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• W2068492010 abstract "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 eIF2α. Here we have clonedDrosophila 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 eIF2Bα confers regulation of eIF2B function in yeast, while eIF2Bε shows guanine nucleotide exchange activity. In common with mammalian eIF2Bε, D. melanogaster eIF2Bε is phosphorylated by glycogen synthase kinase-3 and casein kinase II. Phosphorylation of partially purifiedD. 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 eIF2α, showing the insect factor is regulated similarly to eIF2B from other species. In S2 cells, serum starvation increases eIF2α phosphorylation, which correlates with inhibition of eIF2B, and both effects are reversed by serum treatment. This shows that eIF2α phosphorylation and eIF2B activity are under dynamic regulation by serum. eIF2α 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. 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 eIF2α. Here we have clonedDrosophila 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 eIF2Bα confers regulation of eIF2B function in yeast, while eIF2Bε shows guanine nucleotide exchange activity. In common with mammalian eIF2Bε, D. melanogaster eIF2Bε is phosphorylated by glycogen synthase kinase-3 and casein kinase II. Phosphorylation of partially purifiedD. 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 eIF2α, showing the insect factor is regulated similarly to eIF2B from other species. In S2 cells, serum starvation increases eIF2α phosphorylation, which correlates with inhibition of eIF2B, and both effects are reversed by serum treatment. This shows that eIF2α phosphorylation and eIF2B activity are under dynamic regulation by serum. eIF2α 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. eukaryotic initiation factor glycogen synthase kinase endoplasmic reticulum glutathione S-transferase human embryonic kidney polymerase chain reaction 3-amino-1,2,4-triazole RNA-activated protein kinase heme-regulated inhibitor pancreatic eukaryotic initiation factor 2α kinase casein kinase II general control nonderepressible 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 (1Hinnebusch A.G. J. Biol. Chem. 1997; 272: 21661-21664Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 2Webb B.L.J. Proud C.G. Int. J. Biochem. Cell Biol. 1998; 29: 1127-1131Crossref Scopus (77) Google Scholar, 3Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 139-172Google Scholar). 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 (α–ε) (2Webb B.L.J. Proud C.G. Int. J. Biochem. Cell Biol. 1998; 29: 1127-1131Crossref Scopus (77) Google Scholar). 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 α-subunit (3Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 139-172Google Scholar). Phosphorylated eIF2α acts as a potent competitive inhibitor of eIF2B, and, as cellular levels of eIF2 generally exceed those of eIF2B, low levels of eIF2α phosphorylation can cause substantial inhibition of eIF2B. Recent data suggest that the α-, β-, and δ-subunits of eIF2B interact with eIF2 and that these subunits are required to sensitize eIF2B to inhibition by this mechanism (4Pavitt G.D. Ramaiah K.V.A. Kimball S.R. Hinnebusch A.G. Genes Dev. 1998; 12: 514-526Crossref PubMed Scopus (213) Google Scholar, 5Pavitt G. Yang W. Hinnebusch A.G. Mol. Cell Biol. 1997; 17: 1298-1313Crossref PubMed Scopus (107) Google Scholar). To date, a number of eIF2α 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 eIF2α 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 (1Hinnebusch A.G. J. Biol. Chem. 1997; 272: 21661-21664Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). GCN2 homologs have been described in mammals and in Drosophila melanogaster (6Shi Y. Vattem K.M. Sood R. Liang J. Stramm L. Wek R.C. Mol. Cell. Biol. 1998; 17: 7499-7509Crossref Google Scholar, 7Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2558) Google Scholar, 8Sood R. Porter A.C. Ma K. Quilliam L.A. Wek R.C. Biochem. J. 2000; 346: 281-293Crossref PubMed Scopus (134) Google Scholar), suggesting analogous regulatory mechanisms may operate in these species. Recently, a new eIF2α kinase, pancreatic eukaryotic initiation factor-2α 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 eIF2α kinases (heme-regulated inhibitor (HRI; Ref. 9Chen J.J. London I.M. Trends Biochem. Sci. 1995; 20: 105-108Abstract Full Text PDF PubMed Scopus (265) Google Scholar), interferon-induced double-stranded RNA-activated protein kinase (PKR; Ref. 10Clemens M.J. Elia A. J. Interferon Cytokine Res. 1997; 17: 503-524Crossref PubMed Scopus (520) Google Scholar), general control nonderepressible 2 (GCN2; Ref. 11Sood R. Porter A.C. Olsen D. Cavener D.R. Wek R.C. Genetics. 2000; 154: 787-801Crossref PubMed Google Scholar), and pancreatic eukaryotic initiation factor 2α kinase (PEK, also termed PERK; Refs. 6Shi Y. Vattem K.M. Sood R. Liang J. Stramm L. Wek R.C. Mol. Cell. Biol. 1998; 17: 7499-7509Crossref Google Scholar and 7Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2558) Google Scholar)), which are generally activated under conditions of cellular stress (e.g. viral infection, disruption of endoplasmic reticulum function, and heme deprivation (Ref. 3Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 139-172Google Scholar)). 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 (12Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (638) Google Scholar). 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 (13Mateu M.G. Maroto F.G. Vicente O. Sierra J.M. Biochim. Biophys. Acta. 1989; 1007: 55-60Crossref PubMed Scopus (5) Google Scholar, 14Mateu M.G. Sierra J.M. Eur. J. Biochem. 1987; 165: 507-513Crossref PubMed Scopus (10) Google Scholar). However, Mateu and colleagues (13Mateu M.G. Maroto F.G. Vicente O. Sierra J.M. Biochim. Biophys. Acta. 1989; 1007: 55-60Crossref PubMed Scopus (5) Google Scholar, 15Mateu M.G. Vicente O. Sierra J.M. Eur. J. Biochem. 1987; 162: 221-229Crossref PubMed Scopus (12) Google Scholar) 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 (14Mateu M.G. Sierra J.M. Eur. J. Biochem. 1987; 165: 507-513Crossref PubMed Scopus (10) Google Scholar). The sequence of eIF2α 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 eIF2α (16Qu S. Cavener D.R. Gene (Amst.). 1994; 140: 239-242Crossref PubMed Scopus (13) Google Scholar). D. melanogaster eIF2α can be phosphorylated in vitro by reticulocyte HRI, and this phosphorylation was shown to inhibit guanine nucleotide exchange by mammalian eIF2B (13Mateu M.G. Maroto F.G. Vicente O. Sierra J.M. Biochim. Biophys. Acta. 1989; 1007: 55-60Crossref PubMed Scopus (5) Google Scholar, 14Mateu M.G. Sierra J.M. Eur. J. Biochem. 1987; 165: 507-513Crossref PubMed Scopus (10) Google Scholar). Phosphorylation of the α-subunit of D. melanogaster eIF2 at residue Ser50 in vivo has never been examined. However, two eIF2α kinases have been identified in D. melanogaster. A D. melanogaster ortholog of theS. cerevisiae GCN2p kinase has been identified and characterized (17Berlanga J.J. Santoyo J. de Haro C. Eur. J. Biochem. 1999; 265: 754-762Crossref PubMed Scopus (221) Google Scholar, 18Olsen D.S. Jordan B. Chen D. Wek R.C. Cavener D.R. Genetics. 1998; 149: 1485-1509Google Scholar). Complementation experiments ingcn2-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, aD. melanogaster ortholog of the mammalian PEK has been identified through sequence homology (8Sood R. Porter A.C. Ma K. Quilliam L.A. Wek R.C. Biochem. J. 2000; 346: 281-293Crossref PubMed Scopus (134) Google Scholar). 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 eIF2α phosphorylation in this organism. Another mechanism by which eIF2B can be regulated in mammalian systems is the phosphorylation of its ε-subunit (eIF2Bε) by glycogen synthase kinase-3β (GSK-3β). The activity of GSK-3β is known to be modulated in response to insulin, which induces the phosphorylation and inactivation of GSK-3β (19Welsh G.I. Proud C.G. Biochem. J. 1993; 294: 625-629Crossref PubMed Scopus (351) Google Scholar, 20Saito Y. Vandenheede J.R. Cohen P. Biochem. J. 1994; 303: 27-31Crossref PubMed Scopus (127) Google Scholar, 21Welsh G.I. Stokes C.M. Wang X. Sakaue H. Ogawa W. Kasuga M. Proud C.G. FEBS Lett. 1997; 410: 418-422Crossref PubMed Scopus (89) Google Scholar). This response occurs concomitantly with the dephosphorylation of the ε-subunit of mammalian eIF2B, at the site of phosphorylation by GSK-3β, causing the activation of eIF2B (22Welsh G.I. Miller C.M. Loughlin A.J. Price N.T. Proud C.G. FEBS Lett. 1998; 421: 125-130Crossref PubMed Scopus (248) Google Scholar). D. melanogaster has a homolog of GSK-3β, Shaggy (23Ruel L. Bourouis M. Heitzler P. Pantesco V. Simpson P. Nature. 1993; 362: 557-560Crossref PubMed Scopus (165) Google Scholar), however, although its role in insulin signaling has not been elucidated. Genetic evidence has indicated that Shaggy acts downstream ofDishevelled in the Wingless pathway inactivatingArmadillo (the D. melanogaster homolog of β-catenin) (24Noordermeer J. Klingensmith J. Perrimon N. Nusse R. Nature. 1994; 367: 80-83Crossref PubMed Scopus (323) Google Scholar), and biochemical evidence suggests Shaggy is downstream of protein kinase C (25Cook D. Fry M.J. Hughes K. Sumpathipala R. Woodgett J.R. Dale T.C. EMBO J. 1996; 15: 4526-4536Crossref PubMed Scopus (344) Google Scholar). A putative phosphorylation site for GSK-3β has been identified in D. melanogaster eIF2Bε, based on sequence homology (26Williams D.D. Marin O. Pinna L.A. Proud C.G. FEBS Lett. 1999; 448: 86-90Crossref PubMed Scopus (19) Google Scholar). 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 eIF2α and eIF2Bε phosphorylation probably exist in D. melanogaster. However, neither nucleotide exchange (eIF2B) activity nor the phosphorylation of eIF2α has been demonstrated in vivo in D. melanogaster. Given the recent discoveries of eIF2α kinases in this species, establishing that eIF2α 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 eIF2α phosphorylation in vitro and in vivo. We identify cDNAs encoding all five subunits of D. melanogaster eIF2B (α, β, γ, δ, and ε), and have cloned cDNAs encoding the α-, β-, γ-, and ε-subunits. We have also characterized the functions of the α- and ε-subunits. We also report that eIF2α phosphorylation occurs in a regulated mannerin vivo, that GSK-3β phosphorylates eIF2Bε in vitro, and that eIF2B activity can be inhibited in vitro by GSK-3β. eIF2B and its regulation in this species appear to be similar to other eukaryotic organisms that have so far been studied. 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 [γ-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 fromPromega, 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-3β 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). 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) (27Schneider I. J. Embryol. Exp. Morph. 1972; 27: 363-365Google Scholar). S2 cell used in guanine nucleotide exchange assays were harvested (at a density of 8 × 106 cells/ml) in lysis buffer containing 100 mmTris, pH 7.6, 50 mm β-glycerophosphate, 0.5 mm sodium orthovanadate (Na3VO4), 1.5 mm EGTA, 0.1% Triton, 0.1 mmdithiothreitol, 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 × 106cells/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. SDS-polyacrylamide gel electrophoresis was performed using gels containing 12.5% acrylamide and 0.1%N,N′-methylene-bis-acrylamide (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). 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 eIF2α (αD eIF2Bα) or an antibody against a peptide with the sequence GMILLSELSpRRRIRIN (where Sp denotes a phosphoseryl residue) corresponding to the phosphorylation site in eIF2α (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. 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 (29Mehta H.B. Woodley C.L. Wahba A.J. J. Biol. Chem. 1983; 258: 3438-3441Abstract Full Text PDF PubMed Google Scholar, 30Kleijn M. Welsh G.I. Scheper G.C. Voorma H.O. Proud C.G. Thomas A.A.M. J. Biol. Chem. 1998; 273: 5536-5541Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). 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 mmdithiothreitol 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 (31Proud C.G. Pain V.M. FEBS Lett. 1982; 143: 55-59Crossref PubMed Scopus (20) Google Scholar). Purification of eIF2, as a substrate for eIF2B assays, was carried out as described (32Oldfield S. Proud C.G. Eur. J. Biochem. 1992; 208: 73-81Crossref PubMed Scopus (71) Google Scholar) 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 mmdithiothreitol, protease inhibitors (leupeptin, aprotinin, pepstatin, and benzamidine; all 1 μg/ml), and 0.1 mmphenylmethylsulfonyl fluoride. Extract was then loaded on to a Fast Flow Q-Sepharose column and eluted using a continuous gradient of KCl (from 100 mm to 1 m in a total of 20 ml). Fractions (0.5 ml) were collected. Sequence homology searches were performed using the BLAST program (33Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) 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 α-subunit, or genomic sequence (from the D. melanogaster sequencing project for the others). The β-subunit sequence is encoded within locus DMC100G10.3 (accession no. AL023874), and the ε-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 γ-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 δ-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 eIF2Bδ (see “Results”). Where appropriate, oligonucleotide primers were then designed and used to PCR amplify cDNAs from the Nicholas Brown cDNA library (34Brown N.H. Kafatos F.C. J. Mol. Biol. 1988; 203: 425-437Crossref PubMed Scopus (510) Google Scholar). 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 α-subunit was the cloned (in frame) into pET28c(+) (Novagen), to produce a His-tagged eIF2Bα, using Nde I andBam HI (sites for cloning were introduced on the oligonucleotide primers). The cDNA sequence encoding the ε-subunit was cloned into pGEX-HA (in frame) using Nde I and Xho I for bacterial expression and pcDNA3.1(−)/Myc-His (Invitrogen) using Eco RI andHin dIII for expression in human embryonic kidney (HEK) 293 cells. HEK 293 cells were transfected using the calcium phosphate method as described previously (35Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (793) Google Scholar). Cells were harvested 3 days after transfection and lysed in the buffer used for lysing S2 cells (described above). In vitro transcription and translation reactions were performed using the T'n'T reticulocyte lysate system (Promega). 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 anA 600 of 1. Cultures were then cooled on ice for 15 min and induced with 0.5 mmisopropyl-1-thio-β-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. cDNA encodingD. melanogaster eIF2Bα open reading frame was cloned usingMlu I and Nhe I into the yeast expression vector pAV1411 (pGAL-GCN2FH (Ref. 36Gomez E. Pavitt G.D. Mol. Cell. Biol. 2000; 20: 3965-3976Crossref PubMed Scopus (84) Google Scholar)) by standard techniques (36Gomez E. Pavitt G.D. Mol. Cell. Biol. 2000; 20: 3965-3976Crossref PubMed Scopus (84) Google Scholar). The resulting plasmid (pDWD eIF2Bα) was transformed into isogenic yeast strains GP3153 (MAT a leu2–3 leu2–113 ura3–52 trp1-Δ63 gcn3Δ::LEU2) and GP3140 (MAT a leu2–3 leu2–113 ura3–52 trp1-Δ63 gcn2Δ) (5Pavitt G. Yang W. Hinnebusch A.G. Mol. Cell Biol. 1997; 17: 1298-1313Crossref PubMed Scopus (107) Google Scholar). These yeast strains were also transformed with control plasmids expressing yeast GCN3 (Ep69) (38Hannig E.M. Hinnebusch A.G. Mol. Cell. Biol. 1988; 8: 4808-4820Crossref PubMed Scopus (56) Google Scholar) and yeast GCN2 (p722) (39Ramirez M. Wek R.C. Vazquez de Aldana C.R. Jackson B.M. Freeman B. Hinnebusch A. Mol. Cell. Biol. 1992; 12: 5801-5815Crossref PubMed Google Scholar). 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. 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. 1 A) clearly show that S2 cell extracts effectively mediate nucleotide exchange on eIF2, allowing bound [3H]GDP to be replaced by GTP. Pretreatment of S2 cell extracts with the eIF2α kinase HRI led to increased phosphorylation of eIF2 as assessed using an antibody specific for the phosphorylated form of eIF2α (Fig. 1 B). The identity of the band as eIF2α was confirmed by comparison with the positions of mammalian and D. melanogaster eIF2α (probed with antibodies specific for the respective proteins). This confirms the earlier finding that D. melanogaster eIF2α is a substrate for HRI (15Mateu M.G. Vicente O. Sierra J.M. Eur. J. Biochem. 1987; 162: 221-229Crossref PubMed Scopus (12) Google Scholar). PKR was also able to phosphorylate eIF2α 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.1 A). Inhibition of eIF2B activity by eIF2α phosphorylation is a property common to both yeast and mammalian eIF2B. When isolated from mammalian cells, eIF2 and eIF2B tend to copurify with one another through a number of ion-exchange steps (32Oldfield S. Proud C.G. Eur. J. Biochem. 1992; 208: 73-81Crossref PubMed Scopus (71) Google Scholar, 40Feldhoff R.C. Karinch A.M. Kimball S.R. Jefferson L.S. Prep. Biochem. 1993; 23: 363-374PubMed Google Scholar, 41Kimball S.R. Everson W.V. Myers L.M. Jefferson L.S. J. Biol. Chem. 1987; 262: 2220-2227Abstract Full Text PDF PubMed Google Scholar). 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 eIF2α. 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-eIF2α antiserum in the region of the gradient corresponding to 350–450 mm KCl at an apparent molecular mass of 38 kDa (Fig.2 A). These fractions also showed eIF2 activity (Fig. 2 B). When fractions in this region of the gradient were assayed for eIF2B activity, nucleotide-exchange activity was observed in fraction 11 (≡ 490 mm KCl), i.e. just aft" @default.
• W2068492010 created "2016-06-24" @default.
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• W2068492010 title "Characterization of the Initiation Factor eIF2B and Its Regulation in Drosophila melanogaster" @default.
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