(Received for publication, January 3, 1997)
From the Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Eukaryotic initiation factor-2B (eIF-2B) is a
guanine nucleotide exchange factor (GEF) that plays a key role in the
regulation of protein synthesis. In this study, we have used the
baculovirus-infected Sf9 insect cell system to express and characterize
the five dissimilar subunits of rat eIF-2B. GEF activity was detected
in extracts of Sf9 cells expressing the -subunit alone and was
greatly increased when all five subunits were coexpressed. In addition,
high GEF activity was observed in extracts containing a four-subunit
complex lacking the
-subunit. Assembly of an eIF-2B holoprotein was
confirmed by coimmunoprecipitation of all five subunits. Gel filtration chromatography revealed that recombinant eIF-2B had the same molecular mass as eIF-2B purified from rat liver and that it did indeed possess
GEF activity. Phosphorylation of the substrate eIF-2 inhibited the GEF
activity of the five-subunit eIF-2B; this inhibition required the
eIF-2B
-subunit. The results demonstrate that eIF-2B
functions as
a regulatory subunit that is not required for GEF activity, but instead
mediates the regulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2B
is necessary and is perhaps sufficient for GEF
activity in vitro.
Protein synthesis in eukaryotic cells is regulated in response to growth factors, hormones, and changes in the external environment (reviewed in Refs. 1 and 2). One of the major control points for protein synthesis is the initiation of mRNA translation, which is mediated by a number of eukaryotic initiation factors (eIFs)1 (recently reviewed in Ref. 3). Translation of mRNA begins with the binding of initiator Met-tRNAi to the 40 S ribosomal subunit and is mediated by eIF-2 as part of the eIF-2·GTP·Met-tRNAi ternary complex. During the initiation process, the GTP bound to eIF-2 is hydrolyzed, and a binary complex consisting of eIF-2 and GDP is released from the 80 S initiation complex. Since eIF-2 has a 100-400-fold higher affinity for GDP than for GTP, the guanine nucleotide exchange factor (GEF) known as eIF-2B is required to regenerate the GTP-bound form of eIF-2, which can then participate in another cycle of translation initiation.
In cells subjected to a variety of stresses, e.g. heat
shock, serum and amino acid deprivation, and heme deprivation in
reticulocytes, protein synthesis is inhibited by a mechanism involving
phosphorylation of the -subunit of eIF-2 (reviewed in Ref. 4).
Phosphorylated eIF-2 efficiently binds to eIF-2B, but cannot undergo
nucleotide exchange and therefore competitively inhibits the GEF
function of eIF-2B, which is present in limiting amounts in all cell
types examined (5-7). In addition, eIF-2B may be regulated by other mechanisms since its activity is modulated under certain conditions in
the absence of a change in phosphorylation of eIF-2
(8-12). The
-subunit of eIF-2B is phosphorylated in vitro by glycogen synthase kinase-3, casein kinase-1, and casein kinase-2, which may
alter the GEF activity of the holoprotein (13-16). In addition, the
activity of eIF-2B appears to be regulated by allosteric effectors, e.g. NADP+, NAD+, and ATP inhibit
guanine nucleotide exchange, whereas NADPH, NADH, and polyamines
activate exchange (17-20).
Most GEFs, such as those stimulating GDP dissociation from the Ras
superfamily of proteins, exist as single subunit proteins (21, 22). In
contrast, eIF-2B is a complex protein consisting of five dissimilar
subunits. The cDNAs for all five subunits of eIF-2B have been
cloned from both mammals (23-26) and yeast (27). Alignment of the
coding regions of mammalian and yeast subunits indicates sequence
homology for the mammalian -,
-,
-,
-, and
-subunits
with the yeast GCN3, GCD7, GCD1, GCD2, and GCD6 subunits, respectively.
Genetic studies in yeast indicate that deletion of the
-subunit of
eIF-2B (GCN3) does not affect viability and therefore is not an
essential component of the GCD complex under normal growth conditions
(28). However, there is controversy over the requirement of the
-subunit for eIF-2B function since a recent report has suggested
that purified rabbit eIF-2B lacking the
-subunit does not have GEF
activity (29).
The current model for translation initiation in eukaryotic cells is formulated from biochemical studies of cell-free systems derived from rabbit reticulocytes and from genetic experiments conducted in yeast. In the study reported here, we set out to determine if the baculovirus expression system could be used as a new approach to study the structure/function relationship of translation initiation factors, many of which are composed of multiple subunits with unknown functions. For example, despite the fact that the mammalian eIF-2B complex has been available in purified form for 15 years and all five yeast and mammalian eIF-2B subunits have now been cloned, relatively little is known about the function of individual subunits or the mechanism of nucleotide exchange. Here we report our studies on the expression and coexpression of rat eIF-2B subunits in Sf9 insect cells using recombinant baculoviruses and the characterization of GEF activity in extracts of cells expressing these proteins.
Mouse monoclonal antibodies against the - and
-subunits of eIF-2B were produced using purified protein for antigen
as described previously (30). For immunoblot analysis, a monoclonal
antibody against the
-subunit of eIF-2B was used in conjunction with
a rabbit anti-FLAG polyclonal antibody (Santa Cruz Biotechnology) or a
mouse anti-FLAG M2 monoclonal antibody (Kodak Scientific Imaging
Systems) to detect the eIF-2B
-subunit and the
-,
-,
-, and
-subunits of eIF-2B harboring an amino-terminal FLAG marker
octapeptide (DYKDDDDK), respectively.
The
full-length clone for the rat eIF-2B (GenBankTM/EMBL
accession number U83914[GenBank]) used in this study was obtained by
screening the Stratagene rat brain Uni ZAPXR cDNA library
(catalogue number 936515) using the rabbit eIF-2B
cDNA (31) as a
probe (kindly provided by Dr. C. G. Proud, University of Kent,
Canterbury, United Kingdom). The complementary DNA clones encoding rat
eIF-2B
-,
-,
-, and
-subunits were previously described
(23-26), and their GenBankTM/EMBL accession numbers are U05821[GenBank],
Z48225[GenBank], U38253[GenBank], and U19511[GenBank], respectively.
To facilitate identification and quantitation of rat eIF-2B subunits in
Sf9 cells, sequences encoding a common 8-amino acid FLAG epitope
(DYKDDDDK) were added to the cDNAs encoding the -,
-,
-,
and
-subunits by polymerase chain reaction using the Pfu
DNA polymerase (Stratagene). The polymerase chain reaction products
were cloned into the SrfI site of the plasmid pCR-Script Cam
SK(+) according to instructions supplied by the manufacturer (Stratagene). The eIF-2B
cDNA had an internal BamHI
site that was removed using the QuikChange site-directed mutagenesis
kit (Stratagene). The primers used to remove the BamHI site
resulted in a single nucleotide substitution that did not alter the
amino acid sequence of the protein. The identities of all clones were confirmed by sequence analysis of the 5
- and 3
-regions using the
reverse primer and T7 primers, which bind to the polylinker region of
the pCR-Script plasmid. The full-length eIF-2B
, eIF-2B
, eIF-2B
, and eIF-2B
cDNAs were next subcloned out of the
pCR-Script plasmid and into the baculovirus transfer vector pAcUW51
(Pharmingen) containing the baculovirus p10 and polyhedrin promoters.
Specifically, the cDNAs for the
- and
-subunits containing
the FLAG epitope were sequentially subcloned into the BamHI
and BglII sites, respectively, of pAcUW51. The cDNAs for
the
- and
-subunits were sequentially subcloned into the
BamHI and BglII sites, respectively, of pAcUW51. A baculovirus transfer vector encoding the FLAG-tagged eIF-2B
cDNA was constructed by isolating an
NcoI/Acc65I fragment encoding the full-length
cDNA fragment from pCR-Script and then ligating the fragment into
the corresponding sites downstream of the polyhedrin promoter of the
pAcSG2 transfer vector (Pharmingen). A baculovirus transfer vector that
expressed the FLAG-tagged
-subunit alone was constructed by
subcloning the eIF-2B
cDNA into the BglII site of
pAcUW51.
Sf9 insect cells (American Type Culture Collection CRL-1711) were placed in culture using Grace's insect medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Hyclone Laboratories), 0.1% Pluronic F-68, 2 mM glutamine, and 20 µg/ml gentamicin. To generate recombinant baculoviruses expressing the combinations of eIF-2B subunits described above, BaculoGold Autographa californica nuclear polyhedrosis virus DNA (BaculoGold AcNPV, Pharmingen) and each of the recombinant transfer vectors were cotransfected into Sf9 cells, and recombinant baculoviruses were isolated by plaque purification as described previously (32). Recombinant plaques expressing eIF-2B subunits were identified by infecting 2 × 106 cells with the plaque supernatant. Cell lysates were prepared 72 h later and screened for protein production by immunoblot analysis.
For expression of eIF-2B subunits, 6 × 106 Sf9 insect cells were plated in 100-mm dishes. The Sf9 cells were then infected with recombinant baculovirus(es) at a multiplicity of infection of 10 in a reduced volume of 3.0 ml for 1 h, fed with 11 ml of medium, and then lysed at 60 h post-infection. As a control, Sf9 insect cells were also infected with wild-type AcNPV baculovirus.
Immunoprecipitation and ImmunoblottingFor
immunoprecipitation assays, 6 × 106 infected cells
were washed once in ice-cold phosphate-buffered saline and then lysed in 700 µl of lysis buffer consisting of 1% Nonidet P-40, 20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM phenymethylsulfonyl fluoride,
0.15 unit/ml aprotinin, and 20 mM leupeptin. Cells were vortexed in lysis buffer and incubated on ice for 15 min. Insoluble material was removed by centrifugation at 4 °C for 10 min at
16,000 × g. Immunoprecipitation assays were performed
by incubating lysates with 250 µl of a monoclonal antibody against
eIF-2B at 4 °C overnight. Goat anti-mouse Biomag IgG beads
(PerSeptive Diagnostics) were used to collect the antigen-antibody
complexes. Prior to use, the beads were washed in 1% nonfat dry milk
in Buffer A (20 mM Tris-HCl, pH 7.4, 5 mM EDTA,
0.04%
-mercaptoethanol, 0.5% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 50 mM NaF, 50 mM
-glycerophosphate, 0.1 mM phenymethylsulfonyl fluoride,
1 mM benzamidine, and 0.5 mM sodium vanadate)
containing 150 mM NaCl. The beads were then captured using
a magnetic stand and washed twice with Buffer A containing 50 mM NaCl and once with Buffer A containing 150 mM NaCl. Protein bound to the beads was eluted in 100 µl
of SDS sample buffer, and the sample was incubated at 100 °C for 5 min. The beads were collected by centrifugation, and proteins in the
sample were separated by electrophoresis as described below.
Immunoprecipitates of cell lysates prepared as described above were
resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and were
electrophoretically transferred to 0.2-µm pore size nitrocellulose membranes (Bio-Rad). Membranes were then blocked with 2% bovine serum
albumin (Fraction V) in Tris-buffered saline, pH 8.0, for 1 h;
washed in TBST (Tris-buffered saline, pH 8.0, containing 0.2% Tween
20); and probed overnight at 4 °C with primary antibody diluted in
TBST. The rabbit and mouse anti-FLAG antibodies were diluted 1:2500
(0.04 µg/ml final concentration) and 1:2000 (1.5 µg/ml final
concentration), respectively. The filters were then washed in TBST,
probed for 1 h with a horseradish peroxidase-coupled secondary
antibody (Amersham Corp.) diluted 1:20,000 in TBST, washed again in
TBST, and developed using enhanced chemiluminescence (Amersham Corp.).
To detect the eIF-2B -subunit, the blots were reprobed with a
monoclonal antibody against rat eIF-2B
diluted 1:100, reprobed with
secondary antibody, and developed as described above.
Infected Sf9 cells (6 × 106) were washed once in ice-cold phosphate-buffered saline and lysed in 1.0 ml of ice-cold extraction buffer (45 mM HEPES, pH 7.4, 0.375 mM magnesium acetate, 0.075 mM EDTA, 95 mM potassium acetate, 2.5 mg/ml digitonin, and 10% (v/v) glycerol). The lysates were clarified by centrifugation at 10,000 × g for 10 min at 4 °C. The resulting supernatants were immediately assayed for eIF-2B activity as determined by the exchange of [3H]GDP bound to eIF-2 for unlabeled GDP as described previously (33). The labeled binary complex eIF-2·[3H]GDP was prepared by incubating tubes containing rat liver eIF-2 (~95% pure (34)) and [3H]GDP (2.5 mM, 10.9 Ci/mmol) in 80 µl of assay buffer (62.5 mM MOPS, pH 7.4, 125 mM KCl, 1.25 mM dithiothreitol, and 0.2 mg/ml bovine serum albumin) at 30 °C for 10 min. The Mg2+ concentration was adjusted to 2 mM, and the binary complex was stored on ice before use.
To measure eIF-2B activity, assay buffer containing a 100-fold excess of GDP, 1.25-40 µl of cell lysate, and 2 mM Mg2+ was added to a tube, followed by 1-2 pmol of labeled binary complex. The mixture was then incubated at 30 °C for 0, 2, 4, or 6 min. The exchange reaction was measured as a decrease in the eIF-2-mediated binding of [3H]GDP to nitrocellulose filters with time.
eIF-2For the
experiments shown in Fig. 6, eIF-2 was phosphorylated using the
eIF-2 kinase HCR by incubating 7.5 µg of pure eIF-2 in 37.5 µl
of HCR kinase buffer (20 mM Tris-HCl, pH 7.4, 100 mM KCl, 10% (v/v) glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 2.5 mM magnesium chloride,
and 0.1 mM ATP) with 7.5 µl (~0.5 µg) of purified
rabbit reticulocyte HCR (35). The mixture was incubated at 37 °C for
30 min, and the reaction was terminated by placing the tube on ice. An
equal volume of kinase assay buffer containing the phosphatase
inhibitors NaF (100 µM final concentration) and microcystin (2 µM final concentration) was added to each
tube. A similar reaction containing eIF-2 without HCR was processed identically for use as the unphosphorylated eIF-2 control. The phosphorylation state of eIF-2
was monitored by isoelectric focusing gel electrophoresis and immunoblot analysis. To measure eIF-2B activity, assay buffer containing phosphorylated or unphosphorylated eIF-2, a 100-fold excess of GDP, 1.25-40 µl of cell lysate, and 2 mM Mg2+ was added to a plastic tube and
incubated at 30 °C for 2 min. The eIF-2·[3H]GDP
complex (1-2 pmol) was then added to each tube, and the mixture was
incubated at 30 °C. Aliquots of the reaction mixture were removed
from the tubes at 0, 2, 4, or 6 min and filtered through nitrocellulose
filters as described above.
Gel Filtration of eIF-2B Complexes
Infected Sf9 cells
(6 × 106) were washed once with ice-cold
phosphate-buffered saline and then lysed in 0.25 ml of ice-cold buffer
consisting of 45 mM HEPES, pH 7.4, 0.375 mM
magnesium acetate, 0.075 mM EDTA, 95 mM
potassium acetate, 2.5 mg/ml digitonin, and 10% (v/v) glycerol. The
extracts were clarified by centrifugation at 10,000 × g for 10 min at 4 °C and loaded onto a Superose 6 gel
filtration column (Pharmacia Biotech Inc.) pre-equilibrated with buffer
consisting of 20 mM HEPES, pH 7.6, 0.1 mM EDTA,
1 mM dithiothreitol, 10% glycerol, and 150 mM
KCl. Fractions (0.5 ml) were collected, and the absorbance at 280 nm
was measured. Aliquots (0.02 ml) of each fraction containing protein
were assayed for GEF activity as described above. To confirm the
presence of eIF-2B subunits in fractions, 25 µl of 2 × SDS
sample buffer were added to 25-µl aliquots of each fraction, and 15 µl of the mixture were resolved by SDS-PAGE and visualized by
immunoblotting with anti-FLAG and anti-eIF-2B antibodies as
described above.
To facilitate
detection, a common epitope consisting of the FLAG octapeptide
(DYKDDDDK) was added to the N terminus of the eIF-2B -,
-,
-,
and
-subunits. Recombinant baculoviruses for each of the FLAG-tagged
eIF-2B subunits were constructed so as to express the individual
subunit cDNAs under the control of either the polyhedrin promoter
or the p10 promoter. To reduce the number of baculoviruses needed to
express all five subunits of eIF-2B in Sf9 cells, single baculoviruses
encoding either the
- and
- or
- and
-subunits were
generated using a transfer vector designed for multigene expression.
Expression of the largest eIF-2B subunit (eIF-2B
) was achieved using
a transfer vector designed for single gene expression.
To investigate the relative expression/coexpression of eIF-2B subunits,
Sf9 cells were (i) singly infected with baculoviruses encoding
individual eIF-2B subunits or (ii) triply infected with baculoviruses
encoding all five eIF-2B subunits. The recombinant eIF-2B proteins
expressed in Sf9 cells 60 h post-infection were resolved by
SDS-PAGE and identified by immunoblot analysis using anti-FLAG and
anti-eIF-2B monoclonal antibodies. Similar amounts of expressed
eIF-2B
-,
-,
-, and
-subunit proteins were observed for Sf9
cells singly infected with baculoviruses encoding the
-subunit (Fig.
1A, lane 1), the
- and
-subunits (lane 2), and the
- and
-subunits
(lane 3). For Sf9 cells infected with all three viruses,
coexpression of proteins for each of the five eIF-2B subunits was
observed by immunoblot analysis (Fig. 1A, lane
4). However, in these experiments, the amount of eIF-2B
produced could not be directly compared with the amount of the other
four subunits because a different antibody was used to detect eIF-2B
than was used to detect the other four subunits. To our knowledge, this
is the first time a complex multisubunit translation initiation factor
has been overexpressed using the baculovirus-infected Sf9 cell
system.
GEF Activity of Rat eIF-2B Subunits Expressed in Sf9 Cells
It
was important to establish whether or not rat eIF-2B subunits expressed
in Sf9 cells exhibited GEF activity. The GDP dissociation assay is a
direct measurement of the GEF activity of eIF-2B since, in the presence
of Mg2+, the rate-limiting step of the nucleotide exchange
reaction is GDP dissociation (36). Cell lysates were prepared from Sf9
cells infected with viruses encoding pairs of subunits or the three viruses encoding all five subunits of eIF-2B. The extracts were then
assayed for GEF activity by determining the percentage of [3H]GDP released from eIF-2·[3H]GDP
binary complexes over a 6-min interval (Fig. 1B). A low basal level of GEF activity was detected in extracts of cells infected
with wild-type AcNPV baculovirus or in extracts of cells infected with
baculoviruses encoding either the - and
- or
- and
-subunits of eIF-2B (Fig. 1B). Significantly more GEF
activity was detected in lysates from cells expressing the eIF-2B
-subunit (i.e. 40% of the bound [3H]GDP
was released from eIF-2 over 6 min) compared with lysates from cells
infected with wild-type AcNPV baculovirus. The amount of GEF activity
detected in extracts of triply infected cells coexpressing all five
eIF-2B subunits was markedly greater (~90% of [3H]GDP
bound to eIF-2 was released) than that in extracts of cells expressing
eIF-2B
alone, suggesting that a functional, five-subunit eIF-2B
complex was formed in the cells.
As mentioned above, extracts of cells expressing the eIF-2B -subunit
alone exhibited enhanced GEF activity compared with extracts of cells
infected with wild-type virus. Therefore, we next determined if the
-subunit was required for the GEF activity observed in extracts of
cells expressing all five subunits of eIF-2B. When cells were doubly
infected with viruses expressing a four-subunit complex lacking
eIF-2B
, no GEF activity was observed (Fig.
2B). In contrast, the rate of GDP exchange
observed in extracts of cells expressing a four-subunit complex lacking
eIF-2B
was greatly enhanced compared with control extracts. These
results demonstrate that the eIF-2B
-subunit is required for the GEF activity of eIF-2B, whereas the eIF-2B
-subunit is not.
GEF Activity in Extracts of Cells Expressing the eIF-2B
The activity observed for the five-subunit eIF-2B complex
in Figs. 1B and 2B was out of the linear range of
the assay, and therefore, it was necessary to titrate lysates to
determine the activity of the -subunit alone relative to the highly
active five- and four-subunit eIF-2B complexes. The results show that 1.25 µl of lysate from cells expressing all five subunits of eIF-2B or only the
-,
-,
-, and
-subunits yielded a rate of
exchange similar to that observed for 40 µl of lysate from cells
expressing the
-subunit alone (Fig. 3). Because the
amount of
-subunit expressed in each case was similar, the results
indicate that the five-subunit and four-subunit (
,
,
, and
) eIF-2B complexes were ~40-fold more active than the eIF-2B
-subunit alone.
Formation of the eIF-2B Holoprotein in Sf9 Cells
The enhanced
rate of GDP exchange observed in extracts of cells expressing all five
subunits of eIF-2B suggested that the coexpressed subunits were
interacting to form a functional multisubunit complex. To examine
eIF-2B complex formation in insect cells, we utilized a monoclonal
antibody against the -subunit of rat eIF-2B previously shown to
immunoprecipitate the five-subunit eIF-2B complex isolated from rat
liver (30). In these studies, extracts of Sf9 cells expressing all five
subunits of eIF-2B were immunoprecipitated using the anti-eIF-2B
monoclonal antibody, and the resulting immunoprecipitates were resolved
by SDS-PAGE. The proteins in the gel were visualized by immunoblotting
with a rabbit polyclonal antibody against the FLAG epitope and reprobed with a monoclonal antibody against eIF-2B
. All five subunits were
effectively coimmunoprecipitated with the anti-eIF-2B
monoclonal antibody (Fig. 4B, lane 1),
indicating that the subunits interact to form a complex in insect
cells. As a control for antibody specificity, Sf9 cells were doubly
infected so as to express the
-,
-,
-, and
-subunits in the
absence of the
-subunit (Fig. 4B, lane 2). In
the absence of the
-subunit, none of the eIF-2B subunits were
immunoprecipitated by the anti-eIF-2B
monoclonal antibody. In
addition, in the absence of the anti-eIF-2B
monoclonal antibody, none of the subunits were detected in the precipitate. Thus, in Sf9
cell extracts containing all five rat eIF-2B subunits, the subunits
associate to form a stable complex that can be detected by
coimmunoprecipitation.
Extracts of triply infected Sf9 cells expressing either four (,
,
, and
) or all five rat eIF-2B subunits were chromatographed on a
Superose 6 gel filtration column to examine the molecular mass and
activity of the recombinant multisubunit complexes. As shown in Fig.
5, GEF activity cofractionated with the multisubunit complex and exhibited an apparent molecular mass similar to that observed for eIF-2B isolated from rat liver. In contrast, when extracts
of cells expressing eIF-2B
alone were fractionated, GEF activity
eluted at a much lower apparent molecular mass than observed for the
protein purified from rat liver. The latter result suggests that the
GEF activity observed in cells expressing eIF-2B
alone is not the
result of rat eIF-2B
binding to the endogenous Sf9 cell eIF-2B
-,
-,
-, and/or
-subunit to form an active complex. Rather, the
results suggest that the eIF-2B
-subunit alone exhibits GEF
activity.
Effect of eIF-2
In vivo, the GEF activity of eIF-2B is
competitively inhibited by phosphorylation of the -subunit of eIF-2
by the protein kinases PKR, HCR, and GCN2. Genetic analyses and
transfection studies with mammalian eIF-2 kinases in yeast indicate
that the inhibitory effects of eIF-2
phosphorylation are mediated in
part by the
-subunit of eIF-2B (37, 38). However, the studies conducted in yeast provide an indirect measurement of eIF-2B activity since the parameter used is the induction of the histidine biosynthetic pathway in response to eIF-2
phosphorylation. The expression of
recombinant eIF-2B subunits in Sf9 cells provided an opportunity, for
the first time, to directly measure the effect of eIF-2
phosphorylation on eIF-2B activity. We therefore examined the effects
of eIF-2
phosphorylation on the activity of four-subunit (
,
,
, and
) and five-subunit rat eIF-2B protein complexes produced in
Sf9 insect cells. GEF assays were conducted on the cell extracts
following preincubation of the extract with either unphosphorylated
eIF-2 or eIF-2 phosphorylated by HCR in vitro. As shown in
Fig. 6, extracts of cells expressing either four eIF-2B
subunits (
,
,
, and
) or the five-subunit complex exhibited
similar rates of GDP exchange, and in both cases, the rate of exchange
was significantly greater than in extracts of cells infected with
wild-type virus. The rate of GDP exchange measured both in extracts of
cells expressing all five subunits and in extracts of cells infected
with wild-type virus was greatly reduced when extracts were
preincubated with phosphorylated eIF-2 compared with preincubation with
unphosphorylated eIF-2 (Fig. 6). In contrast, the activity of the
eIF-2B complex lacking the
-subunit was not inhibited by
phosphorylated eIF-2. The amount of eIF-2
in the phosphorylated form
at the end of the assay was the same for each condition (data not
shown), demonstrating that the lack of inhibition observed with cell
extracts expressing the four-subunit eIF-2B complex (
,
,
, and
) was not the result of dephosphorylation of eIF-2
during the
assay. The lack of inhibition of the four-subunit eIF-2B complex by
phosphorylated eIF-2 demonstrates, for the first time, a regulatory
role for the eIF-2B
-subunit using a biochemical assay.
Sf9 cells are useful for the production of biologically active recombinant proteins because the proteins they express appear to undergo proper folding and correct post-translational modifications (39). Therefore, we were interested in testing the utility of Sf9 cells to study eIF-2B as the insect cell system has been used successfully to produce active recombinant GEFs for the Ras superfamily of proteins (40, 41). An important feature of Sf9 cells is that they can be simultaneously infected with multiple viruses, and this allows the coexpression of multiple gene products. In the study described here, we focused on expressing all five subunits of rat eIF-2B in insect cells via baculovirus infection.
The multisubunit structure of eIF-2B is more complex than most GEFs,
which consist of predominantly single subunit proteins. The complex
structure of eIF-2B suggests that it may contain several distinct
structural and functional domains. We found that expression of the
eIF-2B -subunit alone resulted in detectable GDP exchange compared
with extracts of cells infected with wild-type virus. In addition, GEF
activity was found to cofractionate with the
-subunit during gel
filtration chromatography. In contrast, we have not been able to detect
any GEF activity following purification of eIF-2B
using an anti-FLAG
immunoaffinity resin (data not shown). It is possible that the
conditions used during the purification resulted in inactivation of the
protein. Nonetheless, we feel that the assumption that eIF-2B
alone
manifests GEF activity must be viewed with caution until further proof
validating the activity of the individual subunit is obtained.
By coinfecting insect cells with a combination of three recombinant
baculoviruses, we were able to simultaneously express all five subunits
of rat eIF-2B in Sf9 insect cells and to show that the expressed
complex exhibited GEF activity. The presence of the eIF-2B -subunit
was required for GEF activity, supporting a catalytic role for this
subunit. This result is consistent with the previous observation (42)
that the guanine nucleotide exchange activity of purified eIF-2B could
be blocked by monoclonal antibodies recognizing the
-subunit. The
formation of an intact eIF-2B complex in coinfected insect cells was
confirmed by coimmunoprecipitation and gel filtration assays.
In this study, we also used the insect cell expression system to
explore the role of eIF-2B in the regulation of eIF-2B activity. Studies in yeast suggest that a four-subunit eIF-2B complex lacking eIF-2B
is functional since deletion of GCN3 (eIF-2B
) has no effect on cell growth under normal conditions (27). In contrast, Craddock and Proud (29) recently reported that purified rabbit eIF-2B
lacking eIF-2B
was not functional and suggested that mammalian eIF-2B may therefore differ from yeast eIF-2B in the requirement for
the
-subunit. By overexpressing combinations of rat eIF-2B subunits
in Sf9 cells, we show that the
-subunit of eIF-2B is not required
for GEF activity. Furthermore, using gel filtration chromatography, we
were able to confirm that the GEF activity in Sf9 cell lysates
containing the four-subunit eIF-2B complex (lacking eIF-2B
) was due
to the formation of an active four-subunit complex. It is not clear why
the
-subunit was missing from certain purified rabbit eIF-2B
preparations reported by Craddock and Proud (29). One interpretation is
that the absence of the
-subunit in the rabbit eIF-2B preparations
might indicate modifications in the other eIF-2B subunits that were not
observed, but that might be inhibiting the activity of the purified
four-subunit eIF-2B complex.
The -subunit of eIF-2 is phosphorylated at Ser51 by
three different protein kinases (PKR, HCR, and GCN2), and this
phosphorylation event is an important mechanism used by cells to
control translation initiation in response to a variety of stresses
(3). In addition, recent evidence suggests that at least one of the
eIF-2
kinases, PKR, may have a potential role as a tumor suppressor
in normal cells and function in the restraint of cell growth (43). In yeast, deletion of the eIF-2B
-subunit (GCN3) suppresses the lethal
effects from a constitutively active GCN2 kinase or the growth
inhibitory effects resulting from high level exogenous expression of
mammalian eIF-2
kinases (HCR and PKR) (37, 44, 45). The genetic and
expression studies in yeast provide indirect evidence that the GEF
activity of eIF-2B lacking the
-subunit is insensitive to
phosphorylation of eIF-2
. Phosphorylated eIF-2 has an increased
affinity for eIF-2B and acts as a competitive inhibitor since the
complex formed does not undergo GDP/GTP exchange. The inhibition of
eIF-2B activity impairs translation initiation by reducing the
availability of ternary complexes
(eIF-2·GTP·Met-tRNAi). In this report, we provide
direct evidence that the effect of eIF-2 phosphorylation is indeed
mediated by the
-subunit of eIF-2B because the GEF activity of a
four-subunit eIF-2B complex lacking eIF-2B
was insensitive to
eIF-2
phosphorylation.
Finally, the baculovirus-infected insect cell expression system may
prove useful for determining the mechanism by which eIF-2B catalyzes
the GEF reaction on eIF-2. At least two possible mechanisms have been
proposed for the eIF-2B-catalyzed guanine nucleotide exchange reaction
(46). One possible model is the substituted enzyme (ping-pong)
mechanism, which involves the sequential release of GDP and the binding
of GTP to eIF-2 catalyzed by eIF-2B. An alternative mechanism is based
upon kinetic analysis of the GDP exchange reaction and involves complex
formation between all four components of the exchange reaction (eIF-2,
GDP, eIF-2B, and GTP). The four-component mechanism implies that eIF-2B
contains a binding site for GTP. Dholakia et al. (47)
reported the binding of a photoreactive 8-azido analog of GTP to the
-subunit of eIF-2B and therefore suggested that eIF-2B is a
GTP-binding protein. The GTP binding data are difficult to reconcile
with the amino acid sequences of yeast or mammalian eIF-2B subunits
since none of the subunits harbor a consensus sequence for GTP binding.
Future studies using different combinations of normal and mutant
versions of individual eIF-2B subunits will be required to further
delineate their functional roles in the guanine nucleotide exchange
reaction.
We are grateful to Lynne Hugendubler for technical assistance.