alterations in their states of phosphorylation which influence their activity and interactions. The chapter by Proud describes the control of eukaryotic initiation factor 2. Its control has been intensively studied as it was a very early example of regulation of translation by protein phosphorylation. It is a target for four mammalian protein kinases which play important roles in regulating translation in response to diverse stress conditions. Recent data, using knock-in or knock-out mouse models, have clearly shown the importance of the kinases for normal physiology. It is also now clear that inherited mutations either in one of these kinases or in an ancillary factor for eIF2 (eIF2B) can lead to severe human diseases, as also discussed by Proud. Clemens discusses the mechanisms by which viruses seek to subvert the cellular protein synthesis machinery to favour expression of their own genetic information and the ways in which host cells strive to prevent this. A key player here is an eIF2 kinase termed PKR. Many of the mechanisms are also relevant in uninfected cells in response to stresses or early in apoptosis. As precursors for protein synthesis, amino acids exert important regulatory effects on the translational machinery. Some are exerted through eIF2, others through a key signaling pathway involving a protein termed the mammalian target of rapamycin, mTOR. Kimball focuses on recent studies that employ intact animals, rather than isolated cells, to explore the roles of amino acids in regulating translation factor activity. The mTOR pathway is currently the focus of intense attention. This reflects both its key roles in cellular and organismal physiology, and the links between this pathway and a range of human diseases including certain types of cancer and a condition termed tuberous sclerosis. Tee and Blenis discuss how studies on tuberous sclerosis have led to an improved understanding of mTOR signaling, and the roles of mTOR in health and disease. The second group of articles concern features of mRNA molecules themselves that are important for the control of their translation. In many cases, these features lie in the 5?- or 3?-untranslated regions (UTRs) of the message. Pickering and Willis review the roles of elements in the 5?-UTRs of certain mRNAs in mediating or controlling their translation. These include internal ribosome entry sites, which were initially found in viral mRNAs and which allow these mRNAs to be efficiently translated in the host cell. The articles by de Moor and colleagues and by Espel concern features found in the 3?-UTRs of specific mRNAs. The first chapter discusses 3?-UTR elements that are important in controlling the translation of specific mRNAs during early development and cell differentiation and during the cell cycle. The resulting translational control processes play critical roles in a diverse array of biological processes. A range of such regulatory elements have been identified, and these work by interacting with specific RNA-binding proteins. This is also true of the distinct 3?-UTR elements described by Espel: these AU-rich elements (AREs) play important roles in regulating the stability and translation of mRNAs encoding pro-inflammatory proteins and tumour-related proteins. As discussed by Espel, particular attention has focused on the role of AREs and ARE-binding proteins in inflammation, and on the signaling pathways involved. As the reader will appreciate from this set of articles, it is now abundantly clear that translational control is crucial for normal development and growth, and that dysregulation of these mechanisms leads to a range of serious human diseases.

Eukaryotic translation initiation factor 2B (eIF2B) is a five-subunit complex that catalyzes guanine nucleotide exchange on eIF2. Phosphorylation of the alpha subunit of eIF2 [creating eIF2(alphaP]) converts eIF2 x GDP from a substrate to an inhibitor of eIF2B. We showed previously that the inhibitory effect of eIF2(alphaP) can be decreased by deletion of the eIF2B alpha subunit (encoded by GCN3) and by point mutations in the beta and delta subunits of eIF2B (encoded by GCD7 and GCD2, respectively). These findings, plus sequence similarities among GCD2, GCD7, and GCN3, led us to propose that these proteins comprise a regulatory domain that interacts with eIF2(alphaP) and mediates the inhibition of eIF2B activity. Supporting this hypothesis, we report here that overexpression of GCD2, GCD7, and GCN3 specifically reduced the inhibitory effect of eIF2(alphaP) on translation initiation in vivo. The excess GCD2, GCD7, and GCN3 were coimmunoprecipitated from cell extracts, providing physical evidence that these three proteins can form a stable subcomplex. Formation of this subcomplex did not compensate for a loss of eIF2B function by mutation and in fact lowered eIF2B activity in strains lacking eIF2(alphaP). These findings indicate that the trimeric subcomplex does not possess guanine nucleotide exchange activity; we propose, instead, that it interacts with eIF2(alphaP) and prevents the latter from inhibiting native eIF2B. Overexpressing only GCD2 and GCD7 also reduced eIF2(alphaP) toxicity, presumably by titrating GCN3 from eIF2B and producing the four-subunit form of eIF2B that is less sensitive to eIF2(alphaP). This interpretation is supported by the fact that overexpressing GCD2 and GCD7 did not reduce eIF2(alphaP) toxicity in a strain lacking GCN3; however, it did suppress the impairment of eIF2B caused by the gcn3c-R104K mutation. An N-terminally truncated GCD2 protein interacted with other eIF2B subunits only when GCD7 and GCN3 were overexpressed, in accordance with the idea that the portion of GCD2 homologous to GCD7 and GCN3 is sufficient for complex formation by these three proteins. Together, our results provide strong evidence that GCN3, GCD7, and the C-terminal half of GCD2 comprise the regulatory domain in eIF2B.

The guanine nucleotide-exchange factor eIF2B mediates the exchange of GDP bound to translation initiation factor eIF2 for GTP. This exchange process is a key regulatory step for the control of translation initiation in eukaryotic organisms. To improve our understanding of the structure, function and regulation of eIF2B, we have obtained and sequenced cDNA species encoding all of its five subunits. Here we report the sequences of eIF2B beta and delta from rat. This paper focuses on sequence similarities between the alpha, beta and delta subunits of mammalian eIF2B. Earlier work showed that the amino acid sequences of the corresponding subunits of eIF2B in the yeast Saccharomyces cerevisiae (GCN3, GCD7 and GCD2) exhibit considerable similarity. We demonstrate that this is also true for the mammalian subunits. Moreover, alignment of the eIF2B alpha, beta and delta sequences from mammals and yeast, along with the sequence of the putative eIF2B alpha subunit from Caenorhabditis elegans and eIF2B delta from Schizosaccharomyces pombe shows that a large number of residues are identical or conserved between the C-terminal regions of all these sequences. This strong sequence conservation points to the likely functional importance of these residues. The implications of this are discussed in the light of results concerning the functions of the subunits of eIF2B in yeast and mammals. Our results also indicate that the large apparent differences in mobility on SDS/PAGE between eIF2B beta and delta subunits from rat and rabbit are not due to differences in their lengths but reflect differences in amino acid composition. We have also examined the relative expression of mRNA species encoding the alpha, beta, delta and epsilon subunits of eIF2B in a range of rat tissues by Northern blot analysis. As might be expected for mRNA species encoding subunits of a heterotrimeric protein, the ratios of expression levels of these subunits to one another did not vary between the different rat tissues examined (with the possible exception of liver). This represents the first analysis of the levels of expression of mRNA species encoding the different subunits of eIF2B.

Mitogenic stimulation of T-lymphocytes causes a rapid activation or protein synthesis, which reflects in part increased expression of many translation components. Their levels, however, rise more slowly than the rate of protein synthesis, indicating an enhancement of the efficiency of their utilization. Initiation factor eIF2B catalyzes a key regulatory step in the initiation of translation, and we have therefore studied its activity following T-cell activation. eIF2B activity rises quickly, increasing as early as 5 min after cell stimulation. This initial phase is followed by an additional slow but substantial increase in eIF2B activity. The level of eIF2B subunits did not change over the initial rapid phase but did increase at later time points. Northern analysis revealed that levels of eIF2B mRNA only rose during the later phase. The rapid activation of EIF2B following mitogenic stimulation of T-cells is therefore mediated by factors other than its own concentration. The largest (epsilon) subunit of eIF2B is a substrate for glycogen synthase kinase-3 (GSK-3), the activity of which rapidly decreases following T-cell activation. Since phosphorylation of eIF2B by GSK-3 appears to inhibit nucleotide exchange in vitro, this provides a potential mechanism by which eIF2B may be activated.

s ingediend voor het Amsterdam Kindersymposium 2013 35 The influence of Vanishing White Matter mutations on oligodendrocyte differentiation S. Dooves (1,2), J.A. van der Kreeke (1,2), P. Leferink (1,2), J.G. Jacobs (1,2), M.S. van der Knaap (1), V.M. Heine (1,2) (1) Center for Children with White Matter Disorders, Pediatrics, VU University Medical Center, Amsterdam (2) Stem Cell Laboratory, VU University/VU University Medical Center, Amsterdam INTRODUCTION Vanishing White Matter disease (VWM) is an autosomal recessive leukoencephalopathy caused by mutations in genes encoding the eukaryotic translation initiation factor 2B (eIF2B). Children are mainly aff ected, who suff er from progressive neurological symptoms as ataxia and die at young age. Previous research indicates that the macroglial cells of the brain (astrocytes and oligodendrocytes) in VWM show a defect in maturation. Currently there is no treatment available. METHODS In order to gain more knowledge about the disease pathology we tested the diff erentiation capability of stem cells derived from VWM mice in culture. Furthermore, a co-culture system is used to test the infl uence of VWM mutated astrocytes on the diff erentiation of WT oligodendrocyte precursor cells (OPCs). RESULTS The fi rst results show that VWM mutated astrocytes have a negative eff ect on the maturation of oligodendrocytes. CONCLUSION Astrocytes with a VWM mutation have a negative infl uence on OPC maturation. Further studies will show if it is possible to rescue this maturation defect. The results will give us more information about the disease mechanisms of VWM and will help in the development of a cell replacement therapy for VWM.

s ingediend voor het Amsterdam Kindersymposium 2013 117 Towards understanding the underlying mechanism of vanishing white matter disease Lisanne E. Wisse, Emiel Polder, M. Clair van de Beek, Marjo S. van der Knaap, Truus E.M. Abbink Department of Pediatrics, Center for Childhood White Matter Disorders, VUmc INTRODUCTION Vanishing white matter disease (VWM) is one of the most prevalent genetic childhood white matter disorders. The disease is characterized by ataxia and spasticity. Diagnosis is performed by MRI, which shows progressive white matter degeneration, aggravated by stress, minor head trauma or fever. The lack of myelin and scar tissue formation in the aff ected area may result from a maturation defect in astrocytes and oligodendrocytes. VWM is caused by recessive mutations in the eukaryotic initiation factor 2B (eIF2B). This enzyme regulates the protein synthesis rate in every cell type and is regulated by various stressors, including heat shock. METHODS Our project aims to unravel the molecular mechanisms that underlie VWM. Although we have acquired increasing insights into the VWM brain pathology, it remains elusive how a defect in eIF2B causes a disease that almost exclusively aff ects the white matter. Aided by two clinically representative disease models, we currently address two unresolved questions. Firstly, we investigate whether relative white matter eIF2B levels deviate from those in other tissues using immunoblotting. Secondly, we address whether aberrant translation regulation of specifi c mRNAs in white matter cells underlies VWM using polysomal profi ling and pulsed isotope labeling. In follow-up assays we will assess the functional importance of the observed diff erences between control and VWM tissues. RESULTS Preliminary results suggest that we can purify polysomes from brain cells and tissue from normal and VWM mice. CONCLUSION Two representative disease models in combination with two robust genome-wide assays allow us to gain further insight into the disease mechanism of VWM.

This review focuses on how cells establish the levels of initiation factors, within the broader context of determining levels of the translational machinery. Most initiation factor polypeptides are moderately abundant proteins with concentrations approaching those of ribosomes. eIF4A and eIF5A are more abundant than ribosomes, whereas eIF4F alpha and eIF2B are considerably less abundant than the other factors. The cloning of cDNAs generates hybridization probes for monitoring the levels and activities of factor mRNAs, and the cloning of their genes is just beginning to provide insight into promoter structures and regulation. Initiation factor gene expression appears to be coordinately regulated in many cases, and preferential synthesis is seen in mitogen-activated T-cells. The gene for eIF2 alpha has been best characterized, and mechanisms that provide for the coordinated synthesis of eIF2 subunits are emerging. Recombinant DNA methods also allow investigators to manipulate the levels of expression of specific factor genes by overexpression or antisense repression. Such approaches provide a means to investigate in vivo the mechanisms of action of the initiation factors and their roles in regulating translation rates.

We show that phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2) by the protein kinase GCN2 mediates translational control of the yeast transcriptional activator GCN4. In vitro, GCN2 specifically phosphorylates the alpha subunit of rabbit or yeast eIF-2. In vivo, phosphorylation of eIF-2 alpha increases in response to amino acid starvation, which is dependent on GCN2. Substitution of Ser-51 with alanine eliminates phosphorylation of eIF-2 alpha by GCN2 in vivo and in vitro and abolishes increased expression of GCN4 and amino acid biosynthetic genes under its control in amino acid-starved cells. The Asp-51 substitution mimics the phosphorylated state and derepresses GCN4 in the absence of GCN2. Thus, an established mechanism for regulating total protein synthesis in mammalian cells mediates gene-specific translational control in yeast.

Insulin rapidly activates protein synthesis by activating components of the translational machinery including eIFs (eukaryotic initiation factors) and eEFs (eukaryotic elongation factors). In the long term, insulin also increases the cellular content of ribosomes to augment the capacity for protein synthesis. The rapid activation of protein synthesis by insulin is mediated primarily through phosphoinositide 3-kinase. This involves the activation of PKB (protein kinase B). In one case, PKB acts to phosphorylate and inactivate glycogen synthase kinase 3, which in turn phosphorylates and inhibits eIF2B. Insulin elicits the dephosphorylation and activation of eIF2B. Since eIF2B is required for recycling of eIF2, a factor required for all cytoplasmic translation initiation events, this will contribute to overall activation of protein synthesis. PKB also phosphorylates the TSC1 (tuberous sclerosis complex 1)-TSC2 complex to relieve its inhibitory action on the mTOR (mammalian target of rapamycin). Inhibition of mTOR by rapamycin markedly impairs insulin-activated protein synthesis. mTOR controls translation initiation and elongation. The cap-binding factor eIF4E can be sequestered in inactive complexes by 4E-BP1 (eIF4E-binding protein 1). Insulin elicits phosphorylation of 4E-BP1 and its release from eIF4E, allowing eIF4E to form initiation factor complexes. Insulin induces dephosphorylation and activation of eEF2 to accelerate elongation. Both effects are blocked by rapamycin. Insulin inactivates eEF2 kinase by increasing its phosphorylation at several mTOR-regulated sites. Insulin also stimulates synthesis of ribosomal proteins by promoting recruitment of their mRNAs into polyribosomes. This is inhibited by rapamycin. Several key questions remain about, for example, the mechanisms by which mTOR controls 4E-BP1 and eEF2 kinase and the control of ribosomal protein translation.

eIF2B (eukaryotic initiation factor 2B) is a multisubunit protein that is required for protein synthesis initiation and its regulation in all eukaryotic cells. Mutations in eIF2B have also recently been found to cause a fatal human disease called CACH (childhood ataxia with central nervous system hypomyelination) or VWM (vanishing white matter disease). This review provides a general background to translation initiation and mechanisms known to control eIF2B function, before describing molecular genetic and biochemical analysis of eIF2B structure and function, integrating work from studies of the yeast and mammalian eIF2B proteins.

Activation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus. Activation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF) 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF) 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F. Both eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.

Guanine nucleotide exchange factor (GEF) is a multisubunit protein involved in the initiation of translation. Although numerous models have been proposed for its mechanism of action, none have been definitive. An assay dependent on GEF activity was developed using highly purified eukaryotic initiation factor 2 (eIF-2) and GEF from Ehrlich cells. GEF was considered in terms of an enzyme whose catalytic function was the exchange of eIF-2-bound [alpha-32P]GDP for unlabeled nucleotide. The turnover number of GEF at 37 degrees C, calculated on the basis of enzyme kinetic methods is 0.027 s, which is consistent with in vivo rates of protein synthesis. Moreover, kinetic data support an enzyme-substituted mechanism as the mode of GEF function. This mechanism proposes the existence of a GEF.eIF-2.GDP complex and excludes the possibility of two guanine nucleotide binding sites on eIF-2. An analogous mechanism has been recently reported for elongation factor Ts, suggesting the importance of this mechanism to protein synthesis. The mechanism of inhibition of GEF function by eIF-2 alpha phosphorylation has also been investigated. It has been generally assumed that the mechanism by which eIF-2(P) traps GEF is an excessively stable complex, from which GEF is released very slowly. Data presented here, however, reveal that eIF-2(P).GDP is a competitive inhibitor of GEF (rather than an irreversible inhibitor) competing with eIF-2.GDP for binding to GEF. Even though the eIF-2(P).GDP.GEF complex dissociates too rapidly to measure, GEF is trapped because it has at least 150-fold greater affinity for eIF-2(P).GDP than for eIF-2.GDP. The implications of competitive inhibition with respect to the mechanism of reversal of inhibition by an eIF-2(P) phosphatase are discussed.

Ovarian failure (OF) at age40 years occurs in approximately 1% of all women. Other than karyotype abnormalities, very few genes are known to be associated with this ovarian dysfunction. We studied eight patients who presented with premature OF and white-matter abnormalities on magnetic resonance imaging. Neurological signs may be absent or present after OF. In seven patients, we report for the first time mutations in three of the five EIF2B genes (EIF2B2, -4, and -5) that were recently shown to cause childhood ataxia with central nervous system hypomyelination/vanishing white-matter disease leukodystrophy. The correlation we observed between the age at onset of the neurological deterioration and the severity of OF suggests a common pathophysiological pathway.

Eukaryotic initiation factor eIF2B mediates a key regulatory step in peptide-chain initiation and is acutely activated by insulin, although it is not clear how. Inhibitors of phosphatidylinositide 3-kinase blocked activation of eIF2B, although rapamycin, which inhibits the p70 S6 kinase pathway, did not. Furthermore, a dominant negative mutant of PI 3-kinase also prevented activation of eIF2B, while a Sos-mutant, which blocks MAP kinase activation, did not. The data demonstrate that a pathway distinct from MAP and p70 S6 kinases regulates eIF2B. Glycogen synthase kinase-3 (GSK-3) phosphorylates and inactivates eIF2B. In all cases, eIF2B and GSK-3 were regulated reciprocally. Dominant negative PI 3-kinase abolished the insulin-induced inhibition of GSK-3. These data strongly support the hypothesis that insulin activates eIF2B through a signalling pathway involving PI 3-kinase and inhibition of GSK-3.

Examination of the M. jannaschii genome sequence revealed the presence of eleven genes encoding proteins implicated in translation initiation, three implicated in elongation, and one implicated in termination-release (Bult et al. 1996xBult, C.J., White, O., Olsen, G.J., Zhou, L., Fleischmann, R.D., Sutton, G.G., Blake, J.A., FitzGerald, L.M., Clayton, R.A., Gocayne, J.D. et al. Science. 1996; 273: 1058–1072Crossref | PubMedSee all ReferencesBult et al. 1996). Surprisingly, of the 11 putative initiation factor proteins, 10 appear homologous to eucaryal initiation factors (see Table 1Table 1). The first is an eIF1A-like protein. In Eucarya, eIF1A binds to free 40S ribosomal subunits and prevents premature reassociation with 60S subunits. Three other proteins are the α, β, and γ subunits of an eIF2-like complex. In Eucarya, eIF2 forms a ternary complex with met-tRNAinit and GTP. The complex binds to the 40S subunit to form a 43S preinitiation complex. Normally, the 43S complex then binds mRNA via its 7 methyl G cap; binding is mediated by the cap recognition factor eIF4E and the organizing factor eIF4G. Archaeal mRNAs do not contain the 5′ end 7 methyl G modification and neither eIF4E nor 4G homologs appear to be present in M. jannaschii.Table 1Translation Initiation Factors Encoded by the Genome of M. jannaschiiGene NumberHomologProposed Function0445eIF1Aantiassociation, binds to 40S subunit0117eIF2α subunitternary complex with met-tRNAinit and GTP0097eIF2β subunit1261eIF2γ subunit1574eIF4AATP-dependent RNA helicase activity1505eIF4AATP-dependent RNA helicase activity0669eIF4AATP-dependent RNA helicase activity1228eIF5stimulates GTP hydrolysis on eIF2 when initiation codon is properly aligned0454eIF2Bα subunitguanine nucleotide exchange factor for eIF20122eIF2Bδ subunit0262IF2 (bacterial)ternary complex with fmet-tRNAinit and GTP (bacterial type containing a guanine nucleotide exchange domain)There are three separate genes encoding members of the eIF4A family. These proteins have ATP-dependent RNA helicase activity; they function to remove secondary structure in eucaryl mRNA and permit scanning for the initiation codon by the 43S preinitiation complex. At the present time, it is uncertain if any or all of the M. jannaschii helicase proteins are required for protein synthesis initiation, if they are used exclusively for this purpose, or if they are employed to unwind or rearrange RNA in other cellular processes. Another initiation factor is homologous to eIF5. In Eucarya, this factor associates with the eIF2 ternary complex on the surface of the 40S subunit; when the initiation codon is properly aligned, it mediates hydrolysis of the GTP bound to eIF2 and facilitates the ejection of the eIF2·GDP and the initiation factors from the 40S subunit. This opens the way for entry of the 60S subunit to form the 80S initiation complex.A common feature of eucaryl eIF2 complexes is that they lack a guanine nucleotide exchange domain and therefore require an extrinsic guanine nucleotide exchange factor. Not surprisingly, two M. jannaschii genes encode homologs to the α and δ subunits of the guanine nucleotide exchange factor eIF2B (see Table 1Table 1). The δ subunit participates directly in the nucleotide exchange reaction whereas the α subunit is involved in regulation of initiation. The M. jannaschii eIF2α subunit contains a conserved serine at position 51, which in Eucarya can be phosphorylated by one of a number of different eIF2-specific protein kinases. The eIF2Bα subunit acts as a sensor for phosphoserine at position 51 of EIF2α and, when present, prevents the guanine nucleotide exchange reaction. This blocks the formation of eIF2·GTP·mettRNAinit ternary complex and prevents further translation initiation. Since both the serine at position 51 of eIF2α and the sensing eIF2Bα subunit are present in M. jannaschii, it is possible that this important exchange cycle is used to regulate protein synthesis initiation, perhaps in response to environmental stresses such as nutrient or energy limitation. No eIF2-specific protein kinases have yet been identified. The three other eucaryal subunits of eIF2B are apparently not present in M. jannaschii.The last initiation factor gene encodes a protein member of the bacterial IF2 family. In protein synthesis, bacterial IF2 carries out the same function as eIF2. However, it possesses a guanine nucleotide exchange domain and therefore does not require a guanine nucleotide exchange factor for regeneration. Is this IF2-like protein involved in initiation of protein synthesis in M. jannaschii or has it been recruited to carry out a separate and unrelated function? Saccharomyces cerevisiae also encodes a protein of this bacterial IF2 family. The gene is essential for yeast viability but its function is unknown. There is no evidence that it is required for protein synthesis. From the above list of factors, it would appear that Archaea utilize a hybrid system for translation initiation; the factors are mostly eucaryal-like whereas the basic mechanism for initiation codon selection in many and perhaps most instances is bacterial, utilizing an interaction of mRNA with the 3′ end of 16S rRNA.There are three genes encoding elongation factor proteins. The first encodes a eucaryl-like EF1α, the second a eucaryl-like EF2 and the third, a bacterial-like EFTu. EF1α forms a ternary complex with aa tRNA and GTP and is responsible for the codon-dependent deposition of aa tRNA into the A site of an elongating ribosome. This protein is expected to require a guanine nucleotide exchange factor as it lacks its own nucleotide recycling domain. No candidate for a recycling protein has been identified in the M. jannaschii genome. The second factor, EF2, carries out GTP-dependent ribosome translocation. This protein possesses a recycling domain and no exchange protein is required. The third bacterial-like factor is a highly specialized Tu factor that deposits selenocysteinyl tRNA into the A site of the ribosome in the presence of a UGA codon and a selenocysteine insertion (SECIS) element somewhere on the mRNA (Wilting et al. 1997xWilting, R., Schorling, S., Persson, B.C., and Bock, A. J. Mol. Biol. 1997; 266: 637–641Crossref | PubMed | Scopus (97)See all ReferencesWilting et al. 1997). In Methanococcus and eukaryotes, this element is never in the coding region but rather is usually located in the 3′ untranslated region of the mRNA. In bacteria, the SECIS element is located immediately 3′ to the UGA selenocysteine codon.A single eucaryal-like release factor capable of recognizing all three translation termination codons has been identified. In Eucarya, a second protein with GTPase activity is also required for efficient recognition of termination codons and hydrolysis of the nascent polypeptide from the p site peptidyl tRNA. The homolog of this auxillary protein has not been identified in M. jannaschii.In summary, the Archaea appear to utilize an intriguing mixture of bacterial and rudimentary eucaryl features in their translation apparatus. Recognizing how these features interface within Archaea will lead to a more thorough understanding of the disparate characteristics of bacterial and eucaryal translation and help to create a universal or general model for translation, applicable to all organisms. High on the list of topics demanding intellectual and detailed biochemical investigations are pre-rRNA processing, ribosomal subunit assembly, and translation initiation. The availability of archaeal genome sequences provides an excellent starting position for these studies but many challenges remain. Where there is uncertainty, there are bound to be surprises.

Eukaryal translation initiation factor 2B (eIF2B) acts as guanine nucleotide exchange factor (GEF) for eIF2 and forms a central target for pathways regulating global protein synthesis. eIF2B consists of five non-identical subunits (α-ϵ), which assemble into a catalytic subcomplex (γ, ϵ) responsible for the GEF activity, and a regulatory subcomplex (α, β, δ) which regulates the GEF activity under stress conditions. Here, we provide new structural and functional insight into the regulatory subcomplex of eIF2B (eIF2B(RSC)). We report the crystal structures of eIF2Bβ and eIF2Bδ from Chaetomium thermophilum as well as the crystal structure of their tetrameric eIF2B(βδ)2 complex. Combined with mutational and biochemical data, we show that eIF2B(RSC) exists as a hexamer in solution, consisting of two eIF2Bβδ heterodimers and one eIF2Bα2 homodimer, which is homologous to homohexameric ribose 1,5-bisphosphate isomerases. This homology is further substantiated by the finding that eIF2Bα specifically binds AMP and GMP as ligands. Based on our data, we propose a model for eIF2B(RSC) and its interactions with eIF2 that is consistent with previous biochemical and genetic data and provides a framework to better understand eIF2B function, the molecular basis for Gcn(-), Gcd(-) and VWM/CACH mutations and the evolutionary history of the eIF2B complex.

The eukaryotic translation initiation factor eIF2B promotes mRNA translation as a guanine nucleotide exchange factor (GEF) for translation initiation factor 2 (eIF2). Endoplasmic reticulum (ER) stress-mediated activation of the kinase PERK and the resultant phosphorylation of eIF2's alpha subunit (eIF2α) attenuates eIF2B GEF activity thereby inducing an integrated stress response (ISR) that defends against protein misfolding in the ER. Mutations in all five subunits of human eIF2B cause an inherited leukoencephalopathy with vanishing white matter (VWM), but the role of the ISR in its pathogenesis remains unclear. Using CRISPR-Cas9 genome editing we introduced the most severe known VWM mutation, EIF2B4A391D, into CHO cells. Compared to isogenic wildtype cells, GEF activity of cells with the VWM mutation was impaired and the mutant cells experienced modest enhancement of the ISR. However, despite their enhanced ISR, imposed by the intrinsic defect in eIF2B, disrupting the inhibitory effect of phosphorylated eIF2α on GEF by a contravening EIF2S1/eIF2αS51A mutation that functions upstream of eIF2B, selectively enfeebled both EIF2B4A391D and the related severe VWM EIF2B4R483W cells. The basis for paradoxical dependence of cells with the VWM mutations on an intact eIF2α genotype remains unclear, as both translation rates and survival from stressors that normally activate the ISR were not reproducibly affected by the VWM mutations. Nonetheless, our findings support an additional layer of complexity in the development of VWM, beyond a hyperactive ISR.

Background Leukoencephalopathy with Vanishing White Matter (VWM) is an autosomal recessive disorder caused by germline mutations in the genes EIF2B1-5, which encode the 5 subunits of the eukaryotic translation initiation factor eIF2B. To date, analysis of the biochemical effects of mutations in the EIF2B2-5 genes has been carried out, but no study has been performed on mutations in the EIF2B1 gene. This gene encodes eIF2Bα, the smallest subunit in eIF2B which has an important role in both the structure and regulation of the eIF2B complex. Methods eIF2B subunits were overexpressed in HEK293 cells and isolated from the resulting cell lysates by affinity chromatography. Formation of the eIF2B complex and binding of its substrate, eIF2, was assessed by western blot. Assays of the guanine nucleotide exchange (GEF) activity were also carried out. Results Of the 5 eIF2Bα mutations studied, we found 3 that showed loss or reduction of binding of eIF2Bα to the rest of the complex, one with increased GEF activity, and one where no effects on activity or complex formation were observed. Conclusions This is the first study on eIF2Bα VWM mutations. We show that some mutations cause expected decreases in GEF activity or complex formation, similar to a majority of observed VWM mutations. However, we also observe some unexpected changes which hint at other effects of these mutations on as yet undescribed functions of eIF2B.

Recessive inherited mutations in any of five subunits of the general protein synthesis factor eIF2B are responsible for a white mater neurodegenerative disease with a large clinical spectrum. The classical form is called Childhood Ataxia with CNS hypomyelination (CACH) or Vanishing White Matter Leukoencephalopathy (VWM). eIF2B-related disorders affect glial cells, despite the fact that eIF2B is a ubiquitous protein that functions as a guanine-nucleotide exchange factor (GEF) for its partner protein eIF2 in the translation initiation process in all eukaryotic cells. Decreased eIF2B activity measured by a GEF assay in patients’ immortalised lymphocytic cells provides a biochemical diagnostic assay but is limited by the availability of eIF2 protein, which is classically purified from a mammalian cell source by column chromatography. Here we describe the generation of a recombinant expression system to produce purified human eIF2 from yeast cells. We demonstrate that human eIF2 can function in yeast cells in place of the equivalent yeast factor. We purify human eIF2 and the C-terminal domain of human eIF2Be using affinity chromatography from engineered yeast cells and find that both function in a GEF assay: the first demonstration that this human eIF2Be domain has GEF function. We show that CACH/VWM mutations within this domain reduce its activity. Finally we demonstrate that the recombinant eIF2 functions similarly to eIF2 purified from rat liver in GEF assays with CACH/VWM eIF2B-mutated patient derived lymphocytic cells.

Local translation is rapidly regulated by extrinsic signals during neural wiring, but its control mechanisms remain elusive. Here we show that the extracellular cue Sema3A induces an initial burst in local translation that precisely controls phosphorylation of the translation initiation factor eIF2α via the unfolded protein response (UPR) kinase PERK. Strikingly, in contrast to canonical UPR signaling, Sema3A-induced eIF2α phosphorylation bypasses global translational repression and underlies an increase in local translation through differential activity of eIF2B mediated by protein phosphatase 1. Ultrasensitive proteomics analysis of axons reveals 75 proteins translationally controlled via the Sema3A-p-eIF2α pathway. These include proteostasis- and actin cytoskeleton-related proteins but not canonical stress markers. Finally, we show that PERK signaling is needed for directional axon migration and visual pathway development in vivo. Thus, our findings reveal a noncanonical eIF2 signaling pathway that controls selective changes in axon translation and is required for neural wiring.

RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α.IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR., RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α.