Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits

The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [14C] eIF-2 or [alpha-32P]GTP, we observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. Our data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The accumulation of eIF-2.GDP on 60 S subunits occurs before binding of Met-tRNAf to 40 S subunits becomes reduced and before protein synthesis becomes inhibited. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Additional RF increases the turnover of eIF-2.GDP on 60 S subunits and 80 S ribosomes to near the control rate by promoting dissociation of eIF-2.GDP but not eIF-2(alpha-P).GDP. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining. The phosphorylation of eIF-2 alpha inhibits polypeptide chain initiation by preventing dissociation of eIF-2.GDP from either free 60 S subunits (thus inhibiting subunit joining directly) or the 60 S subunit component of an 80 S initiation complex (thereby blocking elongation and resulting in the dissociation of the 80 S complex).     

Modulation of the eukaryotic initiation factor 2 alpha-subunit kinase PERK by tyrosine phosphorylation

The endoplasmic reticulum (ER)-resident protein kinase PERK attenuates protein synthesis in response to ER stress through the phosphorylation of translation initiation factor eIF2alpha at serine 51. ER stress induces PERK autophosphorylation at several serine/threonine residues, a process that is required for kinase activation and phosphorylation of eIF2alpha. Herein, we demonstrate that PERK also possesses tyrosine kinase activity. Specifically, we show that PERK is capable of autophosphorylating on tyrosine residues in vitro and in vivo. We further show that tyrosine 615, which is embedded in a highly conserved region of the kinase domain of PERK, is essential for autocatalytic activity. That is, mutation of Tyr-615 to phenylalanine compromises the autophosphorylation capacity of PERK and the phosphorylation of eIF2alpha in vitro and in vivo. The Y615F mutation also impairs the ability of PERK to induce translation of ATF4. Immunoblot analyses with a phosphospecific antibody confirm the phosphorylation of PERK at Tyr-615 both in vitro and in vivo. Thus, our data classify PERK as a dual specificity kinase whose regulation by tyrosine phosphorylation contributes to its optimal activation in response to ER stress.     

Structure and phosphorylation of eukaryotic initiation factor 2. Casein kinase 2 and protein kinase C phosphorylate distinct but adjacent sites in the beta-subunit

Eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes can be phosphorylated on its beta-subunit by two different protein kinases, protein kinase C and casein kinase 2. Phosphorylation by these kinases is additive, suggesting that they phosphorylate different sites (serine residues) in eIF-2 beta. Two-dimensional peptide mapping of the phosphopeptides generated from labelled eIF-2 beta by digestion with trypsin, cyanogen bromide or Staphylococcus aureus V8 proteinase showed that protein kinase C and casein kinase 2 phosphorylated distinct and different sites in this protein. This conclusion was supported by the results of analysis of the phosphopeptides on reverse-phase chromatography. Analysis of the phosphopeptides derived from eIF-2 beta labelled by both kinases together strongly suggested that the sites labelled by protein kinase C and casein kinase 2 are adjacent in the primary sequence. These data are discussed in the light of the present understanding of the sequence specificity of the kinases. Rat liver eIF-2 beta was also found to be a substrate for protein kinase C and casein kinase 2, which were again shown to label different serine residues.     

Specific phosphorylation of eukaryotic initiation factor 2 alpha, eIF-2 alpha, by bovine adrenocortical cyclic nucleotide-independent protein kinase, PK 380

Recently, we characterized a novel cyclic nucleotide-independent protein kinase, PK 380, from bovine adrenal cortex (Y. Kuroda and R. K. Sharma (1980) Biochem. Biophys. Res. Commun.96, 601–610). PK 380 is independent of cyclic AMP, cyclic GMP, calcium, and calcium-calmodulin for its activity. It does not phosphorylate any of the commonly used exogenous substrates but phosphorylates an endogenous 120,000-dalton peptide. In the present study we demonstrate that PK 380 specifically phosphorylates the serine residue of eukaryotic initiation factor 2α, eIF-2α. PK 380 can be differentiated from two other protein kinases, hemin-controled repressor (HCR) or double-stranded RNA-activated inhibitor (dsRI), that are also known to phosphorylate eIF-2α. Unlike the activity of HCR, PK 380 activity is independent of hemin (5–10 μm) and dependent on sulfhydryl groups. Poly(I) · poly(C), which is known to activate dsRI, does not affect the activity of PK 380. The possibility exists that the physiological substrate of PK 380 is eIF-2α. Thus, this novel protein kinase could play an important role in the translational control processes of adrenocortical cell.     

Glucosamine-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is mediated by the protein kinase R-like endoplasmic-reticulum associated kinase

In diabetic animals, enhanced production of vascular endothelial growth factor is thought to be a major contributor to the development of diabetic retinopathy. In the present study, glucosamine-treated R28 retinal neuronal cells were used as an experimental model system to explore the possible involvement of the hexosamine biosynthetic pathway in the diabetes-induced changes in mRNA translation. Glucosamine treatment enhanced vascular endothelial growth factor production subsequent to changes in phosphorylation of the alpha-subunit of eukaryotic initiation factor 2, with no change in vascular endothelial growth factor mRNA content. Possible mechanisms through which glucosamine might act to increase eukaryotic initiation factor 2alpha phosphorylation include enhanced O-linked glycosylation of protein kinase or phosphatase regulatory proteins and/or induction of oxidative stress. However, increasing global protein O-glycosylation through inhibition of O-beta-N-acetylglucosaminidase did not mimic the effect of glucosamine on eukaryotic initiation factor 2alpha phosphorylation. Likewise, attenuating glucosamine-induced oxidative stress with two different antioxidants did not reduce glucosamine-induced eukaryotic initiation factor 2alpha phosphorylation. Glucosamine treatment was also found to promote eukaryotic initiation factor 2alpha phosphorylation in wild-type mouse embryonic fibroblasts, but not in mouse embryonic fibroblasts lacking the eukaryotic initiation factor 2alpha kinase referred to as RNA-dependent protein kinase-like endoplasmic-reticulum associated kinase, implicating the kinase in the glucosamine-induced increase in eukaryotic initiation factor 2alpha phosphorylation. Overall, the results are consistent with glucosamine causing activation of RNA-dependent protein kinase-like endoplasmic-reticulum associated kinase, which phosphorylates eukaryotic initiation factor 2alpha and consequently upregulates translation of mRNAs encoding specific proteins, such as vascular endothelial growth factor.     

Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2alpha (eIF2alpha) kinase.

Four distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases phosphorylate eIF2alpha at S51 and regulate protein synthesis in response to various environmental stresses. These are the hemin-regulated inhibitor (HRI), the interferon-inducible dsRNA-dependent kinase (PKR), the endoplasmic reticulum (ER)-resident kinase (PERK) and the GCN2 protein kinase. Whereas HRI and PKR appear to be restricted to mammalian cells, GCN2 and PERK seem to be widely distributed in eukaryotes. In this study, we have characterized the second eIF2alpha kinase found in Drosophila, a PERK homologue (DPERK). Expression of DPERK is developmentally regulated. During embryogenesis, DPERK expression becomes concentrated in the endodermal cells of the gut and in the germ line precursor cells. Recombinant wild-type DPERK, but not the inactive DPERK-K671R mutant, exhibited an autokinase activity, specifically phosphorylated Drosophila eIF2alpha at S50, and functionally replaced the endogenous Saccharomyces cerevisiae GCN2. The full length protein, when expressed in 293T cells, located in the ER-enriched fraction, and its subcellular localization changed with deletion of different N-terminal fragments. Kinase activity assays with these DPERK deletion mutants suggested that DPERK localization facilitates its in vivo function. Similar to mammalian PERK, DPERK forms oligomers in vivo and DPERK activity appears to be regulated by ER stress. Furthermore, the stable complexes between wild-type DPERK and DPERK-K671R mutant were mediated through the N terminus of the proteins and exhibited an in vitro eIF2alpha kinase activity.     

Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2alpha kinase.

In eukaryotic cells, protein synthesis is regulated in response to various environmental stresses by phosphorylating the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha). Three different eIF2alpha kinases have been identified in mammalian cells, the heme-regulated inhibitor (HRI), the interferon-inducible RNA-dependent kinase (PKR) and the endoplasmic reticulum-resident kinase (PERK). A fourth eIF2alpha kinase, termed GCN2, was previously characterized from Saccharomyces cerevisiae, Drosophila melanogaster and Neurospora crassa. Here we describe the cloning of a mouse GCN2 cDNA (MGCN2), which represents the first mammalian GCN2 homolog. MGCN2 has a conserved motif, N-terminal to the kinase subdomain V, and a large insert of 139 amino acids located between subdomains IV and V that are characteristic of the known eIF2alpha kinases. Furthermore, MGCN2 contains a class II aminoacyl-tRNA synthetase domain and a degenerate kinase segment, downstream and upstream of the eIF2alpha kinase domain, respectively, and both are singular features of GCN2 protein kinases. MGCN2 mRNA is expressed as a single message of approximately 5.5 kb in a wide range of different tissues, with the highest levels in the liver and the brain. Specific polyclonal anti-(MGCN2) immunoprecipitated an eIF2alpha kinase activity and recognized a 190 kDa phosphoprotein in Western blots from either mouse liver or MGCN2-transfected 293 cell extracts. Interestingly, serum starvation increased eIF2alpha phosphorylation in MGCN2-transfected human 293T cells. This finding provides evidence that GCN2 is the unique eIF2alpha kinase present in all eukaryotes from yeast to mammals and underscores the role of MGCN2 kinase in translational control and its potential physiological significance.     

Further characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Immunochemical characterization and phosphorylation by casein kinase II

Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Translation and phosphorylation of wheat germ lysate: phosphorylation of wheat germ initiation factor 2 by casein kinase II and in N-ethylmaleimide-treated lysates

Previously, we observed that N-ethylmaleimide (NEM), a thiol-alkylating agent, was found to stimulate the phosphorylation of several proteins in translating wheat germ (WG) lysates, including the phosphorylation of alpha, the p41-42 doublet subunit, and beta, the p36 subunit, of the WG initiation factor 2 (eIF2). We find now that NEM increases phosphorylation of several proteins significantly in lysates which are moderate or low in their translation compared to optimally active lysates. Heat treatment, which stimulates oxidation of protein sulfhydryls, decreases the translation and phosphorylation ability of WG lysates. The decrease in phosphorylation, but not translation, that occurs in heat-treated lysates is prevented very efficiently by NEM and partially by reducing agents such as dithiothreitol (DTT) and GSH. DTT prevents, however, completely the loss of sulfhydryl content of heat-treated WG lysates and does not at all prevent heat-induced inhibition of translation. In contrast, DTT prevents completely the diamide-induced translational inhibition and also the loss of sulfhydryl content. These findings therefore suggest that in addition to the maintenance of sulfhydryl groups, heat-labile proteins and their interactions with other proteins play an important role in overall translation and phosphorylation. It is also observed here that heat treatment stimulates the phosphorylation of rabbit reticulocyte eIF2 alpha but not the alpha subunit (p41-42 doublet) of WG eIF2. A phosphospecific anti-eIF2 alpha antibody recognizes the WG eIF2 alpha(P) that is phosphorylated by an authentic eIF2 alpha kinase such as double-stranded RNA-dependent protein kinase, but it is unable to recognize the eIF2 alpha that is phosphorylated in NEM-treated lysates. These findings therefore suggest that phosphorylation of WG eIF2 alpha in NEM-treated lysates occurs on a site different from the serine 51 residue that is phosphorylated by authentic eIF2 alpha kinases. In addition, it also suggests that WG eIF2 alpha, unlike reticulocyte eIF2 alpha, is phosphorylated by eIF2 alpha kinases and also by other kinases. Consistent with this idea, it has been observed here that casein kinase II (CKII) phosphorylates WG eIF2 alpha and the phosphorylation is enhanced by NEM in vitro and in lysates. The phosphopeptide analysis suggests that WG eIF2 alpha has separate phosphorylation sites for CKII and heme-regulated eIF2 alpha kinase (a well-characterized mammalian eIF2 alpha kinase), and NEM-induced phosphorylation in WG lysates resembles CKII-mediated phosphorylation.     

The use of [14C]eukaryotic initiation factor 2 to measure the endogenous pool size of eukaryotic initiation factor 2 in rabbit reticulocyte lysate.

[14C]Eukaryotic initiation factor 2 (eIF-2), obtained by reductive methylation of the purified initiation factor, was shown to be active in the unfractionated reticulocyte lysate. This allowed a direct measurement of the endogenous pool size of eIF-2 in rabbit reticulocyte lysate according to the principle of isotope dilution. A value of 20 to 30 pmol/ml of lysate was obtained. Although translational inhibition resulting from hemin deficiency appears to be characterized by a change from catalytic to stoichiometric utilization of eIF-2, the pool size of eIF-2 is too small to account for the normal period of protein synthesis before the onset of translation inhibition. This suggests, therefore, that additional events to eIF-2 alpha phosphorylation may be required for translational inhibition.     

Involvement of double-stranded RNA-dependent protein kinase and phosphorylation of eukaryotic initiation factor-2alpha in neuronal degeneration

Inhibition of protein translation plays an important role in apoptosis. While double-stranded RNA-dependent protein kinase (PKR) is named as it is activated by double-stranded RNA produced by virus, its activation induces an inhibition of protein translation and apoptosis via the phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha). PKR is also a stress kinase and its levels increase during ageing. Here we show that PKR activation and eIF2alpha phosphorylation play a significant role in apoptosis of neuroblastoma cells and primary neuronal cultures induced by the beta-amyloid (Abeta) peptides, the calcium ionophore A23187 and flavonoids. The phosphorylation of eIF2alpha and the number of apoptotic cells were enhanced in over-expressed wild-type PKR neuroblastoma cells exposed to Abeta peptide, while dominant-negative PKR reduced eIF2alpha phosphorylation and apoptosis induced by Abeta peptide. Primary cultured neurons from PKR knockout mice were also less sensitive to Abeta peptide toxicity. Activation of PKR and eIF2alpha pathway by Abeta peptide are triggered by an increase in intracellular calcium because the intracellular calcium chelator BAPTA-AM significantly reduced PKR phosphorylation. Taken together, these results reveal that PKR and eIF2alpha phosphorylation could be involved in the molecular signalling events leading to neuronal apoptosis and death and could be a new target in neuroprotection.     

A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2alpha

A family of protein kinases regulates translation in response to different cellular stresses by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). In yeast, an eIF-2alpha kinase, GCN2, functions in translational control in response to amino acid starvation. It is thought that uncharged tRNA that accumulates during amino acid limitation binds to sequences in GCN2 homologous to histidyl-tRNA synthetase (HisRS) enzymes, leading to enhanced kinase catalytic activity. Given that starvation for amino acids also stimulates phosphorylation of eIF-2alpha in mammalian cells, we searched for and identified a GCN2 homologue in mice. We cloned three different cDNAs encoding mouse GCN2 isoforms, derived from a single gene, that vary in their amino-terminal sequences. Like their yeast counterpart, the mouse GCN2 isoforms contain HisRS-related sequences juxtaposed to the kinase catalytic domain. While GCN2 mRNA was found in all mouse tissues examined, the isoforms appear to be differentially expressed. Mouse GCN2 expressed in yeast was found to inhibit growth by hyperphosphorylation of eIF-2alpha, requiring both the kinase catalytic domain and the HisRS-related sequences. Additionally, lysates prepared from yeast expressing mGCN2 were found to phosphorylate recombinant eIF-2alpha substrate. Mouse GCN2 activity in both the in vivo and in vitro assays required the presence of serine-51, the known regulatory phosphorylation site in eIF-2alpha. Together, our studies identify a new mammalian eIF-2alpha kinase, GCN2, that can mediate translational control.     

In vivo regulation of protein synthesis by phosphorylation of the alpha subunit of wheat eukaryotic initiation factor 2

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.     

Structure of the heme-regulated eukaryotic initiation factor 2 alpha kinase. End group analysis and phosphoaminoacid content

The Mr 80,000 subunit of the inhibitor of protein synthesis that is activated in heme deficiency was purified from SDS gels. Radiolabelled reagents were used to determine the end terminal residues: the N-terminal residue was found to be only glutamine, and the C-terminal residue only valine. Phosphoaminoacids determination revealed both phosphoserine and phosphothreonine.     

The NH2-terminal sequence of the alpha and gamma subunits of eukaryotic initiation factor 2 and the phosphorylation site for the heme-regulated eIF-2 alpha kinase

Rabbit reticulocyte eukaryotic initiation factor 2 was phosphorylated with the heme-regulated alpha subunit of eukaryotic initiation factor 2 kinase, and then the individual subunits were resolved by reversed-phase high performance liquid chromatography. Phosphorylated and unphosphorylated forms of the alpha subunit also were well resolved. The NH2-terminal sequences of intact alpha and gamma subunits were determined. No sequence was obtained from the beta subunit, suggesting that it may have a blocked NH2-terminus. Overlapping tryptic and chymotryptic phosphopeptides from the NH2-terminal sequence of the alpha subunit of eukaryotic initiation factor 2 were used to establish the order of amino acids 1-52 and localized the phosphorylation site within the sequence: -Leu-Leu-Ser48-Glu-Leu-Ser51-. Subdigestion of a tryptic fragment with chymotrypsin generated only phosphopeptides that appeared to terminate at leucine 50, indicating phosphorylation at serine 48.     

Nuclear factor 90 is a substrate and regulator of the eukaryotic initiation factor 2 kinase double-stranded RNA-activated protein kinase.

Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNA-independent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNA-dependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed.     

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation.     

Regulation of protein synthesis in rabbit reticulocyte lysate. Glucose 6-phosphate is required to maintain the activity of eukaryotic initiation factor (eIF)-2B by a mechanism that is independent of the phosphorylation of eIF-2 alpha

Previous studies from other laboratories, using rabbit reticulocyte lysate filtered through Sephadex G-25 or G-50, have demonstrated that glucose 6-phosphate is required to maintain active rates of translation, but its mechanism of action is currently unsettled. We have tested whether glucose 6-phosphate is required to prevent activation of the hemin-controlled translational repressor and the phosphorylation of the smallest or alpha subunit of eukaryotic initiation factor 2 (eIF-2). We have found that antibody to the hemin-controlled translational repressor can completely restore protein synthesis in reticulocyte lysate, filtered through Sephadex G-25, that is incubated in the absence of hemin and presence of glucose 6-phosphate, but cannot restore protein synthesis in such lysate incubated in the presence of hemin and absence of glucose 6-phosphate. We have also found, using a modification of the method of Matts and London [1984) J. Biol. Chem. 259, 6708-6711) to measure the ability of gel-filtered lysate to dissociate and exchange GDP from eIF-2.GDP, that this endogenous eIF-2B activity is reduced to the same low level in the presence of hemin and absence of glucose 6-phosphate as it is in the absence of hemin and presence of glucose 6-phosphate. Although there is a low level of phosphorylation of eIF-2 alpha in gel-filtered lysate given hemin but no glucose 6-phosphate, it cannot account for the loss of eIF-2B activity, since this phosphorylation is removed by antibody to the hemin-controlled translational repressor or isocitrate, which do not restore protein synthesis or eIF-2B activity, and not by fructose 1,6-diphosphate, which does partially restore protein synthesis and eIF-2B activity. These findings suggest that sugar phosphates may exert a direct effect on eIF-2B and may be required for its proper function. Additional support for this conclusion is our finding that protein synthesis and eIF-2B activity in partially hemin-deficient lysate can be restored by high levels of glucose 6-phosphate or fructose 1,6-diphosphate without a reduction in the level of phosphorylated eIF-2 alpha, suggesting that such levels of sugar phosphate may permit restoration of normal function with a limiting amount of eIF-2B.