Characterization of the Initiation Factor eIF2B and Its Regulation in Drosophila melanogaster*,

Daniel D. WilliamsDagger , Graham D. Pavitt§, and Christopher G. ProudDagger

From the Dagger  School of Life Sciences, Medical Sciences Institute/Wellcome Trust Biocentre Complex, University of Dundee, Dundee DD1 5EH and the § Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), Manchester M60 1QD, United Kingdom

Received for publication, September 1, 2000, and in revised form, October 30, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Eukaryotic initiation factor (eIF) 2B catalyzes a key regulatory step in the initiation of mRNA translation. eIF2B is well characterized in mammals and in yeast, although little is known about it in other eukaryotes. eIF2B is a hetropentamer which mediates the exchange of GDP for GTP on eIF2. In mammals and yeast, its activity is regulated by phosphorylation of eIF2alpha . Here we have cloned Drosophila melanogaster cDNAs encoding polypeptides showing substantial similarity to eIF2B subunits from yeast and mammals. They also exhibit the other conserved features of these proteins. D. melanogaster eIF2Balpha confers regulation of eIF2B function in yeast, while eIF2Bepsilon shows guanine nucleotide exchange activity. In common with mammalian eIF2Bepsilon , D. melanogaster eIF2Bepsilon is phosphorylated by glycogen synthase kinase-3 and casein kinase II. Phosphorylation of partially purified D. melanogaster eIF2B by glycogen synthase kinase-3 inhibits its activity. Extracts of D. melanogaster S2 Schneider cells display eIF2B activity, which is inhibited by phosphorylation of eIF2alpha , showing the insect factor is regulated similarly to eIF2B from other species. In S2 cells, serum starvation increases eIF2alpha phosphorylation, which correlates with inhibition of eIF2B, and both effects are reversed by serum treatment. This shows that eIF2alpha phosphorylation and eIF2B activity are under dynamic regulation by serum. eIF2alpha phosphorylation is also increased by endoplasmic reticulum stress in S2 cells. These are the first data concerning the structure, function or control of eIF2B from D. melanogaster.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The binding of the initiator Met-tRNAi to the 40 S ribosomal subunit is a key control point in the initiation of mRNA translation in both Saccharomyces cerevisiae and mammals (1-3). This step is mediated by eukaryotic initiation factor (eIF)1 2, a heterotrimeric GTP-binding protein, which, when liganded with GTP, is able to bind the initiator Met-tRNA to form a ternary complex (eIF2·GTP·Met-tRNAi). This complex then binds to the 40S ribosomal subunit, forming the 43S pre-initiation complex that then interacts with other initiation factors on the mRNA to allow selection of the translation start codon. The GTP molecule is hydrolyzed late in the initiation process releasing eIF2 as a relatively stable and inactive binary complex (eIF2·GDP). At physiological magnesium concentrations, the affinity of eIF2 for GDP is high and recycling of eIF2 to the active GTP-bound state requires a further protein factor, eIF2B, a guanine nucleotide-exchange factor, which in yeast and mammals is composed of five nonidentical subunits (alpha -epsilon ) (2).

The activity of eIF2B is limiting for peptide chain initiation and is regulated under a variety of conditions. The best characterized mechanism of regulation, which is known to operate both in yeast and in mammalian cells, is the inhibition of eIF2B by the phosphorylation of its substrate, eIF2, at a highly conserved serine residue (Ser51 in mammals) in its alpha -subunit (3). Phosphorylated eIF2alpha acts as a potent competitive inhibitor of eIF2B, and, as cellular levels of eIF2 generally exceed those of eIF2B, low levels of eIF2alpha phosphorylation can cause substantial inhibition of eIF2B. Recent data suggest that the alpha -, beta -, and delta -subunits of eIF2B interact with eIF2 and that these subunits are required to sensitize eIF2B to inhibition by this mechanism (4, 5).

To date, a number of eIF2alpha kinases have been studied in yeast and mammals. In S. cerevisiae, general control nonderepressible 2 (GCN2), is activated under conditions of amino acid deprivation, leading to increased phosphorylation of eIF2alpha and partial inhibition of global protein synthesis. However, translation of the mRNA for the transcriptional activator GCN4 is actually enhanced by virtue of a set of short upstream open reading frames in its 5'-leader region (1). GCN2 homologs have been described in mammals and in Drosophila melanogaster (6-8), suggesting analogous regulatory mechanisms may operate in these species. Recently, a new eIF2alpha kinase, pancreatic eukaryotic initiation factor-2alpha kinase (PEK), was identified in mammalian pancreatic cells and characterized as a membrane-bound protein localized in the lumen of the endoplasmic reticulum (ER). This kinase has been implicated in the control of translation in response to ER stresses such as improper protein folding. In total, mammalian cells possess at least four eIF2alpha kinases (heme-regulated inhibitor (HRI; Ref. 9), interferon-induced double-stranded RNA-activated protein kinase (PKR; Ref. 10), general control nonderepressible 2 (GCN2; Ref. 11), and pancreatic eukaryotic initiation factor 2alpha kinase (PEK, also termed PERK; Refs. 6 and 7)), which are generally activated under conditions of cellular stress (e.g. viral infection, disruption of endoplasmic reticulum function, and heme deprivation (Ref. 3)).

The initiation phase of translation and the role of eIF2 and its regulation have been studied intensively in S. cerevisiae and also in mammalian systems (12). However, relatively little work has focused on other metazoans. In D. melanogaster the evidence that a mechanism of guanine nucleotide exchange exists remains equivocal. Previous studies indicated that D. melanogaster eIF2 can be purified as a stable binary complex with GDP. The affinity of eIF2 for GDP at physiological magnesium concentrations was such that a mechanism of catalysis would be required to form the active eIF2·GTP complex (13, 14). However, Mateu and colleagues (13, 15) reported that nucleotide exchange on D. melanogaster eIF2 was independent of an exchange factor under several conditions. Moreover, no guanine nucleotide exchange activity for eIF2 in D. melanogaster was detected in embryos (14).

The sequence of eIF2alpha from D. melanogaster does, however, contain a seryl residue (Ser50) in the position corresponding to Ser51 in mammals and in a very similar sequence context. Indeed, this residue and the surrounding 19 amino acids are conserved with those found at the phosphorylation site (Ser51) in mammalian and S. cerevisiae eIF2alpha (16). D. melanogaster eIF2alpha can be phosphorylated in vitro by reticulocyte HRI, and this phosphorylation was shown to inhibit guanine nucleotide exchange by mammalian eIF2B (13, 14). Phosphorylation of the alpha -subunit of D. melanogaster eIF2 at residue Ser50 in vivo has never been examined. However, two eIF2alpha kinases have been identified in D. melanogaster. A D. melanogaster ortholog of the S. cerevisiae GCN2p kinase has been identified and characterized (17, 18). Complementation experiments in gcn2-deleted strains of S. cerevisiae have confirmed that D. melanogaster GCN2 (dGCN2) is a functional homolog of GCN2p. Expression studies show that dGCN2 is expressed in a developmentally regulated manner and is restricted to the central nervous system during later stages of development. More recently, a D. melanogaster ortholog of the mammalian PEK has been identified through sequence homology (8). However, the physiological conditions and mechanism by which these kinases function in vivo in D. melanogaster remain unclear, especially in the absence of information about eIF2B and its regulation in this species. Thus, it was unclear whether D. melanogaster possessed or required a factor equivalent to eIF2B or whether this process was truly regulated by eIF2alpha phosphorylation in this organism.

Another mechanism by which eIF2B can be regulated in mammalian systems is the phosphorylation of its epsilon -subunit (eIF2Bepsilon ) by glycogen synthase kinase-3beta (GSK-3beta ). The activity of GSK-3beta is known to be modulated in response to insulin, which induces the phosphorylation and inactivation of GSK-3beta (19-21). This response occurs concomitantly with the dephosphorylation of the epsilon -subunit of mammalian eIF2B, at the site of phosphorylation by GSK-3beta , causing the activation of eIF2B (22). D. melanogaster has a homolog of GSK-3beta , Shaggy (23), however, although its role in insulin signaling has not been elucidated. Genetic evidence has indicated that Shaggy acts downstream of Dishevelled in the Wingless pathway inactivating Armadillo (the D. melanogaster homolog of beta -catenin) (24), and biochemical evidence suggests Shaggy is downstream of protein kinase C (25). A putative phosphorylation site for GSK-3beta has been identified in D. melanogaster eIF2Bepsilon , based on sequence homology (26). However, the role of phosphorylation of this site has not been studied.

Together, these data suggest that a mechanism of guanine nucleotide exchange and its regulation by eIF2alpha and eIF2Bepsilon phosphorylation probably exist in D. melanogaster. However, neither nucleotide exchange (eIF2B) activity nor the phosphorylation of eIF2alpha has been demonstrated in vivo in D. melanogaster. Given the recent discoveries of eIF2alpha kinases in this species, establishing that eIF2alpha phosphorylation is a regulatory mechanism in the initiation of translation, in fruit flies, was an important priority. In this study we report the presence of guanine nucleotide exchange activity in D. melanogaster S2 cell lines and its regulation by eIF2alpha phosphorylation in vitro and in vivo. We identify cDNAs encoding all five subunits of D. melanogaster eIF2B (alpha , beta , gamma , delta , and epsilon ), and have cloned cDNAs encoding the alpha -, beta -, gamma -, and epsilon -subunits. We have also characterized the functions of the alpha - and epsilon -subunits. We also report that eIF2alpha phosphorylation occurs in a regulated manner in vivo, that GSK-3beta phosphorylates eIF2Bepsilon in vitro, and that eIF2B activity can be inhibited in vitro by GSK-3beta . eIF2B and its regulation in this species appear to be similar to other eukaryotic organisms that have so far been studied.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and Biochemicals-- Chemicals and biochemicals were obtained from BDH (Poole, Dorset, United Kingdom (UK)) and Sigma-Aldrich (Gillingham, Dorset, UK), unless stated otherwise. [3H]GDP (10 Ci/mmol), [35S]methionine (1000 Ci/mmol), and [gamma -32P]ATP (10 mCi/ml) were from Amersham International. X-ray film was obtained from Konica Corp.. Nitrocellulose filters (0.45-µm pore size) were obtained from Whatman. Restriction enzymes and DNA polymerases were obtained from Promega, and oligonucleotide primers were obtained from MWG Biotech. Recombinant HRI was kindly provided by Dr. J.-J.. Chen (Harvard-Massachusetts Institute of Technology, Cambridge, MA) and the vector encoding PKR was kindly provided by Dr. T. Dever (National Institutes of Health, Bethesda, MD). Recombinant GSK-3beta was provided by Dr. A. Paterson (University of Dundee, Dundee, UK) and the vector pGEX-HA was kindly provided by Dr. N. Helps (University of Dundee, Dundee, UK).

Cell Culture, Treatment, and Lysis-- D. melanogaster Schneider (S2) cells were grown in 75-ml tissue culture flasks in DES expression medium with L-glutamine (Invitrogen, Netherlands), containing 10% heat-inactivated fetal calf serum (Life Technologies, Paisley, UK) (27). S2 cell used in guanine nucleotide exchange assays were harvested (at a density of 8 × 106 cells/ml) in lysis buffer containing 100 mM Tris, pH 7.6, 50 mM beta -glycerophosphate, 0.5 mM sodium orthovanadate (Na3VO4), 1.5 mM EGTA, 0.1% Triton, 0.1 mM dithiothreitol, 1 µg/ml microcystin, protease inhibitors (leupeptin, aprotinin, pepstatin, and benzamidine; all 1 µg/ml), and 0.1 mM phenylmethylsulfonyl fluoride. For starvation experiments, cells were starved by placing them into medium containing no serum when they reached a density of 3 × 106 cells/ml and lysed or recovered at the times indicated. For experiments involving ER stresses, thapsigargin (1 µg/ml) in dimethyl sulfoxide or tunicamycin (1 µM) also in dimethyl sulfoxide was added to the cells 12 h prior to lysis; control cells were incubated for the same time in the same concentration of dimethyl sulfoxide.

Gel Electrophoresis and Immunoblotting-- SDS-polyacrylamide gel electrophoresis was performed using gels containing 12.5% acrylamide and 0.1% N,N'-methylene-bis-acrylamide (28). Gels were either stained with Coomassie Blue and dried or transferred to polyvinylidene difluoride membranes (Immobilon, Millipore) and subjected to immunoblotting. For Western blotting, samples of total extracts from D. melanogaster S2 cells were subjected to SDS-polyacrylamide gel electrophoresis and probed with either an antibody raised against the peptide CQFDPEKEFNHKGSGAGR corresponding to residues 313-330 of D. melanogaster eIF2alpha (alpha DeIF2Balpha ) or an antibody against a peptide with the sequence GMILLSELSpRRRIRIN (where Sp denotes a phosphoseryl residue) corresponding to the phosphorylation site in eIF2alpha (New England Biolabs). Anti-His and anti-Myc antibodies (both Sigma-Aldrich) were used as indicated in the figure legends. Antibody-antigen complexes were detected using ECL (Amersham Pharmacia Biotech) and horseradish peroxidase-conjugated sheep, rabbit, or mouse secondary antisera.

Assays for Translation Factors-- Guanine nucleotide exchange (eIF2B) activity was determined by measuring the loss of [3H]GDP from pre-formed mammalian eIF2·[3H]GDP binary complexes, in the presence of GTP, in an assay similar to that described (29, 30). More specifically, formation of the binary complex was achieved by incubating 570 nM purified eIF2 with 7.2 µM [3H]GDP in 20 mM Tris-HCl, pH 7.6, 100 mM KCl, 1 mg/ml bovine serum albumin, and 1 mM dithiothreitol for 20 min at 30 °C (~1 pmol of of eIF2 binds 1 pmol of [3H]GDP). Assays were carried out, following the addition of 20 mM Tris-HCl, pH 7.6, 100 mM KCl, 1 mM MgCl2, and 200 µM GTP, at 30 °C. D. melanogaster S2 cell extract (45 µg of protein) was used in each assay, unless otherwise stated. Following a given time, a sample was removed and diluted in 1 ml of ice-cold 50 mM Tris-HCl, pH 7.6, 90 mM KCl, and 5 mM Mg(CH3CO2)2 and filtered through nitrocellulose. Filters were washed in the same buffer and dried, and associated radioactivity was determined by scintillation counting.

Formation of [eIF2·GTP·Met-tRNAi] (ternary) complexes was assayed as described previously using [35S]methionyl-tRNA (31).

Isolation of eIF2 and eIF2B-- Purification of eIF2, as a substrate for eIF2B assays, was carried out as described (32) except that HeLa cell extracts were used as the source instead of rabbit reticulocyte lysate. The partial purification of D. melanogaster eIF2·eIF2B complex was also performed in a similar manner. 5 liters of S2 cells were grown to a density of 6 × 106 cells/ml and harvested by centrifugation at 480 × g. The cells were then lysed mechanically in lysis buffer containing 20 mM HEPES/KOH, pH 7.6, 0.5% glycerol, 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, protease inhibitors (leupeptin, aprotinin, pepstatin, and benzamidine; all 1 µg/ml), and 0.1 mM phenylmethylsulfonyl fluoride. Extract was then loaded on to a Fast Flow Q-Sepharose column and eluted using a continuous gradient of KCl (from 100 mM TOM in a total of 20 ml). Fractions (0.5 ml) were collected.

DNA Cloning and Sequence Analysis and Transfections-- Sequence homology searches were performed using the BLAST program (33) in the D. melanogaster data base Flybase (available via the World Wide Web) to obtain sequence data for the eIF2B subunits. These searches revealed either expressed sequence-tagged cDNA encoding a partial sequence (HL01112) for the alpha -subunit, or genomic sequence (from the D. melanogaster sequencing project for the others). The beta -subunit sequence is encoded within locus DMC100G10.3 (accession no. AL023874), and the epsilon -subunit is encoded within the cosmid clone 86E4. Unfortunately, these sequences were either partial sequences or contained introns. To obtain full-length sequence, oligonucleotide primers were designed to the 5' end of the known sequence and used with a T7 primer (at the 3' end of the cDNA library used) to amplify cDNAs encoding the full-length sequence. These were then cloned into the vector pGEMTeasy by ligation of the A and T nucleotides on the 3' and 5' ends of the PCR product and the vector. The sequence for the gamma -subunit was also identified using BLAST searches, which revealed an expressed sequence tag encoding what initially seemed to be a partial sequence (GM07434, accession no. AA696313). However, when sequenced, the full-length coding region of this protein was revealed. The sequences encoding the putative delta -subunit of D. melanogaster eIF2B was found by searching the recently completed D. melanogaster genomic sequence; however, cDNA encoding this subunit has not been acquired. This has revealed a full-length sequence encoding 626 amino acid residues with homology to mammalian and yeast eIF2Bdelta (see "Results").

Where appropriate, oligonucleotide primers were then designed and used to PCR amplify cDNAs from the Nicholas Brown cDNA library (34). PCR reaction products were then gel purified and subcloned into a pGEMTeasy vector using a TA cloning kit (Promega). cDNAs were sequenced on both strands by dideoxy chain termination method using the ABI PRISM dye terminator cycle sequence ready reaction kit with AmpliTaq DNA polymerase FS and the Automatic Sequencer system 373A (Applied Biosystems). For bacterial expression the cDNA sequence for the alpha -subunit was the cloned (in frame) into pET28c(+) (Novagen), to produce a His-tagged eIF2Balpha , using NdeI and BamHI (sites for cloning were introduced on the oligonucleotide primers). The cDNA sequence encoding the epsilon -subunit was cloned into pGEX-HA (in frame) using NdeI and XhoI for bacterial expression and pcDNA3.1(-)/Myc-His (Invitrogen) using EcoRI and HindIII for expression in human embryonic kidney (HEK) 293 cells. HEK 293 cells were transfected using the calcium phosphate method as described previously (35). Cells were harvested 3 days after transfection and lysed in the buffer used for lysing S2 cells (described above).

Transcription and Translation of cDNA Sequences-- In vitro transcription and translation reactions were performed using the T'n'T reticulocyte lysate system (Promega).

Expression of Recombinant Proteins-- Escherichia coli (BL21 DE3 or JM109) transformed with the appropriate vector was grown at 37 °C overnight in LB containing 100 µg/ml ampicillin. They were then diluted 1/10 and grown to an A600 of 1. Cultures were then cooled on ice for 15 min and induced with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside for 5 h. Cells were harvested by centrifugation at 3500 × g and lysed in 20 mM Tris-HCl, pH 7.6, 200 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, protease inhibitors (leupeptin, aprotinin, pepstatin, and benzamidine; all 1 µg/ml), 0.1 mM phenylmethylsulfonyl fluoride, and 0.2 g/ml lysozyme for 30 min on ice. To ensure lysis and shear any DNA, the cells were sonicated. Purification of recombinant proteins was performed using either nickel-nitrilotriacetic acid-agarose (Qiagen) or glutathione-Sepharose 4B (Amersham Pharmacia Biotech). Proteins were used the same day.

Analysis of DeIF2Balpha Function in Yeast-- cDNA encoding D. melanogaster eIF2Balpha open reading frame was cloned using MluI and NheI into the yeast expression vector pAV1411 (pGAL-GCN2FH (Ref. 36)) by standard techniques (36). The resulting plasmid (pDWDeIF2Balpha ) was transformed into isogenic yeast strains GP3153 (MATa leu2-3 leu2-113 ura3-52 trp1-Delta 63 gcn3Delta ::LEU2) and GP3140 (MATa leu2-3 leu2-113 ura3-52 trp1-Delta 63 gcn2Delta ) (5). These yeast strains were also transformed with control plasmids expressing yeast GCN3 (Ep69) (38) and yeast GCN2 (p722) (39). Strains containing each plasmid were grown at 30 °C to confluence on SGal medium (10% galactose) supplemented with leucine (2 mM) and tryptophan (1 mM) and replica-plated to the same medium and to SGal medium additionally supplemented with 25 mM 3-amino-1,2,4-triazole (3-AT) and incubated at 30 °C for an additional 3 days.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Schneider Cell Extracts Display eIF2B Activity, Which Is Inhibited by Phosphorylation of eIF2alpha -- Previous data concerning the existence of eIF2B in D. melanogaster were equivocal. Thus, to assess whether D. melanogaster cells contain a protein with eIF2B activity, we assayed extracts of S2 Schneider cells for their ability to catalyze guanine nucleotide exchange on eIF2 using complexes containing mammalian eIF2 and [3H]GDP as substrate. The data (Fig. 1A) clearly show that S2 cell extracts effectively mediate nucleotide exchange on eIF2, allowing bound [3H]GDP to be replaced by GTP.



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Fig. 1.   eIF2B activity in D. melanogaster Schneider cell extracts and its inhibition by eIF2alpha phosphorylation. A, guanine nucleotide exchange assay showing eIF2B activity in Schneider cell extracts (45 µg of protein) when incubated with a binary complex of [3H]GDP and purified mammalian eIF2 for the times shown. Where indicated samples were pretreated with ATP and MgCl2 and HRI prior to the assay. Nucleotide exchange assays were performed at 30 °C in duplicate and replicated four times. (Shown are data ± S.E. where n = 4.) B, Western blot of purified mammalian eIF2 (treated ± HRI, lanes 1 and 2, respectively) and D. melanogaster (S2) Schneider cell extract (45 µg of protein, also treated ± HRI, lanes 3 and 4, respectively) probed with anti-phospho-eIF2alpha antibody (alpha eIF2alpha ). Purified mammalian eIF2 (lane 5) and D. melanogaster Schneider cell extract (lane 6) probed with the D. melanogaster eIF2alpha -specific antibody (alpha DeIF2alpha ) is also shown. Phosphorylation of eIF2alpha was performed with 50 µM ATP and 0.5 mM MgCl2 at 30 °C for 10 min, and controls were incubated using the same conditions in the absence of HRI.

Pretreatment of S2 cell extracts with the eIF2alpha kinase HRI led to increased phosphorylation of eIF2 as assessed using an antibody specific for the phosphorylated form of eIF2alpha (Fig. 1B). The identity of the band as eIF2alpha was confirmed by comparison with the positions of mammalian and D. melanogaster eIF2alpha (probed with antibodies specific for the respective proteins). This confirms the earlier finding that D. melanogaster eIF2alpha is a substrate for HRI (15). PKR was also able to phosphorylate eIF2alpha in extracts of Schneider cells (data not shown).

When eIF2B assays were performed with cell extract that had been pretreated with HRI, eIF2B activity was markedly reduced (Fig. 1A). Inhibition of eIF2B activity by eIF2alpha phosphorylation is a property common to both yeast and mammalian eIF2B.

Isolation of eIF2 and eIF2B from Schneider Cells-- When isolated from mammalian cells, eIF2 and eIF2B tend to copurify with one another through a number of ion-exchange steps (32, 40, 41). To characterize further the corresponding factors from D. melanogaster, Schneider cell extracts were subjected to ion-exchange chromatography on an Mono-Q column, which was developed with a salt gradient from 0.1 to 1.0 M KCl. Fractions were subjected to immunoblotting with the antibody to D. melanogaster eIF2alpha . They were also assayed both for eIF2 activity (measured as formation of ternary complexes) with [35S]Met-tRNAi in the presence of GTP and for eIF2B activity using the mammalian eIF2·[3H]GDP complex as substrate.

Western blotting revealed a strong signal with the anti-eIF2alpha antiserum in the region of the gradient corresponding to 350-450 mM KCl at an apparent molecular mass of 38 kDa (Fig. 2A). These fractions also showed eIF2 activity (Fig. 2B). When fractions in this region of the gradient were assayed for eIF2B activity, nucleotide-exchange activity was observed in fraction 11 (triple-bond  490 mM KCl), i.e. just after the peak of eIF2 protein detected by immunoblotting (Fig. 2C). This behavior is similar to that of mammalian eIF2 and eIF2B on Mono-Q as purification produces two pools of eIF2 (due to the excess of eIF2 over eIF2B) consisting of eIF2 (eluted first) and eIF2·eIF2B as a complex (eluted slightly later) (32). These data suggest that the chromatographic behavior of D. melanogaster eIF2 and eIF2B is similar to that of their mammalian counterparts and provides evidence that, as in mammals and yeast, these two proteins copurify through ion-exchange chromatography.



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Fig. 2.   Copurification of eIF2 and eIF2B from D. melanogaster Schneider cell extracts. Protein from Schneider cell extracts was partially purified on a Mono-Q-Sepharose column, which was developed with a KCl gradient ranging from 100 mM to 1 M (as described under "Materials and Methods"). A, fractions were then subjected to Western blotting using an antibody directed against D. melanogaster eIF2alpha . B, to determine the activity of eIF2 in each fraction, ternary complex formation was assayed by measuring the binding of [35S]methionyl-tRNAi to eIF2 (20 min at 30 °C) as described under "Materials and Methods." C, the eIF2B activity of each fraction was determined using the guanine nucleotide exchange assay as described under "Materials and Methods," except each assay was performed for 10 min at 30 °C. All fractions are labeled according to the KCl concentration at which they were eluted. The above data represent two separate purifications.

Identification and Cloning of D. melanogaster cDNAs with Homology to Subunits of eIF2B from Other Eukaryotes-- The above data strongly suggested that D. melanogaster cells contain a factor equivalent to eIF2B from other eukaryotes. To identify sequences encoding potential eIF2B subunits from this species, we searched D. melanogaster nucleotide sequence data bases with the sequences corresponding to subunits of yeast and mammalian eIF2B. This revealed the presence of genomic or expressed sequence tag sequences with high levels of identity to all five subunits of eIF2B (alpha -epsilon ) (Tables I and II, and supplementary data available on-line). This strongly supports the initial conclusion that D. melanogaster does possess a protein homologous to the eIF2B complex in yeast and mammals.


                              
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Table I
Properties of the eIF2B subunits from D. melanogaster


                              
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Table II
Sequence identity (similarity) between eIF2B subunits of different species

The D. melanogaster subunits are generally similar in size to the corresponding subunits from yeast and from mammals (Table I). Their sequences show high identity to those of the mammalian proteins (about 50% in each case, Table II) and slightly lower identity (but still high similarity) to the yeast polypeptides (identity 29-37%, similarity around 50%). It has already been noted that the sequences of these five subunits show mutual sequence similarity in yeast (42-44) and mammals (45). This is also the case in D. melanogaster (Fig. 3). In addition to the D. melanogaster sequences for eIF2B, reported here, other putative sequences have become available for a range of other species. When D. melanogaster eIF2B sequences are aligned with these other sequences, it is very striking that certain residues are completely conserved, or only very conservatively replaced, across plants (Arabidopsis thaliana), budding and fission yeast, lower animals (Caenorhabditis elegans), insects (D. melanogaster), and mammals (only one sequence included here of the several known to avoid artificially "biasing" the appearance of the alignment).



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Fig. 3.   Characterization of cDNA sequences encoding the subunits of D. melanogaster eIF2B. Features of the amino acid sequences of eIF2Balpha , eIF2Bbeta , and eIF2Bdelta (A) and of eIF2Bgamma and eIF2Bepsilon (B) are shown schematically. The patterns show regions of sequence similarity between these proteins, orthologs from other species, and with other protein families as indicated by the key. Amino acid residue numbers at the boundaries of these regions are shown for the D. melanogaster proteins. Full amino acid sequence information including alignments with the sequences from other species can be found in the supplementary data (available on-line). C, in vitro transcription and translation of cDNA sequences encoding the alpha -, beta -, gamma -, and epsilon -subunits of D. melanogaster eIF2B. In vitro transcription and translation reactions were carried out as described under "Materials and Methods." Translation products labeled with [35S]methionine were resolved on a 12.5% SDS-polyacrylamide gel, which was treated with Amplify (Amersham Pharmacia Biotech) and exposed to the autoradiographic film overnight. Migration of the subunits was compared with broad range protein markers from Bio-Rad, their positions being shown on the left.

In the case of the alpha -, beta -, and delta -subunits, there are 11 positions in which all 12 sequences have the same residue, and another 11 where the residue is always a hydrophobic one (Leu, Ile, Val, Phe, or Met). This feature of the sequences of these subunits of eIF2B has been noted before (45) but is shown here for more species. These residues do show the rough periodicity of seven, which one would expect for residues involved in interactions between helical regions of the proteins, which might, e.g., be involved in complex assembly. The most highly conserved residues in these sequences fall into three clusters. One is near the N terminus, where the alpha  sequences show similarity to one another not that is shared with beta  or delta . The second is a large region (about 130 residues) toward the center of each sequence, which contains many of the highly conserved residues, especially the hydrophobic ones. Not immediately evident from this alignment is the high similarity between the different sequences for the individual subunits (alpha , beta , and delta ) within this region. Finally, there is a highly conserved region almost at the C terminus of the protein (28 residues), which shows marked identities/similarities across all three sequences from all species. Within this region, the sequences for any one subunit also show a high degree of identity. For eIF2Balpha , 18/28 residues are identical/conservatively replaced in this region; for eIF2Bbeta , the figure is 20/28; and, for eIF2Bdelta , 24/28.

The sequences of the gamma - and epsilon -subunits of eIF2B also show mutual similarities within a given species. Koonin (46) identified within eIF2Bepsilon three motifs similar to those found in many nucleotidyl transferases: (i) a variant of the phosphate-binding loop (P-loop), (ii) a version of the magnesium-binding site of such proteins, and (iii) more C-terminal than the first two, a region containing imperfectly repeated units usually with a hydrophobic residue in the first position. All three motifs are well conserved in the gamma - and epsilon -subunits of eIF2B from D. melanogaster (Fig. 3 and supplementary data available on-line). Within the third of these regions, the most C-terminal one, the positions of the hydrophobic residues in other species are also occupied by hydrophobic residues in D. melanogaster eIF2Bepsilon . The observation (made for the mammalian and yeast sequences of eIF2Bepsilon ) that the Ile residues within this region are often followed by Gly is not, however, a consistent feature of the D. melanogaster sequence, where only two such pairs are found. The conserved triplet NFD (residues 249-251 of yeast eIF2Bepsilon , noted by Gomez and Pavitt (Ref. 36)) is also found in D. melanogaster eIF2Bepsilon . This therefore remains as the only completely conserved triplet sequence in eIF2Bepsilon sequences. Mutations at the Asn and Phe residues within this triplet impair the activity of yeast eIF2B (36).

eIF2Bepsilon from mammals contains a conserved phosphorylation site for glycogen synthase kinase-3 (GSK-3) located at Ser540 in the rabbit sequence (22) (shown bold here, ELDSRAGSPQL). Four residues C-terminal to this is a second seryl residue (underlined), which undergoes phosphorylation and probably serves as a "priming" site for phosphorylation by GSK-3 (47, 48). The D. melanogaster sequence contains a seryl residue in the position corresponding to the GSK-3 site in mammals, but the priming site is occupied by Thr in D. melanogaster (EDASRAVTPLP). We have shown previously that phospho-threonyl residues can efficiently prime GSK-3 mediated phosphorylation, at least when peptides are used as substrates (26).

Characterization of Proteins Encoded by the D. melanogaster cDNAs for eIF2B Homologs-- cDNAs encoding the putative subunits of D. melanogaster eIF2B were cloned into pGEM3Z (alpha , beta , and epsilon ) or pBluescript (gamma ), and the polypeptides encoded were studied by in vitro coupled transcription/translation, using [35S]methionine to label the synthesized polypeptides, which were then resolved on SDS-polyacrylamide gel electrophoresis. As shown in Fig. 3C, each cDNA gave rise to a single major translation product, and no product was observed for the empty vector control (data not shown). The cDNA clones for eIF2Balpha , -beta , -gamma , and -epsilon produced polypeptides of ~34, 39, 50, and 72 kDa, which compare well with the molecular masses expected for these polypeptides (Table I). Since no clone was available for the delta -subunit, we were unable to perform a similar analysis for this polypeptide.

eIF2Bepsilon is thought to be the (principal) catalytic subunit of the eIF2B complex (4, 36, 49). To test whether the D. melanogaster homolog of eIF2Bepsilon actually possessed guanine nucleotide exchange activity, we expressed it both as a hexahistidine- and Myc-tagged protein in HEK 293 cells or as a glutathione S-transferase (GST) fusion protein in E. coli. eIF2Bepsilon and, as a control, eIF2Balpha were each expressed separately with Myc-His tags in 293 cells (Fig. 4A). To determine whether the epsilon -subunit of D. melanogaster eIF2B possessed guanine nucleotide-exchange activity, samples of extracts from HEK 293 cells expressing eIF2Bepsilon and corresponding controls, transfected with empty vector or a vector encoding the noncatalytic alpha -subunit of eIF2B, were analyzed in our standard exchange assay (Fig. 4B). Samples from cells expressing eIF2Bepsilon showed substantially enhanced exchange activity relative to the control (empty vector). Cells expressing eIF2Balpha showed no increase in nucleotide-exchange activity relative to cells transfected with the empty vector, confirming that eIF2Balpha itself is inactive in nucleotide exchange.



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Fig. 4.   Activity of D. melanogaster eIF2Bepsilon expressed in HEK 293 and E. coli. A, Western blot showing the expression of Myc-His-tagged D. melanogaster eIF2Balpha (used as the control, lane 2) and Myc-His-tagged eIF2Bepsilon in transfected HEK 293 cells (probed with the anti-Myc antibody, lane 3). Cells were also transfected with an empty vector (pcDNA 3.1, lane 1) as a control. B, guanine nucleotide exchange assay showing that 293 cell lysates from cells expressing the D. melanogaster Myc-His-tagged eIF2Bepsilon have increased activity over the controls (which contain endogenous eIF2B). Assays in this panel were performed as described under "Materials and Methods," except they were at 10 °C for the times indicated using 50 µg of lysate protein. (Shown are data ± S.E. where n = 5.) C, 12.5% SDS-polyacrylamide gel showing purified GST-tagged recombinant D. melanogaster eIF2Bepsilon (lane 1, purification as described under "Materials and Methods") and GST (lane 2, from the empty vector). D, guanine nucleotide exchange assay showing the purified recombinant D. melanogaster eIF2Bepsilon has activity. These assays were performed using 50 µg of lysate at 30 °C for the time indicated. Assays in this panel are averages of two experiments performed in duplicate.

D. melanogaster eIF2Bepsilon was also expressed in E. coli as a GST fusion protein, and purified using glutathione-Sepharose 4B (see Fig. 4C). Samples were subjected to our standard assays for GDP/GTP exchange, and substantial activity was observed, while none was seen for cells transformed with empty vector, i.e. expressing only GST (Fig. 4D). This confirms that the epsilon -subunit of eIF2B from D. melanogaster itself has exchange activity as concluded previously.

As noted above, the sequence of D. melanogaster eIF2Bepsilon contains a seryl residue in a similar to position of the GSK-3 site in mammalian eIF2Bepsilon and this is followed by a threonyl residue at the +4 position which, when phosphorylated can "prime" the phosphorylation of the more N-terminal residue by GSK-3beta at least when synthetic peptides based on this sequence are studied. The priming site in both mammals and D. melanogaster eIF2Bepsilon is followed by a prolyl residue, and it seems likely that it is a target for a proline-directed protein kinase. To date, however, we have not been able to identify the kinase responsible for phosphorylating the priming site in mammalian eIF2Bepsilon ; a number of proline-directed kinases such as members of the mitogen-activated protein kinase and cdc2 families have been tested, but none was able to phosphorylate this site. However, preparations of GSK-3beta made in Spodoptera frugiperda cells appear to be contaminated with a kinase which can catalyze phosphorylation of this site.2 We therefore used such preparations of GSK-3beta to test whether they could phosphorylate D. melanogaster eIF2Bepsilon which had been expressed in E. coli. As shown in Fig. 5A, GSK-3beta catalyzed phosphorylation of the D. melanogaster eIF2Bepsilon . No phosphorylation was observed when GSK-3beta was omitted from the reactions. However, phosphorylation by GSK-3 was substoichiometric, even when extended incubation periods were used, possibly because the amount of the priming kinase present is too low to permit more efficient phosphorylation. To test whether this phosphorylation by GSK-3beta inhibited the activity of D. melanogaster eIF2B, as is the case with mammalian eIF2B, D. melanogaster eIF2B was purified using a Mono-Q column to remove any endogenous Shaggy and incubated with GSK-3beta prior to using it in a guanine nucleotide exchange assay. Following treatment with GSK-3beta , the activity of this partially purified D. melanogaster eIF2B was inhibited by ~50% (Fig. 6) indicating that, in vitro, D. melanogaster eIF2B activity is impaired by GSK-3beta -mediated phosphorylation.



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Fig. 5.   Phosphorylation of recombinant D. melanogaster eIF2Bepsilon by GSK-3beta and CK-II. Recombinant D. melanogaster GST-eIF2Bepsilon expressed in E. coli was purified using glutathione-Sepharose. eIF2Bepsilon was then cleaved from the beads using thrombin to give the full-length protein. This protein was then incubated with 125 µM ATP (including 1 µCi of [gamma -32P]ATP), 25 mM MgCl2, and the indicated kinase for the times shown at 30 °C. Control incubations lacked added kinase as indicated. The samples were then resolved on 12.5% SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue. Gels were dried and exposed to x-ray film overnight. Panel A shows the phosphorylation of D. melanogaster eIF2Bepsilon by 1 unit of GSK-3beta . Panel B shows the phosphorylation by 3 units of CK-II. Each phosphorylation experiment was repeated three times with similar results. (One unit is the amount of protein kinase transferring 1 nmol of phosphate/min to the standard substrate under standard assay conditions.)



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Fig. 6.   Phosphorylation of partially purified D. melanogaster eIF2B by GSK-3beta inhibits eIF2B activity. Guanine nucleotide exchange assay using partially purified D. melanogaster eIF2B phosphorylated using 1 unit of GSK-3beta (1 unit is the amount of protein kinase transferring 1 nmol of phosphate/min to the standard substrate under standard assay conditions), 125 µM ATP, and 25 mM MgCl2 incubated at 30 °C for 10 min. Partially purified eIF2B incubated with ATP and MgCl2 under the same conditions was used as a control. (Shown are data ± S.E. where n = 3.)

Mammalian eIF2Bepsilon is also phosphorylated by casein kinase II (CK-II, Refs. 32 and 50). We therefore tested whether CK-II could also phosphorylate the recombinant D. melanogaster eIF2Bepsilon made in E. coli. Recombinant CK-II catalyzed phosphorylation of fly eIF2Bepsilon in vitro (Fig. 5B). Phosphorylation of mammalian eIF2Bepsilon by CK-II appears to occur at seryl residues within an acidic region at the C terminus of the protein.3 D. melanogaster eIF2Bepsilon contains a similar acidic region at its C terminus (DDQSSEEDDDEEDDD), and it is likely that CK-II phosphorylates one or both seryl residues within this region.

Complementation of Yeast GCN3 by D. melanogaster eIF2Balpha -- In yeast cells, amino acid deprivation leads to the activation of the eIF2alpha kinase GCN2. In addition to reducing overall translation initiation, phosphorylation of eIF2alpha induces translation of GCN4 mRNA (51). GCN4 is a transcriptional activator of multiple amino acid biosynthetic genes; therefore, induction of GCN4 translation is required for growth in the presence of 3-AT, an inhibitor of histidine biosynthesis. Thus, deletion of GCN2 makes yeast cells sensitive to 3-AT (3-ATS). Deletion of GCN3, the yeast eIF2Balpha subunit, also causes a 3-ATS phenotype as it apparently renders yeast eIF2B insensitive to the normally inhibitory effects of eIF2alpha phosphorylation on eIF2B-catalyzed guanine-nucleotide exchange (4). It has been shown previously that mammalian (rat) eIF2Balpha can substitute for GCN3 in vivo in yeast (52). We therefore asked whether expression of the D. melanogaster protein could complement the GCN4-dependent defect in histidine biosynthesis caused by deletion of GCN3.

The D. melanogaster eIF2Balpha cDNA was cloned downstream of a yeast galactose inducible promoter on a high copy number plasmid and introduced into yeast strains deleted for GCN3 or GCN2. In parallel, plasmids bearing yeast GCN3 or GCN2 were also transformed into these two strains. In the gcn3Delta strain, D. melanogaster eIF2Balpha conferred resistance to 3-AT equivalent to that shown by GCN3. The control plasmid bearing GCN2 remained 3-ATS (Fig. 7, gcn3Delta panel labeled SGal+3-AT). All transformed strains grew equivalently in the absence of starvation (Fig. 7, panels labeled SGal). These results indicate that D. melanogaster eIF2Balpha can substitute for GCN3 in yeast eIF2B and restore the normal response to amino acid starvation. DeIF2Balpha , like GCN3, did not confer 3-AT resistance in the gcn2Delta strain (Fig. 7, right panel). This shows that 3-AT resistance conferred by D. melanogaster eIF2Balpha is dependent on phosphorylation of eIF2alpha by GCN2 rather than the result of a bypass caused by interfering with the activity of eIF2 or eIF2B.



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Fig. 7.   Genetic complementation of GCN3 in Saccharomyces cerevisiae. Expression of D. melanogaster eIF2Balpha complements gcn3Delta allowing growth of yeast cells on histidine starvation medium. Plasmids expressing DeIF2Balpha , GCN3, or GCN2 were transformed into yeast strains deleted for gcn3Delta (GP3153, left panels) and gcn2Delta (GP3140, right panels). Cells were grown to confluence on SGal medium and replica plated to SGal medium and to SGal supplemented with 25 mM 3-AT (as indicated). Plates were incubated at 30 °C for 3 days.

The Phosphorylation of eIF2alpha and eIF2B Activity Are Regulated in S2 Schneider Cells-- When extracts from 72-h serum-starved cells were assayed for eIF2B activity, none was detected (Fig. 8B), whereas substantial activity was seen in extracts from control cells analyzed in parallel. When S2 cells were starved of serum for 72 h, the level of phosphorylation of eIF2alpha increased markedly as revealed using the antibody against eIF2alpha phosphorylated at Ser50 (Fig. 8A, lane 5 as compared with lane 4). Taken together, these results suggest that the removal of serum causes the inhibition of eIF2B activity through the phosphorylation of its substrate eIF2 and also that there may be an eIF2alpha kinase in S2 cells that is activated under conditions of serum deprivation. To study this further, we subjected S2 cells to differing periods of serum withdrawal up to 72 h, and then used the anti-phosphorylated eIF2alpha antibody to assess the level of eIF2alpha phosphorylation. The data clearly showed that, even after 12 h without serum, the level of eIF2alpha phosphorylation was substantially increased compared with the serum-fed control (Fig. 8C), taking into account the signal from a loading control blot performed using an antiserum that detects eIF2alpha irrespective of its state of phosphorylation (Fig. 8C). Consistent with this increase in eIF2alpha phosphorylation, we observed a marked decrease in eIF2B activity at all times of serum withdrawal (Fig. 8D). Readdition of fresh serum for 1 h caused substantial reactivation of eIF2B (Fig. 8E). Serum treatment for 1 h clearly caused a marked reduction in the level of phosphorylation of eIF2alpha , which may account for the observed increase in eIF2B activity.



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Fig. 8.   Regulation of eIF2alpha phosphorylation and eIF2B activity by serum in D. melanogaster Schneider cells. In vivo phosphorylation of eIF2alpha inhibits eIF2B activity. A, Western blot of purified mammalian eIF2 (± HRI, lanes 1 and 2, respectively) and Schneider cell extract (45 µg of protein) ± HRI (lanes 3 and 4, respectively), subjected to serum starvation (72 h, lane 5) or heat-shocked (37 °C for 30 min, lane 6), which was probed with the anti-phospho-eIF2alpha antibody. Phosphorylation of purified eIF2alpha was performed with 50 µM ATP and 0.5 mM MgCl2 at 30 °C for 10 min, and controls were incubated using the same conditions in the absence of HRI. B, guanine nucleotide exchange assay (using S2 cell extract) showing inhibition of eIF2B activity in extracts from serum-starved Schneider cells (45 µg of protein/assay). Nucleotide exchange assays were performed at 30 °C in duplicate and replicated four times. (Shown are data ± S.E. where n = 4.) C, D. melanogaster Schneider cells were starved of serum for 12, 24, 48, or 72 h (as indicated). They were then rescued from starvation by re-addition of serum (10%) 1 h before cells were lysed. Western blot shows the changes in phosphorylation of eIF2alpha upon starvation (-), over the times indicated, and following re-addition (+) of serum (for 1 h). Phosphorylated eIF2alpha was detected using the anti-phospho-eIF2alpha antibody. The lower panels show a Western blot of the samples used in C probed with the D. melanogaster eIF2alpha -specific antibody to show equal loading of protein. 50 µg of cell lysate protein was loaded in each lane. D, guanine nucleotide exchange assay showing the effect of serum starvation (for the times indicated) on eIF2B activity in Schneider cell lysates. E, guanine nucleotide exchange assay showing that readdition of serum for 1 h increases eIF2B activity in serum-starved S2 cells. Assays were performed as described under "Materials and Methods" at 30 °C for 10 min. All assays were performed in duplicate and replicated four times. (Shown are data ± S.E. where n = 4.)

The Phosphorylation of eIF2alpha Increases in Response to ER Stress in D. melanogaster S2 Cells-- The recent identification by sequence homology of the eIF2alpha kinase, PEK, in D. melanogaster (8) and the characterization of its homolog in mammals (7) prompted us to investigate whether eIF2alpha phosphorylation occurred during endoplasmic reticulum stress in D. melanogaster. D. melanogaster S2 cells were incubated for 12 h in the presence and absence of thapsigargin and tunicamycin (agents that interfere with ER function) and then subjected to immunoblotting using the anti-phospho-eIF2alpha antibody. Fig. 9 clearly shows that eIF2alpha phosphorylation is increased upon addition of these agents when compared with control cells.



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Fig. 9.   Endoplasmic reticulum stress induces eIF2alpha phosphorylation in D. melanogaster S2 cells. Western blot showing the effects of thapsigargin and tunicamycin on eIF2alpha phosphorylation in serum-fed D. melanogaster S2 cells (lanes 4 and 5, respectively). Control cell lysate (lane 1), cell lysate from cells starved of serum for 72 h (lane 2), and cells incubated in dimethyl sulfoxide (lane 3) are also shown. The top panel shows eIF2alpha phosphorylation (blot probed with anti-phospho-eIF2alpha antibody), and the lower panel shows eIF2 (blot probed with the D. melanogaster anti-eIF2alpha -specific antibody) as a loading control. (Cell treatment was as described under "Materials and Methods.").



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented here clearly demonstrate that D. melanogaster cells possess eIF2B activity and that this organism contains genes encoding proteins very similar to all five subunits of eIF2B previously identified in other eukaryotes such as yeast and mammals. In particular, we show that the putative alpha -subunit of D. melanogaster eIF2B can functionally replace the endogenous yeast protein in budding yeast and confer regulation on the yeast eIF2B complex by phosphorylation of eIF2alpha , and that the D. melanogaster eIF2Bepsilon protein, when expressed in bacteria or in mammalian cells, catalyzes guanine nucleotide exchange on eIF2. We can therefore conclude that these genes do encode authentic subunits of eIF2B. Furthermore, treatment of D. melanogaster S2 cell extracts with an eIF2alpha kinase to phosphorylate the endogenous eIF2alpha caused near complete inhibition of nucleotide exchange on exogenous eIF2. This shows that phosphorylation of D. melanogaster eIF2 on its alpha -subunit inhibits eIF2B activity as is also the case for eIF2B from budding yeast and from mammals. In addition, when eIF2alpha phosphorylation was increased within S2 cells as a consequence of serum starvation, inhibition of eIF2B activity was again observed. Thus, D. melanogaster possesses an eIF2B complex, which can be regulated by eIF2alpha phosphorylation in vivo. The sequence data also strongly indicate that the subunits of the complex are closely related to the corresponding orthologs from other eukaryotes. Chefalo et al. (53) have previously reported that the eIF2B activity detected in extracts of Sf9 (S. frugiperda) cells was inhibited when the eIF2 kinase HRI was expressed in those cells. This suggested that eIF2B in insect cells was sensitive to inhibition by phosphorylation of eIF2, consistent with our present data.

The earlier data (15) suggested that D. melanogaster did not possess an eIF2B-like factor. This was based partly on the fact that, under some conditions (e.g. at elevated temperature or using partially purified eIF2 preparations), GDP/GTP exchange occurred freely on D. melanogaster eIF2, without a requirement for eIF2B. Consistent with this, under these conditions, exchange was not affected by raising the concentration of Mg-ions (15). It is not clear why temperature should have this effect of rendering exchange independent of eIF2B, but in the case of the partially purified material it is very likely that these effects were due to the presence in the eIF2 fractions of eIF2B, which copurifies with eIF2 from D. melanogaster cells (this work) and those of other species (see, e.g., Ref. 32). The presence of eIF2B in the substrate eIF2 would overcome the inhibitory effect of magnesium ions and eliminate the need for added eIF2B. The presence of sufficient (e.g. stoichiometric) amounts of eIF2B could also explain the observation that phosphorylation of eIF2alpha did not inhibit exchange under the specific conditions mentioned.

The epsilon -subunit of D. melanogaster eIF2B contains a consensus sequence for phosphorylation by GSK-3 (as is also the case for the factor from all mammals for which eIF2Bepsilon has so far been sequenced). We show that D. melanogaster eIF2Bepsilon is indeed phosphorylated by GSK-3 in vitro, and that the activity of partially purified D. melanogaster eIF2B is inhibited by in vitro phosphorylation by GSK-3beta . The ability of GSK-3 to phosphorylate eIF2Bepsilon that has been expressed in E. coli seems surprising given that it should not be phosphorylated in the priming site, which is normally a prerequisite for phosphorylation of eIF2Bepsilon (47, 48) and several other substrates by GSK-3 (54-56). However, this is also seen for the mammalian factor expressed in this system and reflects the presence in our GSK-3 preparations (expressed in the baculovirus system) of a kinase that can phosphorylate the priming site of the mammalian protein.4 The D. melanogaster GSK-3 homolog Shaggy has a similar substrate specificity to mammalian GSK-3 (26); in particular, it can efficiently phosphorylate peptides based on the sequence around the putative GSK-3 site in D. melanogaster eIF2Bepsilon . Shaggy is known to play key roles during development in D. melanogaster and other metazoans, and it is possible that some of its effects are exerted via phosphorylation of eIF2B and the regulation of the translation of specific mRNAs. This regulation would be analogous to the control of GCN4 expression in yeast, which involves modulation of the translation of its mRNA mediated by changes in eIF2B activity. It is known that there are upstream open reading frames in some mRNAs in D. melanogaster (see, e.g., Ref. 37).

Our data also show that the activity of eIF2B is subject to regulation in response to serum in S2 cells. Serum withdrawal causes a marked decline in its activity concomitant with an increase in the phosphorylation of eIF2alpha . Resupplying serum led to an increase in eIF2B activity and a fall in eIF2alpha phosphorylation. The data suggest that the changes in eIF2B activity are due to alterations in eIF2alpha phosphorylation, although, as serum restoration did not completely restore eIF2B activity, other regulatory events may also be involved. It is also a possibility that cells starved for long periods of time contain less eIF2B. This would also explain why complete restoration was not achieved. These are the first data showing regulation of eIF2B activity or eIF2alpha phosphorylation in invertebrates.

These findings suggest that serum may regulate the activity of an eIF2alpha kinase in S2 cells. Two eIF2alpha kinases have been described from D. melanogaster, dGCN2 (18) and dPEK (8), and the recently published genome sequence from this organism suggests that they are the only ones. The data of Berlanga et al. (17) indicated that mammalian GCN2 may be activated by serum withdrawal, at least when it is overexpressed in human embryonic kidney 293 cells, suggesting that dGCN2 activity might also be controlled by serum in S2 cells.

We have also reported here that ER stresses induced by thapsigargin, which causes the release of calcium from the ER, and tunicamycin, an inhibitor of protein glycosylation, do cause an increase in eIF2alpha phosphorylation. These data suggest a role for dPEK in D. melanogaster in inhibiting translation in response to ER stress. However, the possibility that only a single eIF2alpha kinase is present and activated by two separate mechanisms cannot be dismissed. Further experimentation will be required to characterize the regulatory roles of these kinases in vivo.

These data demonstrate, using a variety of approaches, that D. melanogaster possesses an eIF2B factor, and regulatory mechanisms to control it, which are similar to those which operate in higher eukaryotes. Subsequent studies will focus on the role of these mechanisms in regulating protein synthesis and thus gene expression in D. melanogaster.


    ACKNOWLEDGEMENTS

We thank Drs. Nick Helps and Andrew Paterson for providing vectors and recombinant GSK-3beta , respectively, and Dr. Jane-Jane Chen for kindly providing HRI.


    FOOTNOTES

* This work was supported by a program grant from the Wellcome Trust (to C. G. P.), a career development award from the Medical Research Council (to G. D. P.), and a studentship from the Biotechnology and Biological Sciences Research Council (to D. D. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains full amino acid sequence information for the D. melanogaster proteins, including alignments with the sequences from other species.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF289100-AF289103.

To whom correspondence should be addressed. Tel.: 44-0-1382-344919; Fax: 44-0-1382-322424; E-mail: c.g.proud@dundee.ac.uk.

Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M008041200

2 Y. L. Woods, P. Cohen, L. E. Campbell, F. E. M. Paulin, and C. G. Proud, unpublished data.

3 X. Wang, unpublished data.

4 Y. L. Woods, L. E. Campbell, P. Cohen, and C. G. Proud, unpublished data.


    ABBREVIATIONS

The abbreviations used are: eIF, eukaryotic initiation factor; GSK, glycogen synthase kinase; ER, endoplasmic reticulum; GST, glutathione S-transferase; HEK, human embryonic kidney; PCR, polymerase chain reaction; 3-AT, 3-amino-1,2,4-triazole; PKR, RNA-activated protein kinase; HRI, heme-regulated inhibitor; PEK, pancreatic eukaryotic initiation factor 2alpha kinase; CK-II, casein kinase II; GCN, general control nonderepressible.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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