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eIF2B facilitates and controls protein synthesis in eukaryotes by mediating guanine nucleotide exchange on its partner eIF2. We combined mass spectrometry (MS) with chemical cross-linking, surface accessibility measurements and homology modelling to define subunit stoichiometry and interactions within eIF2B and eIF2. Although it is generally accepted that eIF2B is a pentamer of five non-identical subunits (α–ε), here we show that eIF2B is a decamer. MS and cross-linking of eIF2B complexes allows us to propose a model for the subunit arrangements within eIF2B where the subunit assembly occurs through catalytic γ- and ε-subunits, with regulatory subunits arranged in asymmetric trimers associated with the core. Cross-links between eIF2 and eIF2B allow modelling of interactions that contribute to nucleotide exchange and its control by eIF2 phosphorylation. Finally, we identify that GTP binds to eIF2Bγ, prompting us to propose a multi-step mechanism for nucleotide exchange., Eukaryotic Initiation Factor 2 (eIF2) initiates protein synthesis aided by its partner eIF2B, which stimulates guanine nucleotide exchange on eIF2. Here, Gordiyenko et al. show that eIF2B exists as a decamer and propose a model for its subunit arrangement that provides new insight into its function.

Leukoencephalopathy with vanishing white matter is a recently defined autosomal recessive disorder. The course is chronic progressive with additional episodes of rapid deterioration, provoked by fever and minor head trauma. A previous study showed that mutations in the genes encoding the epsilon- or the beta-subunit of the eukaryotic translation initiation factor eIF2B, a complex consisting of five subunits, cause the disease in most patients. Seven unsolved patients remained. The unsolved patients were investigated by mutation analysis of the genes encoding the alpha-, gamma-, and delta-subunit of eIF2B and the gene encoding the alpha-subunit of eIF2, because phosphorylation of this latter subunit regulates eIF2B activity. Mutations were found in the genes encoding the alpha- (1 patient), gamma- (2 patients), and delta-subunits (2 patients) of eIF2B, but no mutations were found in the gene encoding the alpha-subunit of eIF2. In 2, both less typical patients, no mutations were found. Mutations in all five genes eIF2B subunit genes can cause VWM. eIF2B is essential for the initiation of translation of RNA into protein and is involved in regulation of the process, especially under circumstances of stress, such as fever. A defect in eIF2B may explain the sensitivity to stress factors in vanishing white matter patients.

There are conflicting reports over the question of whether the displacement by GTP of GDP bound to eIF2 catalyzed by eIF2B follows a substituted enzyme mechanism, as is believed to be the case for other guanine nucleotide exchange factors, or is a sequential mechanism. Analysis of data recently provided by Williams et al. (J. Biol. Chem. 276, 24697–24703, 2001) showing displacement by eIF2B of GDP bound to eIF2 in the absence of displacing nucleotide appears to offer a way of resolving the dispute and suggests that both mechanisms may be operative.

The effects of neurotrophic factors on translational activation were investigated in cortical neurons. Brain-derived neurotrophic factor (BDNF) increased protein synthesis within 30 min, whereas insulin produced a weaker enhancement of protein synthesis. BDNF-triggered protein synthesis was inhibited by LY294002, PD98059, and rapamycin, whereas the effect of insulin was unaffected by PD98059. To explore the mechanisms underlying this effect, the protein phosphorylation cascades that lead to the activation of translation initiation in neurons were examined. BDNF induced the phosphorylation of both eukaryote initiation factor (eIF) 4E and its binding protein (eIF4E-binding protein-1). The former reaction was inhibited by PD98059, whereas the latter was inhibited by LY294002 or rapamycin. In agreement, BDNF induced the phosphorylation of mammalian TOR (target of rapamycin) and enhanced its kinase activity toward eIF4E-binding protein-1. In contrast, insulin failed to activate MAPK and did not induce the phosphorylation of eIF4E. Since BDNF and insulin increased the activity of eIF2B and eIF2, the only difference between them was eIF4E phosphorylation. Thus, this may explain the lower activity of insulin in potentiating neuronal protein synthesis. These results suggest strongly that BDNF simultaneously activates multiple signaling cascades consisting of phosphatidylinositol 3-kinase, mammalian TOR, and MAPK to enhance translation initiation in neurons.

Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNA(Met) to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its alpha subunit [eIF2(alphaP)] acts as competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, impairing formation of the ternary complex and thereby inhibiting translation initiation. eIF2B is comprised of catalytic and regulatory subcomplexes harboring independent eIF2 binding sites; however, it was unknown whether the alpha subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2alpha (glutathione S-transferase [GST]-SUI2) bound to the eIF2B regulatory subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction also occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(alphaP) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations in SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(alphaP) also impaired binding of phosphorylated GST-SUI2 to the eIF2B regulatory subunits. These findings provide strong evidence that tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex is crucial for the inhibition of eIF2B and attendant downregulation of protein synthesis exerted by eIF2(alphaP). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the beta and gamma subunits of eIF2 in the manner required for GDP-GTP exchange.

The alpha-subunit of eukaryotic initiation factor eIF2 is a preferred substrate for the double-stranded RNA-activated protein kinase, PKR. Phosphorylation of eIF2alpha converts the factor from a substrate into a competitive inhibitor of the guanine nucleotide exchange factor, eIF2B, leading to a decline in mRNA translation. Early studies provided evidence implicating PKR as the kinase that phosphorylates eIF2alpha under conditions of cell stress such as the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum, i.e., the unfolded protein response (UPR). However, the recent identification of a trans-microsomal membrane eIF2alpha kinase, termed PEK or PERK, suggests that this kinase, and not PKR, might be the kinase that is activated by misfolded protein accumulation. Similarly, genetic studies in yeast provide compelling evidence that a kinase termed GCN2 phosphorylates eIF2alpha in response to amino acid deprivation. However, no direct evidence showing activation of the mammalian homologue of GCN2 by amino acid deprivation has been reported. In the present study, we find that in fibroblasts treated with agents that promote the UPR, protein synthesis is inhibited as a result of a decrease in eIF2B activity. Furthermore, the reduction in eIF2B activity is associated with enhanced phosphorylation of eIF2alpha. Importantly, the magnitude of the change in each parameter is identical in wildtype cells and in fibroblasts containing a chromosomal deletion in the PKR gene (PKR-KO cells). In a similar manner, we find that during amino acid deprivation the inhibition of protein synthesis and extent of increase in eIF2alpha phosphorylation are identical in wildtype and PKR-KO cells. Overall, the results show that PKR is not required for increased eIF2alpha phosphorylation or inhibition of protein synthesis under conditions promoting the UPR or in response to amino acid deprivation.

PRK1, a receptor-like kinase that is expressed in pollen, pollen tubes, and ovaries, has been shown to play important roles in pollen development and embryo sac development in Petunia inflata. We have used the kinase domain of PRK1 as a bait in the yeast two-hybrid system to identify PRK1-interacting proteins. The screening resulted in isolation of a cDNA encoding a protein highly homologous to the human and yeast beta-subunit of translation initiation factor 2B (eIF2B-beta), which was designated NeIF2Bbeta. eIF2B is a guanine nucleotide exchange protein that functions in the regulation of translation in eukaryotic cells. Deletion mutants of NeIF2Bbeta were analyzed for their interaction with PRK1, and the results suggested that the N-terminal half of NeIF2Bbeta, especially the region between residue 103 and 235, is important for the interaction. This protein association was confirmed by in vitro binding assay of the recombinant NeIF2Bbeta and PRK1 proteins. Despite high sequence homology between NeIF2Bbeta and its yeast counterpart, the NeIF2Bbeta cDNA could not rescue the phenotype of the yeast mutant strain lacking the GCD7 gene encoding eIF2B-beta, when transferred into the mutant strain.

Translational control by specific eIF2alpha phosphorylation on serine 51 has been characterized in all eukaryotes with the significant exception of plants. In order to evaluate the capability of plant eIF2alpha to functionally control translation, the wild type (51S) and a nonphosphorylatable mutant (51A) of wheat eIF2alpha were expressed in a yeast genetic system. Expression of either wheat protein did not handicap growth under conditions that repress the eIF2alpha phosphorylation pathway. However, under conditions that induce specific eIF2alpha phosphorylation only strains expressing wheat 51S were able to grow between 2 and 4 days. Growth was dependent upon activity of yeast eIF2alpha kinase GCN2 and resulted in the increased translation of GCN4. The association between plant eIF2alpha and yeast eIF2B is supported by their specific coimmunoprecipitation from transgenic yeast cells. These data support the similarity among eukaryotic translational initiation processes and strengthen the concept that plants may contain an eIF2alpha phosphorylation pathway.

Chronic septic abscess formation causes an inhibition of protein synthesis in gastrocnemius that is not observed in rats with a sterile abscess. The inhibition is associated with an impaired translation initiation. The present study was designed to investigate the effects of sepsis on phosphorylation and availability of eukaryotic initiation factor (eIF)4E in gastrocnemius 5 days after induction of a sterile or septic abscess. Neither sepsis nor sterile inflammation altered the extent of eIF4E phosphorylation. Moreover, no changes in the amount of the binding protein 4E-BP1 associated with eIF4E or in the phosphorylation of 4E-BP1 were observed during sepsis or sterile inflammation. In contrast, sepsis and sterile inflammation caused a reduction in the relative amount of eIF4G bound to eIF4E compared with controls. The diminished amount of eIF4G bound to eIF4E was not the result of a reduced abundance of eIF4E. Sepsis, but not sterile inflammation, caused an increase in the cellular abundance of eIF4E. The results provide evidence that alterations in the eIF4E system are probably not rate controlling for the synthesis of total, mixed proteins in gastrocnemius during sepsis. Instead, on the basis of our previous studies, changes in eIF2B appear to be responsible for limiting protein synthesis in skeletal muscle during sepsis.

Phosphorylation of serine 51 residue on the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) inhibits the guanine nucleotide exchange (GNE) activity of eIF2B, presumably, by forming a tight complex with eIF2B. Inhibition of the GNE activity of eIF2B leads to impairment in eIF2 recycling and protein synthesis. We have partially purified the wild-type (wt) and mutants of eIF2alpha in which the serine 51 residue was replaced with alanine (51A mutant) or aspartic acid (51D mutant) in the baculovirus system. Analysis of these mutants has provided novel insight into the role of 51 serine in the interaction between eIF2 and eIF2B. Neither mutant was phosphorylated in vitro. Both mutants decreased eIF2alpha phosphorylation occurring in hemin and poly(IC)-treated reticulocyte lysates due to the activation of double-stranded RNA-dependent protein kinase (PKR). However, addition of 51D, but not 51A mutant eIF2alpha protein promoted inhibition of the GNE activity of eIF2B in hemin-supplemented rabbit reticulocyte lysates in which relatively little or no endogenous eIF2alpha phosphorylation occurred. The 51D mutant enhanced the inhibition in GNE activity of eIF2B that occurred in hemin and poly(IC)-treated reticulocyte lysates where PKR is active. Our results show that the increased interaction between eIF2 and eIF2B protein, occurring in reticulocyte lysates due to increased eIF2alpha phosphorylation, is decreased significantly by the addition of mutant 51A protein but not 51D. Consistent with the idea that mutant 51D protein behaves like a phosphorylated eIF2alpha, addition of this partially purified recombinant subunit, but not 51A or wt eIF2alpha, increases the interaction between eIF2 and 2B proteins in actively translating hemin-supplemented lysates. These findings support the idea that phosphorylation of the serine 51 residue in eIF2alpha promotes complex formation between eIF2alpha(P) and eIF2B and thereby inhibits the GNE activity of eIF2B.

Epidermal and nerve growth factors (EGF and NGF) activate protein synthesis and initiation factor eIF2B in rat phaeochromocytoma (PC12) cells. The activation of protein synthesis by EGF or NGF depends upon extracellular regulated kinase kinase (MEK)/extracellular regulated kinase signalling. Here we show that PD98059, an inhibitor of MEK activation, blocks the activation of eIF2B by EGF or NGF. It is known that eIF2B activity can be inhibited by phosphorylation at Ser535 in its epsilon-subunit by glycogen synthase kinase (GSK)-3. We find that inactivation of GSK-3 by EGF or NGF is blocked by PD98059. However, neither EGF nor NGF caused a detectable change in phosphorylation of Ser535 of eIF2Bepsilon. Thus, the EGF- and NGF-induced activation of eIF2B in PC12 cells involves regulatory mechanisms distinct from dephosphorylation of the GSK-3 site.

The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2alpha to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2alpha and inducibly express the eIF2alpha dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2alpha resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2alpha on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2alpha reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2alpha and the 51A mutant with mammalian eIF2gamma and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2alpha is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.

Eukaryotic translation initiation factor 2B (eIF2B) is a guanine nucleotide exchange factor (GEF) that converts its substrate, eIF2, from an inactive eIF2-GDP binary complex to eIF2-GTP. This active complex binds charged initiator tRNAMet (Met-tRNAiMet) forming a ternary complex that interacts with eIF3 and the 40S ribosomal subunit. Following addition of mRNA, associated initiation factors, and the 60S ribosomal subunit, the G-protein cycle is completed by hydrolysis of eIF2-bound GTP and the release of eIF2-GDP from the ribosome (reviewed in references 14, 31, and 42). Thus, the functions of eIF2 and eIF2B are believed to be similar to those of the small GTPases and exchange factors, respectively, of the RAS superfamily (4). Recent three-dimensional structure determinations have demonstrated that while the nucleotide binding domains of the G proteins are very similar, GEF structures differ markedly from one another, each employing different amino acid motifs to drive the release of GDP (8, 41). eIF2 and eIF2B are complex proteins of three (α to γ) and five (α to ɛ) nonidentical subunits, respectively. The subunit complexity of eIF2B reflects, at least in part, its novel mechanism of regulation. Four protein kinases, called PKR, HCR (HRI), PERK (PEK), and GCN2, specifically phosphorylate the seryl residue at position 51 of the α subunit of eIF2 (eIF2α) under different stress conditions (12, 23, 40). Phosphorylation of eIF2α at this site converts eIF2 from a substrate into an inhibitor of eIF2B (33, 38), thus inhibiting global translation initiation. In the yeast Saccharomyces cerevisiae, the protein kinase GCN2 phosphorylates eIF2α in response to amino acid or purine starvation. Under moderate amino acid starvation conditions, the level of phosphorylated eIF2 produced is not sufficient to inhibit total protein synthesis; however, it specifically enhances translation of GCN4 mRNA, which encodes a transcriptional regulator of amino acid biosynthetic genes (24). GCN4 translation is inversely coupled to ternary complex concentration and thus to eIF2B activity by the presence of inhibitory short open reading frames in the 5′ leader of its mRNA. Recently, homologues of GCN2 have been identified in Drosophila melanogaster (32) and mammals (2), indicating that this kinase may be universally conserved in eukaryotes. By using both genetic and biochemical methods, it has been demonstrated that three subunits of S. cerevisiae eIF2B (α, β, and δ encoded by GCN3, GCD7, and GCD2, respectively) act together to mediate regulation of eIF2B activity in response to phosphorylation of its substrate, eIF2 (33, 34, 43). We also found that the ɛ subunit of eIF2B, encoded by GCD6 in yeast, is a catalytic subunit of eIF2B: the ability of extracts from yeast cells overexpressing eIF2Bɛ alone to dissociate GDP from eIF2-GDP binary complexes was higher than that of nonoverexpressing cell extracts (33). Interestingly, eIF2Bɛ catalyzed nucleotide exchange at a reduced rate compared with that of the five-subunit eIF2B complex. Others have obtained similar results expressing mammalian eIF2Bɛ cDNA in insect cells (18). In addition, we showed that the ɛ and γ subunits can form an eIF2B catalytic subcomplex in the absence of the other three subunits. This γɛ catalytic subcomplex promoted release of GDP from eIF2-GDP at a higher rate than ɛ alone and could also bind stably to eIF2 (33), but in contrast to the full five-subunit complex, nucleotide exchange and binding of this subcomplex to eIF2 were not affected by the phosphorylation of eIF2α. In this study, we decided to follow up on our observation that eIF2Bɛ showed catalytic activity to determine what regions or residues of this polypeptide are important for its GEF activity. Examination of the primary sequence of eIF2Bɛ reveals no significant sequence identity with any other GEF. However, eIF2Bɛ does share significant sequence similarity with eIF2Bγ (5, 35) (see Fig. ​Fig.1),1), to which it binds, forming the eIF2B catalytic subcomplex (33). In addition, eIF2Bγ and eIF2Bɛ both share extended sequence similarity with two other protein families found mainly in bacteria–nucleoside triphosphate (NTP)-hexose pyrophosphorylases and acyltransferases (see Fig. ​Fig.1A).1A). It has been proposed that the region of similarity with the bacterial NTP-hexose pyrophosphorylase family represents a nucleotide binding domain composed of a modified P-loop and magnesium ion coordinating region (28), suggesting a role for nucleotide binding by eIF2B in the guanine nucleotide exchange reaction. Finally, it has been shown recently that the sequence motif shared between the extreme C termini of eIF2Bɛ and eIF5 (a potential GTPase-activating protein for eIF2) (28) provides a binding site in both proteins for the β subunit of their common substrate eIF2 (1). FIG. 1 Genetic characterization of novel mutations in yeast eIF2Bɛ. (A) eIF2Bγ and eIF2Bɛ subunits encoded by yeast genes GCD1 and GCD6 are shown schematically from N to C termini. The patterns indicate regions of significant sequence ... We show here that the C-terminal region of eIF2Bɛ is responsible for binding to the substrate eIF2 and contains the catalytic domain for GEF activity. Missense alleles in which single conserved amino acids within this region were changed dramatically reduce the GEF activity of eIF2Bɛ without affecting eIF2 binding, indicating that different residues are responsible for these two functions. In contrast, the N-terminal half of eIF2Bɛ is required for its interactions with the other eIF2B subunits. Missense alleles, where single conserved residues have been altered, in this region of the gene affect the stimulation of eIF2B activity observed upon eIF2B complex formation without detectably altering binding to eIF2. The implications of these results for eIF2B function are discussed.

The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode. This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5' end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome. Here we provide a comparative sequence analysis of the beta subunit of eIF2 and its archaeal counterpart (aIF2beta). aIF2beta differs from eIF2beta in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B. The remaining sequences are highly conserved, and are shared with eIF5. Previously isolated mutations in the yeast eIF2beta, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2beta, but not in eIF5. We show that the sequence of eIF2beta homologous to aIF2beta is sufficient for binding eIF2gamma, the only subunit with which it interacts, and comprises, at the most, 78 residues. eIF5 does not interact with eIF2gamma, despite its similarity with eIF2beta, probably because of a gap in homology in this region. These observations have implications for the evolution of the mechanism of translation initiation.

Background: Acute alcohol intoxication in rats decreases protein synthesis in skeletal muscle and, to a lesser extent, in liver. The purpose of the present study was to examine potential mechanisms for the inhibitory effect of acute ethanol exposure. Methods: Rats were injected intraperitoneally with either ethanol (75 mmol/kg) or saline, and tissues were examined 2.5 hr later. Rates of protein synthesis in vivo were determined by [ 3 H]phenylalanine incorporation into protein, and various eukaryotic initiation factors (eIFs) were quantitated by Western blot analysis to identify possible mechanisms for regulating translation. Results: Protein synthesis in gastrocnemius and liver was decreased (39% and 21%, respectively) after alcohol administration, compared with saline-injected control animals. Alcohol administration did not alter tissue RNA content but diminished translational efficiency in muscle (43%) and liver (24%). Hepatic eIF2B activity was decreased 24% in alcohol-treated rats, and this was associated with a 95% increase in eIF2α phosphorylation. However, alcohol did not alter the amount of 4E-binding protein 1 (4E-BP1) bound to eIF4E, eIF4E bound to eIF4G, or the phosphorylation state of either 4E-BP1 or eIF4E. In contrast to liver, neither eIF2B activity nor the phosphorylation of eIF2a was affected in muscle of alcohol-treated rats. However, acute alcohol intoxication increased binding of 4E-BP1 to eIF4E (113%). decreased the amount of eIF4E bound to cIF4G (81%), and decreased the amount of 4E-BP1 in the phosphorylated γ-form (77%). The plasma concentrations of insulin and insulin-like growth factor-I were unchanged by alcohol, but muscle insulin-like growth factor-I messenger ribonucleic acid abundance was decreased 35%. Conclusions: These data suggest that acute alcohol intoxication decreases translation initiation and protein synthesis in liver and muscle via different mechanisms. Changes in eIF2B appear to predominate in liver, whereas alterations in eIF4E availability appear more critical in skeletal muscle for controlling translation initiation.

Mitogens induce the coordinated activation of a number of anabolic events which culminate in cell growth and division (41). Recent studies have defined the distinction between growth and proliferation, demonstrating the dominance of growth in this process (42, 58). An important component of the growth response is the generation of new translational machinery, required to accommodate the increased demand for additional proteins (56). The enhanced expression of protein synthetic components, most notably ribosomal proteins and elongation factors, is largely controlled at the translational level (5, 39). The transcripts for ribosomal proteins and elongation factors are characterized by an oligopyrimidine tract, termed 5′TOP, at their translational start site (5, 39). More importantly, it has been shown that the translational upregulation of these transcripts is mediated, in part, by the activation of the 40S ribosomal protein S6 kinase (S6K1) (32), presumably through the increased phosphorylation of S6 (33, 34). The importance of S6K1 in cell growth was initially inferred from the microinjection of neutralizing antibodies into cells (38, 50) and the use of the immunosuppressant rapamycin, each inhibiting mitogen-induced S6K1 activation and impeding cell growth (33, 34). Recently, the significance of S6K1 in cell growth has been emphasized by the generation of S6K1-deficient mice, which are significantly reduced in size (55), and by the discovery of a new, highly homologous S6 kinase, S6K2 (28, 55). The signal transduction pathway which mediates S6K1 activation has received considerable attention because of its implied importance in the growth response (25, 47). Early studies demonstrated that mitogen-induced S6K1 activation is initiated at a specific growth factor receptor docking site distinct from that utilized by the mitogen-activated protein kinase-Ras signaling pathway (40). The use of the inhibitory fungal metabolite wortmannin and platelet-derived growth factor (PDGF) receptor mutants led to the identification of phosphatidylinositide-3OH kinase (PI3K) as the effector which initiates downstream signaling from the receptor (17). The activation of S6K1 appears to be mediated in a hierarchical manner, initiated by the phosphorylation of a set of sites in its autoinhibitory domain (24) that facilitate subsequent phosphorylation at T389 in the adjacent linker domain. These two sets of initial phosphorylation events act in a synergistic manner to regulate T229 phosphorylation in the catalytic domain and thus kinase activation (24). Except for the recent identification of phosphoinositide-dependent protein kinase 1 (PDK1) as the S6K1-T229 kinase (4, 49), little is known concerning the identity of the other kinases which regulate the additional phosphorylation of S6K1 (49). The key step in the activation process is T389 phosphorylation, which unlike the phosphorylation of T229, appears to be positively regulated by a wortmannin-sensitive PI3K-dependent input (23). Although the S6K1-T389 kinase has yet to be identified, recent studies have suggested that this step is mediated by PI3K through the activation of protein kinase B (PKB) (15). Like S6K1, PKB activation is mediated by PI3K through the increased phosphorylation of T308 and S473, the sites homologous to T229 and T389 in S6K1, respectively (1). The dependence of PKB activation on PI3K as well as its wortmannin sensitivity can be circumvented by constitutively anchoring PKB to the membrane (6). This leads to increased levels of T308 and S473 phosphorylation, highly activates PKB (6), and induces S6K1 activation (15). These findings have led to the hypothesis that PKB mediates PI3K-induced S6K1 activation. Membrane-targeted alleles of PKB also induce the phosphorylation of two additional signaling components which regulate the translational machinery. One is glycogen synthase kinase 3 (GSK-3), whose direct phosphorylation by PKB leads to its inactivation (21). A key substrate of GSK-3 is the ɛ subunit of protein synthesis initiation factor eIF2B (65), whose phosphorylation suppresses eIF2B GDP-GTP exchange activity, inhibiting translation (65). The inhibition of GSK-3 activity by PKB increases eIF2B GDP-GTP exchange activity, raising the amount of active eIF2-GTP and leading to increased rates of global translation (19). The second downstream target of PKB is eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) (27, 36, 53, 60). The phosphorylation of 4E-BP1 disrupts its interaction with the mRNA m7G cap-binding protein eIF4E, allowing eIF4E to form a productive initiation complex (44). Unlike GSK-3, the effects of PKB on increased 4E-BP1 phosphorylation are indirect and may be mediated by a second kinase, possibly mTOR, the target of rapamycin (14, 53), or a kinase tightly bound to mTOR (43). Indeed, 4E-BP1 is thought to reside on the same rapamycin-sensitive PI3K-dependent signaling pathway as S6K1 (64). Although highly activated alleles of PKB lead to S6K1 activation, the same is also true for activated oncogenic alleles of Ras (10). However, the activation of wild-type Ras by mitogens is neither sufficient nor necessary to bring about S6K1 activation (40). This raised the possibility that highly activated alleles of PKB or constitutive membrane localization may not reflect wild-type PKB signaling. To test the role of PKB signaling in S6K1 activation we have compared the abilities of specific variants of PKB to activate and confer wortmannin resistance on S6K1, as well as the ability of interfering mutants to block this response. In parallel, we have used GSK-3β inactivation and 4E-BP1 phosphorylation to independently monitor the effects of the different PKB alleles on rapamycin-sensitive and -insensitive PKB downstream signaling. The results demonstrate that constitutive membrane localization of active PKB is essential for its ability to signal S6K1 and that the pathway leading to 4E-BP1 phosphorylation, but surprisingly not S6K1 activation, is dominantly regulated by PKB.

eIF2 plays a central role in the maintenance of what is generally considered a rate-limiting step in mRNA translation. In this step, eIF2 binds GTP and Met-tRNAi and transfers Met-tRNAi to the 40S ribosomal subunit. At the end of the initiation process, GTP bound to eIF2 is hydrolyzed to GDP and the eIF2.GDP complex is released from the ribosome. The exchange of GDP bound to eIF2 for GTP is a prerequisite to binding Met-tRNAi and is mediated by a second initiation factor, eIF2B. In what is probably the best-characterized mechanism for the regulation of mRNA translation, phosphorylation of eIF2 on its smallest, or alpha-, subunit converts eIF2 from a substrate of eIF2B into a competitive inhibitor. Thus, phosphorylation of eIF2 alpha effectively prevents formation of the eIF2.GTP.Met-tRNAi complex and inhibits global protein synthesis. Phosphorylation of eIF2 alpha occurs under a variety of conditions including viral infection, apoptosis, nutrient deprivation, heme-deprivation, and certain stresses.

A drug that affects memory targets a constituent of a cellular stress response mechanism [Also see Report by Sekine et al. ]

The guanine nucleotide exchange activity of eIF2B plays a key regulatory role in the translation initiation phase of protein synthesis. The activity is markedly inhibited when the substrate, i. e. eIF2, is phosphorylated on Ser51 of its alpha-subunit. Genetic studies in yeast implicate the alpha-, beta-, and delta-subunits of eIF2B in mediating the inhibition by substrate phosphorylation. However, the mechanism involved in the inhibition has not been defined biochemically. In the present study, we have coexpressed the five subunits of rat eIF2B in Sf9 cells using the baculovirus system and have purified the recombinant holoprotein to >90% homogeneity. We have also expressed and purified a four-subunit eIF2B complex lacking the alpha-subunit. Both the five- and four-subunit forms of eIF2B exhibit similar rates of guanine nucleotide exchange activity using unphosphorylated eIF2 as substrate. The five-subunit form is inhibited by preincubation with phosphorylated eIF2 (eIF2(alphaP)) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. In contrast, eIF2B lacking the alpha-subunit is insensitive to inhibition by eIF2(alphaP) and is able to exchange guanine nucleotide using eIF2(alphaP) as substrate at a faster rate compared with five-subunit eIF2B. Finally, a double point mutation in the delta-subunit of eIF2B has been identified that results in insensitivity to inhibition by eIF2(alphaP) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. The results provide the first direct biochemical evidence that the alpha- and delta-subunits of eIF2B are involved in mediating the effect of substrate phosphorylation.

Regulation of eukaryotic translation initiation is a process that requires collaboration between multiple proteins. The cap-binding factor eukaryotic initiation factor (eIF)4E, its binding protein 4E-BP1, and the guanine-nucleotide-exchange factor eIF2B play important roles in the regulation of the rate of protein synthesis. This review describes the regulation of the activity of these three proteins and the signal-transduction pathways involved therein.

The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2alpha) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2alpha at Ser51. In the present study, rat eIF2alpha was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2alpha kinase activity and was rapidly phosphorylated by the eIF2alpha kinases HCR and PKR. A variant of eIF2alpha in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2alpha was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2alpha has been developed. Use of the wildtype and variant forms of eIF2alpha to measure eIF2alpha kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.

eIF2B is a heteropentameric guanine-nucleotide exchange factor essential for protein synthesis initiation in eukaryotes. Its activity is inhibited in response to starvation or stress by phosphorylation of the alpha subunit of its substrate, translation initiation factor eIF2, resulting in reduced rates of translation and cell growth. We have used an in vitro nucleotide-exchange assay to show that wild-type yeast eIF2B is inhibited by phosphorylated eIF2 [eIF2(alphaP)] and to characterize eIF2B regulatory mutations that render translation initiation insensitive to eIF2 phosphorylation in vivo. Unlike wild-type eIF2B, eIF2B complexes with mutated GCN3 or GCD7 subunits efficiently catalyzed GDP exchange using eIF2(alphaP) as a substrate. Using an affinity-binding assay, we show that an eIF2B subcomplex of the GCN3, GCD7, and GCD2 subunits binds to eIF2 and has a higher affinity for eIF2(alphaP), but it lacks nucleotide-exchange activity. In contrast, the GCD1 and GCD6 subunits form an eIF2B subcomplex that binds equally to eIF2 and eIF2(alphaP). Remarkably, this second subcomplex has higher nucleotide-exchange activity than wild-type eIF2B that is not inhibited by eIF2(alphaP). The identification of regulatory and catalytic eIF2B subcomplexes leads us to propose that binding of eIF2(alphaP) to the regulatory subcomplex prevents a productive interaction with the catalytic subcomplex, thereby inhibiting nucleotide exchange.

Eukaryotic initiation factor eIF2B catalyses a key regulatory step in mRNA translation. eIF2B and total protein synthesis are acutely activated by insulin, and this requires phosphatidylinositol 3-kinase (PI 3-kinase). The epsilon-subunit of eIF2B is phosphorylated by glycogen synthase kinase-3 (GSK-3), which is inactivated by insulin in a PI 3-kinase-dependent manner. Here we identify the phosphorylation site in eIF2Bepsilon as Ser540 and show that treatment of eIF2B with GSK-3 inhibits its activity. Ser540 is phosphorylated in intact cells and undergoes dephosphorylation in response to insulin. This is blocked by PI 3-kinase inhibitors. Insulin-induced dephosphorylation of this inhibitory site in eIF2B seems likely to be important in the overall activation of translation by this hormone.

Modulation of protein/protein interaction is an important mechanism involved in regulation of translation initiation. Specifically, regulation of the interaction of eIF2 with the guanine nucleotide exchange factor, eIF2B, is a key mechanism for controlling translation under a variety of conditions. Phosphorylation of the alpha-subunit of eIF2 converts the protein into a competitive inhibitor of eIF2B by causing an increase in the binding affinity of eIF2B for eIF2. Consequently, it has been assumed that the alpha-subunit of eIF2 is directly involved in binding to eIF2B. In the present study, eIF2 was found to bind only to the delta- and epsilon-subunits of eIF2B, and eIF2B was shown to bind only to the beta-subunit of eIF2 by far-Western blot analysis. The binding site on eIF2beta for either the eIF2B holoprotein, or the isolated delta- or epsilon-subunits of eIF2B was shown to be located within approximately 70 amino acids of the C terminus of the protein. Phosphorylation of the alpha-subunit of eIF2 did not promote binding of eIF2B to the isolated subunit. However, it did cause an increase in the affinity of eIF2B for eIF2. Finally, phosphorylation by protein kinase A of the beta-subunit of eIF2 in the C-terminal portion of the protein increased the guanine nucleotide exchange activity of eIF2B, whereas phosphorylation by casein kinase II or protein kinase C was without effect.

The eukaryotic initiation factor 2B (eIF2B) serves an essential recycling function in protein synthesis. As the guanine nucleotide exchange factor for eIF2, it recycles eIF2 from a GDP to a GTP bound form that is competent for translation initiation. Stress-dependent controls target this eIF2B-recycling step allowing a reprogramming of the global gene expression profile. In addition, a human disease, leukoencephalopathy with vanishing white matter (VWM), is caused by mutations in the eIF2B subunit genes. Recently, we have found that the eIF2B guanine nucleotide exchange factor resides in a specific cytoplasmic focus in the yeast, Saccharmoyces cerevisiae. eIF2B is a resident feature of this focus, whereas eIF2 shuttles to and fro. Moreover, the in vivo rate of eIF2 shuttling correlates with changes in guanine nucleotide exchange activity implicating this large cytoplasmic focus as a site of guanine nucleotide exchange. In this perspective, we discuss these findings in the wider context of the assortment of guanine nucleotide exchange factors.

Vanishing white matter disease is an inherited disorder, most frequently affecting children, caused by mutations in the genes encoding the subunits of the eIF2B complex. This disease is primarily diagnosed by magnetic resonance imaging (MRI). The diagnosis is confirmed when mutations are found in one of the eIF2B genes. Pathological hallmarks are foamy oligodendrocytes, dysmorphic astrocytes, and the presence of high numbers of glia progenitors that fail to mature. The selective involvement of the white matter is unexplained, and a causal role for reduced eIF2B activity is debatable.

A new quantitative strategy has generated a comprehensive rate control map for protein synthesis in exponentially growing yeast cells. This analysis reveals the modularity of the system as well as highly non-stoichiometric relationships between components., A ‘genetic titration' method has generated a map of the in vivo rate control properties of components of the protein synthesis machinery in Saccharomyces cerevisiae and has been used to parameterize a new comprehensive model of the translation pathway. The translation machinery is found to be a highly modular system in functional terms yet the intracellular concentrations of its components range from a few thousand to one million molecules per cell. This approach identifies non-intuitive features of the system such as the strongest rate control being exercised by high abundance elongation factors. The rate control analysis allows us to identify a surprising fine-control function for duplicated translation factor genes., Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNAi to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.

Insulin acutely stimulates protein synthesis in mammalian cells, and this involves activation of the process of mRNA translation. mRNA translation is a complex multi-step process mediated by proteins termed translation factors. Several translation factors are regulated in response to insulin, often as a consequence of changes in their states of phosphorylation. The initiation factor eIF4E binds to the cap structure at the 5ʹ-end of the mRNA and mediates assembly of an initiation-factor complex termed eIF4F. Assembly of this complex can be regulated by eIF4E-binding proteins (4E-BPs), which inhibit eIF4F complex assembly. Insulin induces phosphorylation of the 4E-BPs, resulting in alleviation of the inhibition. This regulatory mechanism is likely to be especially important for the control of the translation of specific mRNAs whose 5ʹ-untranslated regions (5ʹ-UTRs) are rich in secondary structure. Translation of another class of mRNAs, those with 5ʹ-UTRs containing polypyrimidine tracts is also activated by insulin and this, like phosphorylation of the 4E-BPs, appears to involve the rapamycin-sensitive signalling pathway which leads to activation of the 70 kDa ribosomal protein S6 kinase (p70 S6 kinase) and the phosphorylation of the ribosomal protein S6. Overall stimulation of translation may involve activation of initiation factor eIF2B, which is required for all initiation events. This effect is dependent upon phosphatidylinositol 3-kinase and may involve the inactivation of glycogen synthase kinase-3 and consequent dephosphorylation of eIF2B, leading to its activation. Peptide-chain elongation can also be activated by insulin, and this is associated with the dephosphorylation and activation of elongation factor eEF2, probably as a consequence of the insulin-induced reduction in eEF2 kinase activity. Thus multiple signalling pathways acting on different steps in translation are involved in the activation of this process by insulin and lead both to general activation of translation and to the selective regulation of specific mRNAs.

Protein synthesis is altered in both skeletal muscle and liver in response to nutritional status with food deprivation being associated with an inhibition of mRNA translation. In the present study, the effect of food-intake on the initiation of mRNA translation was examined in rats fasted for 18-h and then refed a complete diet. Fasting and refeeding caused alterations in translation initiation in both skeletal muscle and liver that were not associated with any detectable changes in the activity of eIF2B or in the phosphorylation state of eIF2 alpha. Instead, alterations in initiation were associated with changes in the phosphorylation state of eIF4E and/or the association of eIF4E with eIF4G as well as the eIF4E binding protein, 4E-BP1. In muscle from fasted rats, the amount of eIF4E present in an inactive complex with 4E-BP1 was increased 5-fold compared to freely fed control animals. One hour after refeeding a complete diet, the amount of 4E-BP1 bound to eIF4E was reduced to freely fed control values. Reduced association of the two proteins was the result of increased phosphorylation of 4E-BP1. Refeeding a complete diet also stimulated the binding of eIF4E to eIF4G to form the active eIF4F complex. In liver, the amount of eIF4E associated with eIF4G, but not the amount of eIF4E associated with 4E-BP1, was altered by fasting and refeeding. Furthermore, in liver, but not in skeletal muscle, fasting and refeeding resulted in modulation of the phosphorylation state of eIF4E. Overall, the results suggest that protein synthesis may be differentially regulated in muscle and liver in response to fasting and refeeding. In muscle, protein synthesis is regulated through modulation of the binding of eIF4E to eIF4G and in liver through modulation of both phosphorylation of eIF4E as well as binding of eIF4E to eIF4G.

Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43 degrees C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures.

Phosphorylation of serine 51 residue in the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) impairs the guanine nucleotide exchange (GNE) activity of eIF2B protein and thereby inhibits protein synthesis in mammalian systems, insects and yeast. It is not known if phosphorylation of plant eIF2 can inhibit an eIF2B-like activity. Interestingly purified wheat germ eIF2 (WG x eIF2) can exchange guanine nucleotides in vitro without the addition of any protein factor like eIF2B. It is not clear if this is due to a contaminant eIF2B-like activity associated with WG x eIF2 or because the affinity of WG x eIF2 for GDP and GTP is not markedly different. Our observations here indicate that the GNE activity of WG x eIF2 is not inhibited upon phosphorylation of the p41-42 doublet subunit in WG x eIF2 by reticulocyte eIF2alpha kinases, or in the presence of reticulocyte eIF2(alphaP) in which serine 51 residue is phosphorylated. Further, addition of WG x eIF2 reduces the inhibition in eIF2B activity, protein synthesis, and also the formation of 15S complex that occurs between reticulocyte eIF2(alphaP) and eIF2B protein in heme-deficient or poly(IC)-treated reticulocyte lysates, presumably by a mechanism of competition between wheat germ and reticulocyte eIF2 for phosphorylation. Unlike reticulocyte eIF2(alphaP), phosphorylated WG x eIF2 is unable to interact with reticulocyte eIF2B to form a 15S complex. The ability of WG x eIF2 to exchange guanine nucleotides independent of an eIF2B like protein and the inability of phosphorylated WG x eIF2 to interact with reticulocyte eIF2B suggests that WG x eIF2 is different from mammalian eIF2 and these differences may have occurred in evolution probably due to some changes in the amino acid sequences around the phosphorylation site in eIF2alpha.

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide exchange factor (GEF) that plays a key role in the regulation of protein synthesis. In this study, we have used the baculovirus-infected Sf9 insect cell system to express and characterize the five dissimilar subunits of rat eIF-2B. GEF activity was detected in extracts of Sf9 cells expressing the epsilon-subunit alone and was greatly increased when all five subunits were coexpressed. In addition, high GEF activity was observed in extracts containing a four-subunit complex lacking the alpha-subunit. Assembly of an eIF-2B holoprotein was confirmed by coimmunoprecipitation of all five subunits. Gel filtration chromatography revealed that recombinant eIF-2B had the same molecular mass as eIF-2B purified from rat liver and that it did indeed possess GEF activity. Phosphorylation of the substrate eIF-2 inhibited the GEF activity of the five-subunit eIF-2B; this inhibition required the eIF-2B alpha-subunit. The results demonstrate that eIF-2Balpha functions as a regulatory subunit that is not required for GEF activity, but instead mediates the regulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2Bepsilon is necessary and is perhaps sufficient for GEF activity in vitro.

eIF2B is a five-subunit guanine nucleotide exchange factor that is negatively regulated by phosphorylation of the alpha subunit of its substrate, eIF2, leading to inhibition of translation initiation. To analyze this regulatory mechanism, we have characterized 29 novel mutations in the homologous eIF2B subunits encoded by GCD2, GCD7, and GCN3 that reduce or abolish inhibition of eIF2B activity by eIF2 phosphorylated on its alpha subunit [eIF2(alphaP)]. Most, if not all, of the mutations decrease sensitivity to eIF2(alphaP) without excluding GCN3, the nonessential subunit, from eIF2B; thus, all three proteins are critical for regulation of eIF2B by eIF2(alphaP). The mutations are clustered at both ends of the homologous region of each subunit, within two segments each of approximately 70 amino acids in length. Several mutations alter residues at equivalent positions in two or all three subunits. These results imply that structurally similar segments in GCD2, GCD7, and GCN3 perform related functions in eIF2B regulation. We propose that these segments form a single domain in eIF2B that makes multiple contacts with the alpha subunit of eIF2, around the phosphorylation site, allowing eIF2B to detect and respond to phosphoserine at residue 51. Most of the eIF2 is phosphorylated in certain mutants, suggesting that these substitutions allow eIF2B to accept phosphorylated eIF2 as a substrate for nucleotide exchange.

Protein synthesis factor eIF2 delivers initiator tRNA to the ribosome. Two proteins regulate its G-protein cycle: eIF5 has both GTPase-accelerating protein (GAP) and GDP dissociation inhibitor (GDI) functions, and eIF2B is the guanine nucleotide exchange factor (GEF). In this study, we used protein-protein interaction and nucleotide exchange assays to monitor the kinetics of eIF2 release from the eIF2GDP/eIF5 GDI complex and determine the effect of eIF2B on this release. We demonstrate that eIF2B has a second activity as a GDI displacement factor (GDF) that can recruit eIF2 from the eIF2GDP/eIF5 GDI complex prior to GEF action. We found that GDF function is dependent on the eIF2Be and eIF2Bg subunits and identified a novel eIF2-eIF2Bg interaction. Furthermore, GDF and GEF activities are shown to be independent. First, eIF2B GDF is insensitive to eIF2a phosphorylation, unlike GEF. Second, we found that eIF2Bg mutations known to disrupt GCN4 translational control significantly impair GDF activity but not GEF function. Our data therefore define an additional step in the protein synthesis initiation pathway that is important for its proper control. We propose a new model to place eIF2B GDF function in the context of efficient eIF2 recycling and its regulation by eIF2 phosphorylation. (Keywords: protein synthesis initiation; G-protein regulators; GEF; GDF; GDI)

Eukaryotic translation initiation is a complex and highly regulated process involving the ribosome, mRNA and proteins called eukaryotic initiation factors (eIFs). The overall aim of translation initiation is to position the ribosome at the initiation codon of the mRNA. eIF2, in its GTP-bound conformation, binds the initiator tRNA (Met-tRNAiMet) and delivers it to the 40S ribosomal subunit. When the anticodon of the tRNA is bound to the initiation codon, the GTP on eIF2 is hydrolysed to GDP. The guanine nucleotide exchange factor (GEF) eIF2B regenerates eIF2-GTP. eIF2 and eIF2B are multisubunit/multidomain protein complexes. Because information regarding the interface between each complex is limited, particularly the interface on the eIF2γ subunit, which binds the guanine-nucleotides and Met-tRNAiMet, interactions between the minimal GEF domain of eIF2Bε, εGEF, and eIF2 were mapped using mutagenesis and an in vitro cysteine cross-linking approach, with the cross-linker Mts-Atf-Biotin.Site-directed mutagenesis (SDM) was used to mutate five N-terminal and five C-terminal surface-exposed εGEF residues to cysteines. The mutant alleles were analysed in Saccharomyces cerevisiae and it was found that the gcd6-R574C allele was lethal and the gcd6-T572C was Gcd-. Further gcd6-R574 mutant alleles were also found to be lethal in yeast but expressed in vivo.εGEF-R574C has dramatically reduced GEF activity in vitro and binding assays showed that this mutant has significantly reduced affinity for eIF2. The εGEF-T572C and εGEF-S576C mutants also have severe and minor eIF2-binding defects respectively, while the C-terminal εGEF-Cys mutants have slightly reduced affinity for eIF2. The N-terminal εGEF-Cys mutants cross-link specifically to eIF2γ, while the C-terminal εGEF-Cys mutants interact predominantly with eIF2β. From the data obtained in this study, we propose a new model for eIF2B-mediated guanine-nucleotide exchange that reduces the importance of eIF2β and suggests εGEF resembles other GEFs in binding primarily to its G protein partner eIF2γ.

Fusel alcohols signal nitrogen scarcity to elicit a range of responses in the yeast Saccharomyces cerevisiae. These alcohols activate pseudohyphal growth and cause rapid inhibition of translation initiation. Previous work from our lab has highlighted that the translation initiation factor eIF2B is a target for this regulation. eIF2B is the guanine nucleotide exchange factor required for recycling eIF2•GDP to eIF2•GTP. The GTP bound form of eIF2 can interact with the Methionyl initiator tRNA to form the ternary complex. Fusel alcohols target eIF2B leading to reduced ternary complex; however the mechanism by which alcohols cause this effect is currently unknown. This study aims to characterize the effects of fusel alcohols on eIF2B and identify post-translational modifications, which may be responsible for translation inhibition.Following purification of eIF2B, a number of novel phosphorylation sites have been identified using mass spectrometry. In particular, phosphorylated serine has been identified at position 131 within yeast eIF2Bδ. Phosphoantibody analysis suggests that the phosphorylation status of this residue differs following fusel alcohol treatment. Mutagenesis experiments are consistent with phosphorylation of this residue being essential for the translational inhibition seen following fusel alcohol exposure. Therefore, phosphorylation of this residue may prime eIF2B for regulation and provide a switch to sensitize the process of translation to particular conditions.

The protective effect of Regulator of G protein Signaling 2 (RGS2) in cardiac hypertrophy is thought to occur through its ability to inhibit the chronic GPCR signaling that promotes pathogenic growth both in vivo and in cultured cardiomyocytes. However, RGS2 is known to have additional functions beyond its activity as a GTPase accelerating protein, such as the ability to bind to eukaryotic initiation factor, eIF2B, and inhibit protein synthesis. The RGS2 eIF2B-interacting domain (RGS2(eb)) was examined for its ability to regulate hypertrophy in neonatal ventricular myocytes. Both full-length RGS2 and RGS2(eb) were able to inhibit agonist-induced cardiomyocyte hypertrophy, but RGS2(eb) had no effect on receptor-mediated inositol phosphate production, cAMP production, or ERK 1/2 activation. These results suggest that the protective effects of RGS2 in cardiac hypertrophy may derive at least in part from its ability to govern protein synthesis.

Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the ‘integrated stress response’ (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders. DOI: http://dx.doi.org/10.7554/eLife.00498.001, eLife digest The synthesis of proteins is an essential step in many biological processes, including memory, and drugs that inhibit protein synthesis are known to impair memory in rodents. It is thought that the brain needs these proteins to convert short-term memories into long-term memories through a process known as consolidation. A protein called EIF2α has a key role in the regulation of protein synthesis, and has also been implicated in memory. EIF2α can be activated as a result of being phosphorylated by any of four protein kinases: these are in turn activated by processes that subject cells to stress, such as viral infection, UV light or—in the case of a kinase known as PERK—the accumulation of unfolded proteins in a cellular organelle called the endoplasmic reticulum. Activation of EIF2α downregulates most protein synthesis inside the cell, but upregulates the production of a small number of key regulatory molecules: these changes help cells to cope with whatever stressful event they have just experienced. To obtain further insight into the cellular stress response, Sidrauski et al. screened a large library of compounds in search of one that inhibits PERK. They identified a molecule—known as ISRIB—which acts downstream of all four protein kinases by reversing the effects of EIF2α phosphorylation. ISRIB is the first molecule shown to have this effect, and thus represents an important tool for investigating the stress response inside cells. When Sidrauski et al. injected ISRIB into mice, the animals showed improved memory: for example, they learnt to locate a hidden platform in a water maze more rapidly than controls. This suggests that ISRIB could be used to explore the mechanisms that underlie memory consolidation, and possibly even as a memory enhancer. Moreover, given that many tumor cells exploit the cellular stress response to aid their own growth, ISRIB may have potential as a novel chemotherapeutic agent. DOI: http://dx.doi.org/10.7554/eLife.00498.002

Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the 'integrated stress response' (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders. DOI:http://dx.doi.org/10.7554/eLife.00498.001.

Eukaryotic initiation factor 2Be (eIF2Be) plays a critical role in the initiation of mRNA translation and its expression and guanine nucleotide exchange activity are major determinants of the rate of protein synthesis. In this work we provide evidence that the catalytic epsilon subunit of eIF2B is subject to ubiquitination and proteasome-mediated degradation. Lysates of C2C12 myoblasts treated with proteasome inhibitor were subjected to sequential immunoprecipitations for eIF2Be followed by ubiquitin. Tandem mass spectrometry (LC–MS/MS) analysis of immunoprecipitated proteins resulted in the identification of five peptides containing ubiquitin (diglycine) modifications on eIF2Be. The specific lysine residues containing the ubiquitin modifications were localized as Lys-56, Lys-98, Lys-136, Lys-212 and Lys-500 (corresponding to the rat protein sequence). In addition three novel phosphorylation sites were identified including Ser-22, Ser-125, and Thr-317. Moreover, peptides corresponding to the amino acid sequence of the E3 ligase NEDD4 were also detected in the LC–MS/MS analysis, and an interaction between endogenous eIF2Be with NEDD4 was confirmed by co-immunoprecipitation.

Peptide sequence data were obtained from rabbit protein synthesis initiation factor subunit eIF2B gamma. Searching the database of expressed sequence tags (dbEST) revealed nucleotide sequences potentially encoding human eIF2B gamma that contained peptides corresponding to those from the rabbit subunit. PCR primers were derived from these sequences and used to generate a probe. This was used to screen a rat skeletal muscle cDNA library, and a clone encoding rat eIF2B gamma was isolated. This cDNA gave a product in coupled transcription/translation that co-migrated with the gamma-subunit of purified eIF2B under SDS/PAGE. The sequence of this rat eIF2B gamma cDNA is reported. The protein sequence shows homology with that of yeast eIF2B gamma (the GCD1 gene product). We have also identified an open reading frame from the Caenorhabditis elegans genome project that probably encodes the gamma-subunit of C. elegans eIF2B. All these sequences show similarity to nucleotidyl- and acyltransferases, as previously reported for GCD1 [Koonin (1995) Protein Sci. 4, 1608-1617], and contain conserved motifs potentially involved in nucleotide binding. They also contain "I-patch' motifs: isoleucine-rich hexamer repeats that have been associated with the binding of acyl groups in bacterial acyltransferases. The roles of these motifs are discussed in relation to the known properties of eIF2B.

Vanishing white matter is a leukoencephalopathy that usually affects young children. Five genes were found recently for this disease, allowing a DNA-based diagnosis. The authors describe six patients homozygous for the Arg113His mutation in eIF2Bε. Only one had a childhood onset; four had a later onset and a protracted disease course; one adult still has no symptoms. Our data suggest that the Arg113His mutation is particularly mild and should be considered in the differential diagnosis of adult diffuse leukoencephalopathies, independent of whether there are associated clinical signs, an episodic course, or MRI shows white matter rarefaction/cystic degeneration.

See corresponding article on page 286. More than 40 y ago, Morgan et al (1, 2) reported that in the isolated perfused rat heart, elevating amino acid concentrations in the perfusate to 5 times the concentrations observed in the plasma of a fasted animal stimulated protein synthesis rates by enhancing the initiation phase of mRNA translation. Since that time, a plethora of studies have focused on delineating the mechanism or mechanisms through which amino acids act to upregulate translation initiation. The results of those studies have identified 2 steps in initiation as targets of amino acid signaling: the binding of initiator methionyl-tRNA (met-tRNAi)4 to the 40S ribosomal subunit to form the 43S preinitiation complex and the binding of mRNA to the 43S preinitiation complex. The binding of met-tRNAi to the 40S ribosomal subunit is mediated by eukaryotic initiation factor (eIF) 2 and regulated by the guanine nucleotide exchange factor (GEF) eIF2B. Both proteins are phosphorylated in cells in culture on specific subunits (eIF2 on its α-subunit and eIF2B on its ϵ-subunit) in response to changes in amino acid concentrations. Specifically, the deprivation of essential amino acids leads to activation of the protein kinase general control non-derepressing 2, which phosphorylates eIF2α, converting it from a substrate into a competitive inhibitor of eIF2B (3). Amino acid deprivation also leads to increased phosphorylation of eIF2B on its catalytic ϵ-subunit, resulting in inhibition of its GEF activity toward eIF2 (4). Together, phosphorylation of eIF2α and eIF2Bϵ leads to repressed rates of translation initiation by perturbing the assembly of the eIF2·GTP·met-tRNAi complex that mediates transfer of met-tRNAi to the 40S ribosomal subunit. The deprivation of essential amino acids also results in development of an impairment in the binding of mRNA to the 43S preinitiation complex, an event mediated by the eIF4F complex composed of eIF4A, eIF4E, and eIF4G (5). For most mRNAs, binding to the 43S preinitiation complex occurs through the interaction of eIF4E with the m7GTP cap at the 5′-end of the message and the subsequent binding of eIF4G to eIF3 that is part of the 43S preinitiation complex. The deprivation of essential amino acids leads to the disassembly of the eIF4F complex by promoting the interaction of eIF4E with eIF4E binding proteins (eg, 4E-BP1) and the interaction of eIF4A with programmed cell death 4 (PDCD4), thereby preventing their interaction with eIF4G. The association of eIF4E with 4E-BP1 and eIF4A with PDCD4 is regulated by phosphorylation of 4E-BP1 and PDCD4 in response to the activation of a protein kinase complex known as mechanistic target of rapamycin complex 1 (mTORC1). Amino acids, and in particular the branched-chain amino acid leucine, activate mTORC1, which leads to phosphorylation of 4E-BP1 and PDCD4, releasing their companion proteins and allowing them to bind to eIF4G to form the active eIF4F complex. Thus, in response to amino acid deprivation, mTORC1 is inhibited and eIF4E and eIF4A are sequestered away from eIF4G, resulting in impaired binding of mRNA to the 43S preinitiation complex. Studies that investigated the effects of amino acids on protein synthesis in vivo have focused primarily on skeletal muscle. In skeletal muscle, oral administration of either leucine alone or a protein-containing meal leads to a stimulation of the rate of protein synthesis concomitant with activation of mTORC1 and increased assembly of the eIF4F complex (eg, references 6 and 7). Treatment with the mTORC1 inhibitor rapamycin blocks both the amino acid–induced stimulation of protein synthesis and activation of mTORC1 signaling in animals and humans (6–8). Notably, neither phosphorylation of eIF2α nor changes in eIF2B GEF activity are typically observed in skeletal muscle in response to feeding or oral administration of leucine (9, 10), suggesting that activation of mTORC1 is the primary target responsible for the stimulation of protein synthesis by amino acids in vivo. In a study reported in this issue of the Journal, Coeffier et al (11) present data showing that enteral delivery of a mixture of protein and maltodextrin stimulates protein synthesis in the duodenal mucosa compared with provision of maltodextrin alone. Surprisingly, they do not observe an elevation in phosphorylation of 2 direct targets of mTORC1, 4E-BP1 and p70S6K1, suggesting that, in contrast to skeletal muscle, mTORC1 was not activated by the protein mixture even though elevated plasma concentrations of the branched-chain amino acids persisted throughout the 5-h infusion period. The authors conclude that enteral delivery of the protein mixture stimulated duodenal mucosa protein synthesis in humans through an mTORC1-independent pathway. If this conclusion is correct, it would represent an important new mechanism in cell biology and one that would stimulate elucidation of potentially novel means by which amino acids modulate protein synthesis. It is therefore imperative that future studies not only confirm the results of the present one but also test possible alternative pathways that might be involved in the effect. For example, in the Coeffier et al study (11), measurements were taken at a single time point, 5 h after the start of enteral delivery of the protein mixture and intravenous infusion of [13C]leucine and [2H]phenylalanine. To exclude the possibility that the protein-induced increase in incorporation of [13C]leucine and [2H]phenylalanine into protein may have occurred transiently at a time when mTORC1 signaling was activated, and that both may have returned to basal values by the time biopsies were taken, a time course analysis would be necessary. Moreover, a study that uses rapamycin to inhibit mTOR signaling could be performed, as has been used by others to assess the role of mTORC1 in the amino acid– and exercise-induced stimulation of protein synthesis in humans (8, 12). It is important that Western blot analysis, rather than dot-blot analysis, be used to assess potential changes in p70S6K1 and mTOR phosphorylation, and changes in protein phosphorylation should be expressed relative to the respective target protein, rather than GAPDH, to account for potential changes in expression. Finally, possible mTORC1-independent pathways, such as those involving eIF2 and eIF2B, which were not assessed in the Coeffier et al study (11), should be evaluated in future studies. It is possible that such measurements were performed and not reported in the present study or are planned for a future report. If not, future studies should address such possibilities to increase confidence that an mTORC1-independent pathway is indeed involved in the observed changes in duodenal mucosa protein synthesis. Overall, the data reported by Coeffier et al (11) are interpreted to support a model in which amino acids act to stimulate protein synthesis in the duodenal mucosa through a mechanism distinct from the established one involving activation of mTORC1. Future studies should aim to confirm the initial conclusion and to identify the mTORC1-independent pathway involved in regulating protein synthesis in the duodenal mucosa.

Background A prerequisite of hypertrophic response of the myocardium is an increase in protein synthesis. A central regulator of translation initiation is Eukaryotic initiation factor 2B (eIF2B). Here we assessed the hypothesis that regulation of protein synthesis via eIF2Bε is essential to cardiac hypertrophic response in vivo. Methods Two transgenic mouse lines were generated with cardiac restricted overexpression of eIF2Bε or its mutant eIF2Bε-eIFS535A, which cannot be inactivated by phosphorylation through GSK-3β. Results (1) Under baseline conditions eIF2Bε transgenic mice showed no difference in cardiac phenotype compared to wild type, whereas in the mutant eIF2Bε-S535A an increase in LV/tibia length (7.5±0.4 mg/mm vs. 6.2±0.2 mg/mm, p

Background Mutations in the bone morphogenetic protein type II receptor (BMPR-II) underlie the majority (>80%) of familial and up to 25% of ‘sporadic’ cases of idiopathic pulmonary arterial hypertension (PAH). Recently, homozygous recessive mutations in GCN2 (also known as EIF2AK4) were identified as causative in a rare cause of PAH, pulmonary veno-occlusive disease (PVOD) and pulmonary capillary haemangiomatosis (PCH). The mechanisms by which GCN2 deficiency contributes to the development of PAH remain unknown. GCN2 is a serine/threonine protein kinase, one of a family of 4 kinases that phosphorylate the α-subunit of the translation initiation factor eIF2α. Phosphorylation of eIF2α at serine 51 results in an inhibition of eIF2B (the eIF2 guanine nucleotide exchange factor), which ultimately leads to loss of eIF2α function. This results in inhibition of protein synthesis while enhancing the translation of a small number of mRNAs encoding proteins involved in the response to cellular stress. Methods We characterised the pulmonary vascular phenotype of the homozygous null Gcn2 ( Gcn2−/− ) and wild-type controls at 6 months of age. This mouse was chosen because it mimics the homozygous loss-of-function genotype of patients with familial PVOD/PCH. In addition, we investigated the phenotype of Gcn2−/− mice crossed with bmpr2 -deficient mice. In complementary in vitro studies, pulmonary artery smooth muscle cells (PASMCs) were extracted from patients with PVOD and idiopathic/heritable PAH and studied with wild-type controls. Findings The Gcn2−/− mouse displayed mild pulmonary hypertension. Mean right ventricular systolic pressure (RVSP) was 28.3 ± 1.2 mmHg in Gcn2−/− mice compared with 24.7 ± 1.1 mmHg in wild-type mice. GCN2−/− mice crossed with bmpr2 -deficient mice demonstrated a further increase in RVSP (32.8 ± 4.1 mmHg). Exposure of human PASMCs derived from a PVOD patient, and patients with mutations in BMPR2, to L-histidinol, which increases the phosphorylation of eIF2α, resulted in proliferation of these cells but had no effect on control PASMCs. Conclusions GCN2 deficiency promotes the development of pulmonary hypertension in mice, an effect that is exaggerated by BMPR2 deficiency. Increased phosphorylation of eIF2α resulted in PASMC proliferation. Further elucidation of these mechanisms may reveal new treatments for this devastating disease.

Using affinity purifications coupled with mass spectrometry and yeast two-hybrid assays, we show the Saccharomyces cerevisiae translation initiation factor complex eukaryotic translation initiation factor 2B (eIF2B) and the very-long-chain fatty acid (VLCFA) synthesis keto-reductase enzyme YBR159W physically interact. The data show that the interaction is specifically between YBR159W and eIF2B and not between other members of the translation initiation or VLCFA pathways. A ybr159wΔ null strain has a slow-growth phenotype and a reduced translation rate but a normal GCN4 response to amino acid starvation. Although YBR159W localizes to the endoplasmic reticulum membrane, subcellular fractionation experiments show that a fraction of eIF2B cofractionates with lipid membranes in a YBR159W-independent manner. We show that a ybr159wΔ yeast strain and other strains with null mutations in the VLCFA pathway cause eIF2B to appear as numerous foci throughout the cytoplasm.