From the 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
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ABSTRACT |
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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 eIF2 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 ( 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 To date, a number of eIF2 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 eIF2 Another mechanism by which eIF2B can be regulated in mammalian systems
is the phosphorylation of its Together, these data suggest that a mechanism of guanine nucleotide
exchange and its regulation by eIF2 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 [ 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 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 eIF2 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 TO 1 M 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
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 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- Analysis of DeIF2B Schneider Cell Extracts Display eIF2B Activity, Which Is Inhibited
by Phosphorylation of eIF2
Pretreatment of S2 cell extracts with the eIF2
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 eIF2 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 eIF2
Western blotting revealed a strong signal with the anti-eIF2 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 (
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).
In the case of the
The sequences of the
eIF2B 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
(
eIF2B
D. melanogaster eIF2B
As noted above, the sequence of D. melanogaster eIF2B
Mammalian eIF2B Complementation of Yeast GCN3 by D. melanogaster eIF2B
The D. melanogaster eIF2B The Phosphorylation of eIF2 The Phosphorylation of eIF2 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 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 eIF2 The 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 eIF2 These findings suggest that serum may regulate the activity of an
eIF2 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 eIF2 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.
. 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 eIF2B
confers regulation of
eIF2B function in yeast, while eIF2B
shows guanine nucleotide
exchange activity. In common with mammalian eIF2B
, D. melanogaster eIF2B
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 eIF2
, showing the insect factor is regulated similarly to eIF2B from other species. In S2 cells, serum starvation increases eIF2
phosphorylation, which correlates with inhibition of
eIF2B, and both effects are reversed by serum treatment. This shows
that eIF2
phosphorylation and eIF2B activity are under dynamic
regulation by serum. eIF2
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-
) (2).
-subunit (3). Phosphorylated
eIF2
acts as a potent competitive inhibitor of eIF2B, and, as
cellular levels of eIF2 generally exceed those of eIF2B, low levels of
eIF2
phosphorylation can cause substantial inhibition of eIF2B.
Recent data suggest that the
-,
-, and
-subunits of eIF2B
interact with eIF2 and that these subunits are required to sensitize
eIF2B to inhibition by this mechanism (4, 5).
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 eIF2
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 eIF2
kinase,
pancreatic eukaryotic initiation factor-2
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 eIF2
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 2
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)).
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 eIF2
(16). D. melanogaster eIF2
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
-subunit of D. melanogaster eIF2
at residue Ser50 in vivo has never been
examined. However, two eIF2
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 eIF2
phosphorylation in this organism.
-subunit (eIF2B
) by glycogen
synthase kinase-3
(GSK-3
). The activity of GSK-3
is known to
be modulated in response to insulin, which induces the phosphorylation
and inactivation of GSK-3
(19-21). This response occurs
concomitantly with the dephosphorylation of the
-subunit of
mammalian eIF2B, at the site of phosphorylation by GSK-3
, causing
the activation of eIF2B (22). D. melanogaster has a homolog
of GSK-3
, 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
-catenin) (24), and biochemical evidence suggests Shaggy
is downstream of protein kinase C (25). A putative phosphorylation site
for GSK-3
has been identified in D. melanogaster
eIF2B
, based on sequence homology (26). However, the role of
phosphorylation of this site has not been studied.
and eIF2B
phosphorylation probably exist in D. melanogaster. However, neither
nucleotide exchange (eIF2B) activity nor the phosphorylation of eIF2
has been demonstrated in vivo in D. melanogaster.
Given the recent discoveries of eIF2
kinases in this species,
establishing that eIF2
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 eIF2
phosphorylation in vitro and in
vivo. We identify cDNAs encoding all five subunits of D. melanogaster eIF2B (
,
,
,
, and
), and have cloned
cDNAs encoding the
-,
-,
-, and
-subunits. We have also
characterized the functions of the
- and
-subunits. We also
report that eIF2
phosphorylation occurs in a regulated manner
in vivo, that GSK-3
phosphorylates eIF2B
in
vitro, and that eIF2B activity can be inhibited in
vitro by GSK-3
. eIF2B and its regulation in this species appear
to be similar to other eukaryotic organisms that have so far been studied.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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-3
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).
-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.
(
DeIF2B
) or an antibody against a peptide with the
sequence GMILLSELSpRRRIRIN (where Sp denotes a phosphoseryl residue)
corresponding to the phosphorylation site in eIF2
(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.
-subunit, or genomic sequence (from the D. melanogaster
sequencing project for the others). The
-subunit sequence is encoded
within locus DMC100G10.3 (accession no. AL023874), and the
-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
-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
-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 eIF2B
(see
"Results").
-subunit was the cloned (in frame) into pET28c(+)
(Novagen), to produce a His-tagged eIF2B
, using NdeI and
BamHI (sites for cloning were introduced on the
oligonucleotide primers). The cDNA sequence encoding the
-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).
-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.
Function in Yeast--
cDNA encoding
D. melanogaster eIF2B
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 (pDWDeIF2B
) was transformed into
isogenic yeast strains GP3153 (MATa leu2-3
leu2-113 ura3-52 trp1-
63
gcn3
::LEU2) and GP3140
(MATa leu2-3 leu2-113 ura3-52
trp1-
63 gcn2
) (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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
--
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
eIF2 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-eIF2
antibody
(
eIF2
). Purified mammalian eIF2 (lane
5) and D. melanogaster Schneider cell extract
(lane 6) probed with the D. melanogaster eIF2
-specific antibody (
DeIF2
) is
also shown. Phosphorylation of eIF2
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.
kinase HRI led to
increased phosphorylation of eIF2 as assessed using an antibody specific for the phosphorylated form of eIF2
(Fig. 1B).
The identity of the band as eIF2
was confirmed by comparison with
the positions of mammalian and D. melanogaster eIF2
(probed with antibodies specific for the respective proteins). This
confirms the earlier finding that D. melanogaster eIF2
is
a substrate for HRI (15). PKR was also able to phosphorylate eIF2
in
extracts of Schneider cells (data not shown).
phosphorylation is a property common to both yeast and mammalian eIF2B.
. 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.
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 (
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.
View larger version (36K):
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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 eIF2 . 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.
-
)
(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.
Properties of the eIF2B subunits from D. melanogaster
Sequence identity (similarity) between eIF2B subunits of different
species
View larger version (42K):
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Fig. 3.
Characterization of cDNA sequences
encoding the subunits of D. melanogaster eIF2B.
Features of the amino acid sequences of eIF2B , eIF2B
, and
eIF2B
(A) and of eIF2B
and eIF2B
(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
-,
-,
-, and
-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.
-,
-, and
-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
sequences show similarity to
one another not that is shared with
or
. 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 (
,
, and
) 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 eIF2B
, 18/28 residues are identical/conservatively replaced in this region; for
eIF2B
, the figure is 20/28; and, for eIF2B
, 24/28.
- and
-subunits of eIF2B also show mutual
similarities within a given species. Koonin (46) identified within
eIF2B
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
- and
-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 eIF2B
. The observation (made for the
mammalian and yeast sequences of eIF2B
) 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 eIF2B
, noted by Gomez and Pavitt (Ref. 36)) is also found in D. melanogaster eIF2B
. This therefore remains as the only
completely conserved triplet sequence in eIF2B
sequences. Mutations
at the Asn and Phe residues within this triplet impair the activity of yeast eIF2B (36).
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).
,
, and
) or pBluescript (
), 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
eIF2B
, -
, -
, and -
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
-subunit, we were unable to perform a similar analysis for this polypeptide.
is thought to be the (principal) catalytic subunit of the
eIF2B complex (4, 36, 49). To test whether the D. melanogaster homolog of eIF2B
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.
eIF2B
and, as a control, eIF2B
were each expressed separately
with Myc-His tags in 293 cells (Fig.
4A). To determine whether the
-subunit of D. melanogaster eIF2B possessed guanine
nucleotide-exchange activity, samples of extracts from HEK 293 cells
expressing eIF2B
and corresponding controls, transfected with empty
vector or a vector encoding the noncatalytic
-subunit of eIF2B, were
analyzed in our standard exchange assay (Fig. 4B). Samples
from cells expressing eIF2B
showed substantially enhanced exchange
activity relative to the control (empty vector). Cells expressing
eIF2B
showed no increase in nucleotide-exchange activity relative to
cells transfected with the empty vector, confirming that eIF2B
itself is inactive in nucleotide exchange.
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Fig. 4.
Activity of D. melanogaster
eIF2B expressed in HEK 293 and E. coli. A, Western blot showing the expression of
Myc-His-tagged D. melanogaster eIF2B
(used as the
control, lane 2) and Myc-His-tagged eIF2B
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 eIF2B
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 eIF2B
(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 eIF2B
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.
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
-subunit of eIF2B from D. melanogaster itself has exchange activity as concluded previously.
contains a seryl residue in a similar to position of the GSK-3 site in
mammalian eIF2B
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-3
at least when synthetic
peptides based on this sequence are studied. The priming site in both
mammals and D. melanogaster eIF2B
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
eIF2B
; 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-3
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-3
to test whether they could phosphorylate
D. melanogaster eIF2B
which had been expressed in
E. coli. As shown in Fig.
5A, GSK-3
catalyzed
phosphorylation of the D. melanogaster eIF2B
. No
phosphorylation was observed when GSK-3
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-3
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-3
prior to using it in a
guanine nucleotide exchange assay. Following treatment with GSK-3
,
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-3
-mediated phosphorylation.
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Fig. 5.
Phosphorylation of recombinant D. melanogaster eIF2B by
GSK-3
and CK-II. Recombinant D. melanogaster GST-eIF2B
expressed in E. coli was
purified using glutathione-Sepharose. eIF2B
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
[
-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 eIF2B
by 1 unit of GSK-3
. 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-3
inhibits eIF2B activity. Guanine nucleotide exchange assay
using partially purified D. melanogaster eIF2B
phosphorylated using 1 unit of GSK-3
(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.)
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 eIF2B
made
in E. coli. Recombinant CK-II catalyzed phosphorylation of fly eIF2B
in vitro (Fig. 5B). Phosphorylation
of mammalian eIF2B
by CK-II appears to occur at seryl residues
within an acidic region at the C terminus of the
protein.3 D. melanogaster eIF2B
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.
--
In
yeast cells, amino acid deprivation leads to the activation of the
eIF2
kinase GCN2. In addition to reducing overall translation initiation, phosphorylation of eIF2
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 eIF2B
subunit, also causes a
3-ATS phenotype as it apparently renders yeast eIF2B
insensitive to the normally inhibitory effects of eIF2
phosphorylation on eIF2B-catalyzed guanine-nucleotide exchange (4). It
has been shown previously that mammalian (rat) eIF2B
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.
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 gcn3
strain, D. melanogaster eIF2B
conferred resistance to 3-AT equivalent to
that shown by GCN3. The control plasmid bearing GCN2 remained
3-ATS (Fig. 7,
gcn3
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 eIF2B
can substitute for
GCN3 in yeast eIF2B and restore the normal response to amino acid
starvation. DeIF2B
, like GCN3, did not confer 3-AT
resistance in the gcn2
strain (Fig. 7, right
panel). This shows that 3-AT resistance conferred by
D. melanogaster eIF2B
is dependent on phosphorylation of
eIF2
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 eIF2B complements gcn3
allowing
growth of yeast cells on histidine starvation medium. Plasmids
expressing DeIF2B
, GCN3, or GCN2 were transformed into
yeast strains deleted for gcn3
(GP3153, left
panels) and gcn2
(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.
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 eIF2
increased markedly as revealed using the
antibody against eIF2
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 eIF2
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 eIF2
antibody to assess the level of eIF2
phosphorylation. The data clearly showed that, even after 12 h
without serum, the level of eIF2
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 eIF2
irrespective of its state of
phosphorylation (Fig. 8C). Consistent with this increase in
eIF2
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 eIF2
, which may account for the observed increase in eIF2B activity.
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Fig. 8.
Regulation of eIF2
phosphorylation and eIF2B activity by serum in D. melanogaster Schneider cells. In vivo
phosphorylation of eIF2
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-eIF2
antibody. Phosphorylation of purified eIF2
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 eIF2
upon starvation
(
), over the times indicated, and following re-addition (+) of serum
(for 1 h). Phosphorylated eIF2
was detected using the
anti-phospho-eIF2
antibody. The lower panels
show a Western blot of the samples used in C probed with the
D. melanogaster eIF2
-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.)
Increases in Response to ER Stress
in D. melanogaster S2 Cells--
The recent identification by sequence
homology of the eIF2
kinase, PEK, in D. melanogaster (8)
and the characterization of its homolog in mammals (7) prompted us to
investigate whether eIF2
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-eIF2
antibody. Fig. 9 clearly shows that
eIF2
phosphorylation is increased upon addition of these agents when
compared with control cells.
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Fig. 9.
Endoplasmic reticulum stress induces
eIF2 phosphorylation in D. melanogaster S2 cells. Western blot showing the effects
of thapsigargin and tunicamycin on eIF2
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 eIF2
phosphorylation (blot
probed with anti-phospho-eIF2
antibody), and the lower
panel shows eIF2 (blot probed with the D. melanogaster anti-eIF2
-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
-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 eIF2
, and that the
D. melanogaster eIF2B
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 eIF2
kinase to phosphorylate the endogenous eIF2
caused near complete inhibition of nucleotide exchange on exogenous
eIF2. This shows that phosphorylation of D. melanogaster
eIF2 on its
-subunit inhibits eIF2B activity as is also the case for
eIF2B from budding yeast and from mammals. In addition, when eIF2
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 eIF2
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.
did not inhibit exchange under the
specific conditions mentioned.
-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 eIF2B
has so far been
sequenced). We show that D. melanogaster eIF2B
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-3
. The ability of GSK-3
to phosphorylate eIF2B
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
eIF2B
(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 eIF2B
. 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).
. Resupplying serum led to an increase in
eIF2B activity and a fall in eIF2
phosphorylation. The data suggest
that the changes in eIF2B activity are due to alterations in eIF2
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 eIF2
phosphorylation in invertebrates.
kinase in S2 cells. Two eIF2
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.
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 eIF2
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.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Nick Helps and Andrew
Paterson for providing vectors and recombinant GSK-3,
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
2 kinase;
CK-II, casein kinase II;
GCN, general control
nonderepressible.
![]() |
REFERENCES |
---|
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---|
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3. | Clemens, M. J. (1996) in Translational Control (Hershey, J. W. B. , Mathews, M. B. , and Sonenberg, N., eds) , pp. 139-172, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
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