From the Department of Cellular and Molecular
Physiology, Pennsylvania State University, College of Medicine,
Hershey, Pennsylvania 17033 and the ¶ Laboratory of Eukaryotic
Gene Regulation, NICHD, National Institutes of Health,
Bethesda, Maryland 20892
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ABSTRACT |
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The guanine nucleotide exchange activity of eIF2B
plays a key regulatory role in the translation initiation phase of
protein synthesis. The activity is markedly inhibited when the
substrate, i.e. eIF2, is phosphorylated on
Ser51 of its -subunit. Genetic studies in yeast
implicate the
-,
-, and
-subunits of eIF2B in mediating the
inhibition by substrate phosphorylation. However, the mechanism
involved in the inhibition has not been defined biochemically. In the
present study, we have coexpressed the five subunits of rat eIF2B in
Sf9 cells using the baculovirus system and have purified the
recombinant holoprotein to >90% homogeneity. We have also expressed
and purified a four-subunit eIF2B complex lacking the
-subunit. Both
the five- and four-subunit forms of eIF2B exhibit similar rates of
guanine nucleotide exchange activity using unphosphorylated eIF2 as
substrate. The five-subunit form is inhibited by preincubation with
phosphorylated eIF2 (eIF2(
P)) and exhibits little exchange activity
when eIF2(
P) is used as substrate. In contrast, eIF2B lacking the
-subunit is insensitive to inhibition by eIF2(
P) and is able to
exchange guanine nucleotide using eIF2(
P) as substrate at a faster
rate compared with five-subunit eIF2B. Finally, a double point mutation
in the
-subunit of eIF2B has been identified that results in
insensitivity to inhibition by eIF2(
P) and exhibits little exchange
activity when eIF2(
P) is used as substrate. The results provide the
first direct biochemical evidence that the
- and
-subunits of
eIF2B are involved in mediating the effect of substrate
phosphorylation.
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INTRODUCTION |
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Regulation of translation initiation plays an important role in the control of gene expression in eukaryotic cells (reviewed in Ref. 1). By modulating different steps in the initiation pathway, regulation of the translation of mRNAs coding for specific classes of proteins as well as regulation of overall mRNA translation can be achieved. The initiation pathway is composed of a number of discrete steps involving at least 12 unique proteins referred to as eukaryotic initiation factors, or eIFs.1 Of the many steps in translation initiation, only two are thought to be important in regulating the process in vivo. The two steps include the sequential binding of first mRNA and then initiator methionyl-tRNAi (met-tRNAi) to the 40 S ribosomal subunit. In the latter step, eIF2 binds to the 40 S ribosomal subunit as a ternary complex with GTP and met-tRNAi. With formation of the 80 S initiation complex, the GTP is hydrolyzed and eIF2 is released as an eIF2·GDP binary complex. The GDP bound to eIF2 must then be exchanged for GTP, a reaction catalyzed by a guanine nucleotide exchange protein, termed eIF2B (reviewed in Ref. 2). In contrast to most guanine nucleotide exchange proteins, which are usually small and consist of a single subunit, eIF2B is composed of five dissimilar subunits present in equimolar amounts in a heteropentameric complex. Although eIF2B has been available in purified form from mammalian cells for 15 years (3), no information is available about the role of the individual subunits in catalyzing the guanine nucleotide exchange reaction. Furthermore, little is known about the role of the individual subunits in mediating regulation of the exchange activity.
The best characterized means of regulating eIF2B activity involves
phosphorylation of the -subunit of its substrate, eIF2, on
Ser51. Phosphorylation of eIF2
converts eIF2 from a
substrate into a competitive inhibitor (reviewed in Ref. 1). Based on
genetic studies in Saccharomyces cerevisiae, it has been
concluded that the eIF2B
-,
-, and
-subunits are important in
mediating the effect of substrate inhibition of eIF2B (4, 5). In
S. cerevisiae deprived of amino acids, eIF2
becomes
phosphorylated, leading to increased translation of a protein termed
GCN4 (reviewed in Refs. 6 and 7). Although not established
experimentally, it has been assumed that eIF2B activity in yeast is
inhibited in response to eIF2
phosphorylation and that the
inhibition of eIF2B activity is responsible for the increased
translation of GCN4 mRNA. This assumption is based on the finding
that deletion of the
-subunit or point mutations identified in the
-,
-, and
-subunits of eIF2B prevent the increase in
translation of GCN4 mRNA in response to amino acid deprivation (4)
without having any effect on cellular growth in nonstarved cells. In
addition, overexpression of the eIF2B
-,
-, and
-subunits
leads to formation of a stable eIF2B subcomplex that overcomes the
inhibitory effect of high level eIF2 phosphorylation, presumably
through a mechanism involving sequestration of the phosphorylated eIF2
by the eIF2B subcomplex (5). The biochemical basis for the apparent
insensitivity to eIF2
phosphorylation in cells expressing mutant
forms of eIF2B subunits is unknown.
In the present study, the five subunits of rat eIF2B were co-expressed
in Sf9 cells using the baculovirus expression system. A
functional, five-subunit eIF2B complex was purified from the cells. In
addition, a four-subunit complex lacking the -subunit was expressed
and purified. Both the four- and five-subunit forms of eIF2B exhibited
similar specific activities, indicating that the
-subunit of the
protein is not required for optimal guanine nucleotide exchange
activity. However, whereas the four-subunit form of eIF2B was not
inhibited by eIF2(
P), the five subunit form was. Furthermore, the
exchange activity using eIF2(
P) as substrate was greater for four-
than five-subunit eIF2B. Finally, the
-subunit containing a double
point mutation corresponding to mutations identified in yeast was
expressed in combination with the other four subunits of eIF2B. The
sensitivity of eIF2B containing the mutant
-subunit to inhibition by
eIF2(
P) was similar to that observed for eIF2B lacking the
-subunit. However, unlike eIF2B lacking the
-subunit, the ability
of eIF2B containing the mutant
-subunit to catalyze GDP exchange
using eIF2(
P) was the same as wild-type eIF2B. Overall, the results
provide the first biochemical evidence of the regulatory role of the
- and
-subunits in mediating inhibition of exchange activity by
substrate phosphorylation. In addition, they show that the
-subunit
of eIF2B is not required for exchange activity.
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MATERIALS AND METHODS |
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Expression of the Five Subunits of Rat eIF2B in Sf9
Cells--
High titer stocks of baculoviruses encoding the wild-type
eIF2B /
,
/
, and
subunits were generated as described
previously (8). For coexpression of eIF2B subunits, 2 × 108 Sf9 insect cells were infected for 1 h in a
reduced volume of 15 ml containing each of the three different virus
stocks at a multiplicity of infection of 2-5 for each virus. The
infected Sf9 cells were then transferred to a 250-ml Erlenmeyer
flask containing 85 ml of SF-900 serum-free medium (Life Technologies,
Inc.) and were maintained in culture at 28 °C using an orbital
shaker (100 rpm). At 72 h after infection, 1.0-ml aliquots were
removed and centrifuged in Eppendorf tubes at 2000 rpm for 3 min and
pellets were stored at
70 °C until lysis.
Construction of the E311K,L315Q Mutant Form of
eIF2B--
Site-directed mutagenesis (Altered Sites mutagenesis
kit, Promega) was utilized to introduce specific changes to the
eIF2B
cDNA. Oligonucleotide GDP140 (5'-AAT TGC CTG AGA TGC
CTG CAC AAT CTT CTT TTG CAC ATA CCG) was used
to change Glu311 and Leu315 to Lys and Gln,
respectively (eIF2B
-E311K,L315Q), generating pAV1057. Plasmid
pAV1153 (eIF2B
-E311K,L315Q) was created by subcloning the mutated
eIF2B
590-base pair BamHI-NdeI fragments from
pAV1057 into identically cleaved J203 (also called
pAc-2B
FLAG/
FLAG). Nucleotide sequencing of the subcloned 590-base
pair fragment confirmed that the plasmid contained only the expected
site-directed nucleotide substitutions (underlined in the above
oligonucleotide sequence).
Purification of Recombinant eIF2B from Sf9 Cells--
The
proteins expressed in Sf9 cells were immunoaffinity purified by
chromatography on a matrix containing an immobilized anti-FLAG monoclonal antibody (Anti-FLAG M2 Affinity Gel; IBI/Kodak). Briefly, the cells were lysed as described previously (8), and the lysate was
centrifuged at 10,000 × g for 10 min at 4 °C. The
supernatant was mixed with 2 ml of affinity matrix for 2 h at
4 °C, and the mixture was then poured into a plastic column. The
column was washed with 30 ml of buffer B, followed by 30 ml of buffer C
(20 mM Tris, pH 8.0, 150 mM NaCl), and the
bound protein was eluted with 200 µg/ml FLAG octapeptide in buffer C. The protein was then concentrated using a Millipore Biomax 50K
centrifugal concentrator and stored in aliquots at 70 °C.
Measurement of eIF2B Activity--
The guanine nucleotide
exchange activity of eIF2B was measured as described previously (9)
using eIF2 purified from rat liver as substrate. In some assays, eIF2B
(0.5 µg) was preincubated for 2 min at 37 °C with 1 µg of either
unphosphorylated eIF2 or eIF2 phosphorylated on Ser51 of
the -subunit using the eIF2
kinase, HCR (10). The guanine nucleotide exchange activity of eIF2B was then measured as the exchange
of [3H]GDP bound to eIF2 for nonradiolabeled GDP with
time.
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RESULTS |
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The five rat eIF2B subunits were coexpressed in Sf9 cells
using the baculovirus expression system as described previously (8).
Each of the recombinant proteins was expressed with an amino-terminal
octapeptide extension referred to as FLAG to aid in purification. As
shown in the left panel of Fig.
1, a single immunoaffinity purification
step utilizing an anti-FLAG monoclonal antibody coupled to a solid
matrix resulted in isolation of an approximately equimolar mixture of
the five eIF2B subunits at a purity of greater than 90%. Likewise,
expression of just the eIF2B -,
-,
-, and
-subunits yielded
a complex lacking the
-subunit (Fig. 1, right panel). The
four-subunit complex lacking the
-subunit will be hereafter referred
to as eIF2B(
).
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The specific activities of eIF2B and eIF2B() were compared in a
guanine nucleotide exchange assay using eIF2·[3H]GDP as
substrate. As shown in Fig. 2, exchange
activities were the same when equal amounts of eIF2B and eIF2B(
)
were added to the assay. The results show that the
-subunit of eIF2B
is not required for exchange activity.
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In a previous study, incubation of Sf9 cell extracts expressing
rat eIF2B with eIF2 phosphorylated with the eIF2 kinase, HCR,
resulted in a decrease in exchange activity compared with extracts
incubated with unphosphorylated eIF2 (8). In contrast, extracts of
cells expressing eIF2B(
) showed no decrease in exchange activity
when incubated with eIF2 phosphorylated on the
-subunit. In the
present study, purified eIF2B and eIF2B(
) were incubated with
either unphosphorylated eIF2 or eIF2(
P) prior to assay. As shown in
the inset to Fig. 3
(lane 1), there was no detectable unphosphorylated eIF2
in the phosphorylated eIF2 preparation. Similarly, no phosphorylated
eIF2
was detected in the unphosphorylated preparation (Fig. 3,
lane 2). In confirmation of the results obtained previously
with crude cell extracts, incubation of purified eIF2B with eIF2(
P)
(Fig. 3, open symbols) prior to assay resulted in a
substantial decrease in exchange activity compared with incubation with
unphosphorylated eIF2 (Fig. 3, closed symbols). In contrast, the activity of eIF2B(
) was nearly unaffected by phosphorylated eIF2.
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The sensitivity of the exchange activity of eIF2B and eIF2B() to
eIF2 phosphorylation was further examined using an
eIF2(
P)·[3H]GDP complex as substrate. As shown in
the top panel of Fig. 4, both
eIF2B and eIF2B(
) catalyzed GDP exchange using phosphorylated eIF2, although the exchange activity using eIF2(
P) as substrate (open symbols) was significantly slower than that observed
using an equimolar amount of unphosphorylated
eIF2·[3H]GDP (closed symbols). In addition,
eIF2B(
) catalyzed GDP exchange using eIF2(
P) as substrate at a
significantly faster rate than did five subunit eIF2B. The exchange
activity observed was not influenced by substrate dephosphorylation
since the amount of eIF2(
P) was the same at the beginning and end of
the assay (Fig. 4, bottom panel). Likewise, the difference
in exchange activity between the four- and five-subunit forms of eIF2B
was not due to substrate dephosphorylation.
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Pavitt et al. (4) recently described nine eIF2B mutations
that yielded a phenotype similar to that observed in cells lacking eIF2B
, i.e. increased eIF2
phosphorylation was not
accompanied by reduced cell growth rates and increased GCN4
translation. The results suggested that, like eIF2B lacking the
-subunit, eIF2B with any one of these point mutations in the
-subunit should be resistant to inhibition by eIF2(
P). Four of
the amino acids that were found to be mutated in yeast eIF2B
are
conserved in the amino acid sequence of the rat protein (4). Of the
conserved residues, substitutions of Glu377 and
Leu381 with Lys and Gln, respectively, yielded a phenotype
exhibiting the least apparent sensitivity to substrate phosphorylation.
When a double mutation was made in yeast eIF2B
combining these two substitutions, the phenotype observed was identical to the phenotype of
the L381Q single mutation. Therefore, in the present study, the same
double mutation was made in rat eIF2B
and the mutant protein was
coexpressed with the other four wild-type subunits. Recombinant eIF2B
containing the mutant
-subunit (referred to hereafter as
eIF2B(
*)) was purified from Sf9 cells as described above for
wild-type eIF2B. Unexpectedly, over 50% of the mutant eIF2B
was
degraded during the purification procedure, even in the presence of a
mixture of eight different protease inhibitors. Because of the
difficulty in obtaining purified eIF2B(
*) with equimolar amounts of
all five subunits, the exchange activity of eIF2B(
*) was assessed in
cell extracts rather than with the purified protein. As shown in the
inset to Fig. 5, no
degradation of eIF2B
occurred during preparation of extracts from
cells expressing either the wild-type or mutant protein. It can also be
seen that the amount of expressed protein was essentially the same for
each of the five. As observed using purified eIF2B, incubation of
extracts of Sf9 cells expressing all five wild-type eIF2B
subunits with eIF2(
P) prior to assay (Fig. 5, open
symbols) significantly reduced exchange activity compared with
extracts incubated with unphosphorylated substrate (Fig. 5,
closed symbols). In addition, preincubation of extracts of
cells expressing eIF2B(
) with either eIF2(
P) or eIF2 resulted
in little difference in exchange activity. Similar to the results
observed for eIF2B(
), the exchange activity of eIF2B(
*) was
only minimally inhibited by preincubation with eIF2(
P). The results
show directly for the first time that both the
- and
-subunits of
eIF2B are important in mediating the inhibition of exchange activity by
eIF2(
P).
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Finally, the exchange activity of eIF2B(*) was examined using
phosphorylated eIF2 as substrate. As seen in Fig.
6, both wild-type eIF2B and eIF2B(
*)
catalyzed GDP exchange using eIF2(
P) as substrate (open
symbols), although the activity was substantially lower using
phosphorylated compared with unphosphorylated eIF2 (closed symbols). However, unlike eIF2B lacking the
-subunit, the
exchange activity using phosphorylated eIF2 as substrate was nearly the same for eIF2B(
*) as for wild-type eIF2B. Thus, although eIF2B(
*) was less sensitive to the inhibitory effect of eIF2(
P) on nucleotide exchange using unphosphorylated eIF2 as substrate (Fig. 5), it remained
largely incapable of using phosphorylated eIF2 as substrate.
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DISCUSSION |
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Regulation of translation initiation through phosphorylation of
the -subunit of eIF2 occurs in response to a variety of stimuli including deprivation of amino acids (10-12), glucose (12), purines (13), or serum (12). Phosphorylation of eIF2
does not directly inhibit formation of either the eIF2·GTP·Met-tRNAi
ternary complex or the 43 S preinitiation complex (i.e. a 40 S ribosomal subunit associated with eIF-1A, eIF-3, and the eIF2 ternary
complex), as these reactions proceed efficiently in vitro
with the phosphorylated factor (14). Instead, phosphorylation of the
-subunit of eIF2 is thought to impede eIF2B activity by sequestering
eIF2B into an inactive complex. Three lines of evidence support this
assumption. (a) eIF2B reportedly does not catalyze GDP
exchange on eIF2(
P) (15); (b) eIF2(
P) displaces
unphosphorylated eIF2 bound to eIF2B but unphosphorylated eIF2 does not
(16); and (c) eIF2(
P) inhibits the activity of eIF2B in
the presence of low concentrations of substrate (i.e.
<10-fold molar excess of eIF2·GDP to eIF2(
P)) but at higher
substrate concentrations the inhibition caused by eIF2(
P) is
negligible (15), suggesting that eIF2(
P) is acting as a competitive
inhibitor of eIF2B.
Phosphorylation of eIF2 plays a critical role in the general control
response in S. cerevisiae. Starvation of S. cerevisiae for any one of at least 10 amino acids leads to
phosphorylation of eIF2
and increased translation of the mRNA
coding for the transcription factor GCN4 (reviewed in Refs. 6 and 7).
The latter effect is dependent upon the presence of eIF2B (reviewed in
Refs. 7 and 17). In yeast, it has been suggested that three of the five
subunits of eIF2B are involved in recognition of the phosphorylation
status of eIF2
. In particular, deletion of eIF2B
has no effect on
cellular growth under nonstarvation conditions (18). However, eIF2B
is required for induction of GCN4 translation under amino acid
starvation conditions (19) and the induction of GCN4 is dependent upon
phosphorylation of eIF2
(20). Moreover, deletion of eIF2B
reduces
the growth-inhibitory effect of high level eIF2 phosphorylation
catalyzed by overexpression of the human double-stranded RNA-activated
eIF2
protein kinase, PKR (21). A more recent study has identified
point mutations in the
-subunit of eIF2B that are even more
effective than deletion of the subunit in reversing the effects of eIF2
phosphorylation on translation and growth (4). These results suggest
that the primary function of the
-subunit of eIF2B is to mediate the
inhibitory effects of eIF2
phosphorylation on exchange activity.
The yeast eIF2B - and
-subunits exhibit regions of amino acid
sequence similarity to eIF2B
(22), suggesting that these other two
subunits might also be involved in regulating the activity of eIF2B in
response to eIF2
phosphorylation. In support of this suggestion,
overexpression in yeast of the eIF2B
-,
-, and
-subunits together results in formation of a stable subcomplex in vivo
whose presence neutralizes the effects of eIF2 phosphorylation on
translation (5). It was proposed that this subcomplex can sequester
eIF2(
P) and permit native five-subunit eIF2B to catalyze guanine
nucleotide exchange on unphosphorylated eIF2. In addition, point
mutations have been identified in both the
- and
-subunits of
yeast eIF2B that result in the expression of the same phenotype as is
observed in cells in which eIF2B
has been deleted, i.e.
phosphorylation of eIF2
does not result in increased translation of
GCN4 (4, 23). However, eIF2B activity has not been measured in extracts of S. cerevisiae deprived of amino acids and the mechanism
involved in the putative change is still speculative.
The mutations identified in yeast eIF2B -,
-, and
-subunits
could lead to the observed phenotype through several distinct mechanisms. For example, the mutations could lead to an overall increase in the specific activity of eIF2B, be permissive for guanine
nucleotide exchange using phosphorylated eIF2 as substrate, or decrease
the affinity of eIF2B for eIF2
(P) such that it is no longer a
competitive inhibitor of eIF2B. In the present study, rat eIF2B lacking
the
-subunit had the same specific activity as the wild-type
protein. Although the specific activity of eIF2B(
*) could not be
directly measured, the rate of guanine nucleotide exchange was similar
in extracts of Sf9 cells expressing the five wild-type eIF2B
subunits and cells expressing a mutant form of the
-subunit in
combination with the remaining four wild-type subunits. Assuming that
the catalytic properties of yeast eIF2B are similar to the rat protein,
the results suggest that the phenotype observed in yeast lacking
eIF2B
or expressing mutant forms of eIF2B
in response to
increased eIF2
phosphorylation is not a result of an overall
increase in the specific activity of eIF2B. In addition, the results of
the present study suggest that, although wild-type eIF2B can catalyze
GDP exchange using eIF2(
P) as substrate and eIF2B(
) catalyzes
GDP exchange using eIF2(
P) as substrate at a faster rate than
wild-type eIF2B, the difference in rate observed between wild-type
eIF2B and eIF2B(
) using eIF2(
P) as substrate may not be
sufficient to account for the phenotype observed in yeast in response
to phosphorylation of eIF2
. In contrast, both eIF2B(
) and
eIF2B(
*) appear to be completely resistant to inhibition by
eIF2(
P) when the proteins are incubated with eIF2(
P) prior to
assay using unphosphorylated eIF2 as substrate. This result suggests
that the affinity for eIF2(
P) is significantly less for the mutant
forms of eIF2B than for the wild-type protein.
In summary, the present study provides the first biochemical
demonstration that both the - and
-subunits of eIF2B play
important roles in regulating the guanine nucleotide exchange activity
of the protein in response to phosphorylation of eIF2
. In
particular, eIF2B lacking the
-subunit or containing a mutant form
of the
-subunit is completely resistant to inhibition by eIF2(
P).
Finally, lack of the
-subunit allows for faster GDP exchange using
eIF2(
P)·[3H]GDP as substrate.
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ACKNOWLEDGEMENT |
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We acknowledge the excellent technical assistance of Lynne Hugendubler.
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FOOTNOTES |
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* This work was supported in part by Grants DK13499 and DK15658 from the National Institutes of Health.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.
§ To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, P. O. Box 850, Hershey, PA 17033. Tel.: 717-531-8970; Fax: 717-531-7667; E-mail: skimball{at}psu.edu.
1 The abbreviations used are: eIF, eukaryotic initiation factor; HCR, heme-controlled repressor.
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REFERENCES |
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