From the Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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Modulation of protein/protein interaction is an
important mechanism involved in regulation of translation initiation.
Specifically, regulation of the interaction of eIF2 with the guanine
nucleotide exchange factor, eIF2B, is a key mechanism for controlling
translation under a variety of conditions. Phosphorylation of the
-subunit of eIF2 converts the protein into a competitive inhibitor
of eIF2B by causing an increase in the binding affinity of eIF2B for
eIF2. Consequently, it has been assumed that the
-subunit of eIF2 is directly involved in binding to eIF2B. In the present study, eIF2 was
found to bind only to the
- and
-subunits of eIF2B, and eIF2B was
shown to bind only to the
-subunit of eIF2 by far-Western blot
analysis. The binding site on eIF2
for either the eIF2B holoprotein,
or the isolated
- or
-subunits of eIF2B was shown to be located
within approximately 70 amino acids of the C terminus of the protein.
Phosphorylation of the
-subunit of eIF2 did not promote binding of
eIF2B to the isolated subunit. However, it did cause an increase in the
affinity of eIF2B for eIF2. Finally, phosphorylation by protein kinase
A of the
-subunit of eIF2 in the C-terminal portion of the protein
increased the guanine nucleotide exchange activity of eIF2B, whereas
phosphorylation by casein kinase II or protein kinase C was without
effect.
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INTRODUCTION |
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The translation initiation phase of protein synthesis is a complex process mediated by a family of at least 12 proteins collectively referred to as eukaryotic initiation factors (reviewed in Refs. 1-3). Regulation of translation initiation plays a crucial role in maintaining protein homeostasis within cells in response to a variety of hormonal, nutritional, and environmental stimuli. One of the most tightly regulated steps in translation initiation involves two multisubunit proteins referred to as eukaryotic initiation factors eIF2 and eIF2B. During initiation, eIF2 binds GTP and initiator methionyl-tRNAi (Met-tRNAi)1, and the eIF2·GTP·Met-tRNAi ternary complex then binds to a 40 S ribosomal subunit. Upon joining of the 60 S ribosomal subunit, the GTP bound to eIF2 is hydrolyzed to GDP, and eIF2 is released from the ribosome as an eIF2·GDP binary complex. Before eIF2 can participate in another cycle of initiation, the GDP bound to the protein must be exchanged for GTP. This guanine nucleotide exchange reaction is mediated by the heteropentameric protein, eIF2B.
One of the ways through which the activity of eIF2B is regulated occurs
through an unusual mechanism involving phosphorylation of one of the
subunits of its substrate eIF2 (reviewed in Ref. 3). The smallest, or
-subunit, of eIF2 is phosphorylated in response to a variety of
cellular stresses including viral infection, heat shock, heavy metals,
and deprivation of amino acids or serum. Previous studies have reported
that the affinity of eIF2B for eIF2(
P) is increased either 2-fold
(4) or 150-fold (5), depending on the method used to measure the
interaction. An obvious assumption from these studies is that the
-subunit of eIF2 would bind to one or more subunits of eIF2B.
However, even though both proteins have been available in purified form
for more than 15 years, the interprotein subunit interactions have
remained undefined. Yet an understanding of the subunit interactions
between the two proteins is vital not only to understanding the basis
for the inhibition of eIF2B by phosphorylation of eIF2
, but also for delineating the mechanism through which eIF2B mediates guanine nucleotide exchange on eIF2.
In the present study, we have identified the interprotein interactions
among the subunits of eIF2 and eIF2B. Surprisingly, eIF2B was found to
bind not to the -subunit of eIF2, but instead only to the
-subunit. Furthermore, eIF2B was found to bind to a domain within
approximately 70 amino acids of the C terminus of eIF2
.
Phosphorylation of eIF2
did not induce binding of eIF2B to the
isolated
-subunit, but did increase the affinity of eIF2B for the
eIF2 holoprotein. In addition, the
- and
-subunits of eIF2B were
identified as the sites for binding of eIF2. Finally, phosphorylation
of the
-subunit of eIF2 by protein kinase A (PKA) increased the
guanine nucleotide exchange activity of eIF2B.
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EXPERIMENTAL PROCEDURES |
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Materials-- ECL detection reagents and horseradish peroxidase-conjugated, sheep anti-mouse Ig were purchased from Amersham Life Sciences, Inc. PVDF membrane was obtained from Bio-Rad. Casein kinase II (CK-II) was purchased from Boehringer Mannheim. PKA and protein kinase C (PKC) were from Promega.
Far-Western Analysis of the Interaction of eIF2 and
eIF2B--
The subunits of eIF2 and eIF2B were separated by
electrophoresis on SDS-polyacrylamide gels as described previously (6). The proteins were then transferred onto PVDF membranes, and the membranes were blocked in 5% non-fat dry milk powder (ICN) in buffer A
(10 mM Tris·HCl, pH 7.5, 0.5 M NaCl, 0.05%
Tween 20) for 1 h. The membranes were incubated with either
radiolabeled eIF2 (for blots containing eIF2B) or radiolabeled eIF2B
(for blots containing eIF2) overnight at 4 °C. Prior to use as a
probe, eIF2 was radiolabeled by phosphorylation using
[32P]ATP and either heme-controlled repressor (HCR) to
phosphorylate the -subunit or PKA to phosphorylate the
-subunit
(7, 8). eIF2B was either radiolabeled, using phosphorylated
[32P]ATP and a mixture of casein kinase I (CK-I) and
glycogen synthase kinase 3 (GSK-3) to phosphorylate the
-subunit (9,
10), or biotinylated, using a kit (ECL Protein Biotinylation Module) from Amersham Life Sciences, Inc. The blots were washed, incubated with
radiolabeled probe, and then dried and exposed to film. Blots probed
with biotinylated eIF2B were incubated with avidin coupled to
horseradish peroxidase and then developed using an ECL detection kit
from Amersham Life Sciences, Inc.
Expression of the -,
-, and
-Subunits of Rat eIF2 in
Sf9 Insect Cells--
Recombinant baculoviruses containing
cDNAs for the
-,
-, and
-subunits of rat eIF2 were
constructed using the Bac-to-Bac expression system (Life Technologies,
Inc.). All constructs were made in the pFastBac1 vector. Each subunit
of eIF2 was cloned with a 10-amino acid modified FLAG peptide
(DYKDDDDKID) at the N terminus after the initial methionine. The
cDNA for the rat eIF2
subunit (generously provided by Dr.
J. W. B. Hershey, University of California) was cloned into
the BamHI and EcoRI sites of the vector. The
cDNA for the rat eIF2
subunit was cloned into the EcoRI and KpnI sites of the vector. The cDNA
for rat eIF2
was obtained by screening a rat brain cDNA library
(Stratagene) using an expressed sequence-tagged clone (EST78998) with
similarity to human eIF2
, which yielded a partial length cDNA.
The 5
RACE system (Life Technologies, Inc.) was then used to obtain
the 5
-end of the cDNA. A full-length cDNA for eIF2
was
reconstructed and cloned into the EcoRI and KpnI
sites of the vector. The baculovirus constructs were transfected into
Sf9 cells according to the Bac-to-Bac protocol (Life
Technologies, Inc.), and recombinant baculovirus was obtained. Viruses
underwent two rounds of amplification before use for protein
production. Sf9 cells were infected at a multiplicity of
infection of 10 or greater, grown in shaker flasks at 27 °C, and
harvested after 2-3 days. 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 in buffer B (20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol,
1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.5% sodium
deoxychlolate, 2 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 0.15 units/ml aprotinin, 20 µM leupeptin, 0.4 µg/ml pepstatin, and 0.2 mM benzamidine), 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 an Amicon Centricon concentrator and stored in
aliquots at
70 °C.
Phosphorylation of the -Subunit of eIF2--
A binary complex
consisting of purified rat liver eIF2 and [3H]GDP was
formed as described previously (12). Approximately 3 µg of the
eIF2·[3H]GDP complex was then phosphorylated at
30 °C with either CK-II (1 milliunit), PKA (300 units), or PKC (83 milliunits) in a reaction mixture containing 40 mM
Tris·HCl, pH 7.0, 11 mM
-mercaptoethanol, 8%
glycerol, 80 µM phenylmethylsulfonyl fluoride, and 100 µM [32P]ATP (4000 mCi/mmol).
When eIF2 was phosphorylated with PKC, reactions additionally contained
40 µM phosphatidylserine and 0.5 mM
CaCl2.
Measurement of eIF2B Activity-- The activity of eIF2B was measured as described previously (12) except that the eIF2·[3H]GDP binary complex was phosphorylated with CK-II, PKA, PKC, or no added kinase and nonradiolabeled ATP as described above.
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RESULTS |
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The eIF2 and eIF2B holoproteins used in these studies were
purified from rat liver to greater than 95% homogeneity (Fig.
1). Far Western blot analysis was used to
identify the subunits of eIF2B to which eIF2 binds and vice versa. In
the first set of studies, the subunits of eIF2B were separated by
polyacrylamide gel electrophoresis and transferred to a PVDF membrane.
The membrane was then probed with eIF2 that had been phosphorylated
using [32P]ATP and either HCR, to phosphorylate the
-subunit of eIF2, or PKA, to phosphorylate the
-subunit. As shown
in Fig. 2, eIF2 that was radiolabeled on
either the
- or
-subunit bound to both the
- and
-subunits
of eIF2B, with binding to the
-subunit being consistently stronger
than to the
-subunit. No binding of eIF2 to the
-,
-, or
-subunits of eIF2B was detected. In the experiments shown in Fig. 2,
the binding of eIF2 phosphorylated with HCR to eIF2B
appeared to be
weaker than that observed for eIF2 phosphorylated with PKA, suggesting
that phosphorylation of eIF2 on the
-subunit decreased the affinity
of eIF2 for eIF2B
. However, this apparent difference was not
consistent from experiment to experiment.
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In similar experiments, the binding of eIF2B to the subunits of eIF2
was examined by far-Western blot analysis. For these studies, an
aliquot of eIF2 was phosphorylated with HCR with nonradiolabeled ATP,
and phosphorylated and unphosphorylated eIF2 were electrophoresed in
parallel lanes on a polyacrylamide gel. The proteins were transferred to a PVDF membrane, and the membrane was probed with eIF2B that had
either been biotinylated or phosphorylated using [32P]ATP
and a mixture of GSK-3 and CK-I. As shown in Fig. 2, eIF2B bound to the
-subunit of eIF2; no detectable binding of eIF2B to either the
-
or
-subunits of eIF2 was observed. Furthermore, both biotinylated
and phosphorylated eIF2B bound exclusively to eIF2
, indicating that
the method used to label eIF2B did not affect the interaction of the
two proteins. It is noteworthy that phosphorylation of eIF2 by HCR did
not promote binding of eIF2B to eIF2
.
Because the finding that eIF2B did not bind to the -subunit of eIF2
was so unexpected, we used a second method to examine the binding of
the individual subunits of eIF2 to eIF2B. In these experiments, the
subunits of eIF2 were individually expressed in Sf9 cells using
the baculovirus system. Each of the subunits was expressed with an
8-amino acid extension, referred to as FLAG, at the N terminus of the
protein to aid in purification and identification. As shown in the
top panel of Fig. 3,
immunoaffinity purification of the FLAG-tagged eIF2 subunits resulted
in isolation of proteins that were greater than 90% homogeneous. The
purified subunits were used to probe wells of a microtitre plate in
which the eIF2B holoprotein had been immobilized. As shown in the
bottom panel of Fig. 3, only the
-subunit of eIF2
exhibited significant binding to eIF2B. Similar to what was observed in
the far-Western blot analysis, phosphorylation of eIF2
by HCR did
not enhance binding of eIF2
to immobilized eIF2B.
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To identify the region of eIF2 to which eIF2B binds, two deletion
mutants of the eIF2
cDNA were constructed where the C-terminal 65 or 133 amino acids (termed 268 and 200, respectively) were removed
from the coding sequence. As shown in Fig.
4, removal of the C-terminal 65 residues
reduced the binding of eIF2
to eIF2B by greater than 80%. No
significant binding of the 200 mutant to eIF2B was detected. In an
attempt to confirm the specificity of the interaction between eIF2B and
the
-subunit of eIF2, the interactions between the wild-type and
deletion mutants of eIF2
and the
-,
-, and
-subunits of
eIF2B were examined. In these experiments, the
-,
-, and
-subunits of eIF2B were expressed in and purified from Sf9
cells using the baculovirus system (13). In confirmation of the results
obtained by far-Western blot analysis, the isolated
- and
-subunits of eIF2B bound to wild-type eIF2
, whereas the
-subunit did not (Fig. 5).
Furthermore, removal of the C-terminal 65 residues of eIF2
reduced
the binding of both eIF2B
and
by greater than 75%. These
results confirm both the specificity of the interaction between the
-subunit of eIF2 and the
- and
-subunits of eIF2B as well as
the importance of the C terminus of eIF2
in the interaction between
eIF2 and eIF2B.
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In an attempt to gain greater insight into the interaction between the
-subunit of eIF2 and eIF2B, the amino acid sequence of eIF2
was
submitted to the Protein Sequence Analysis server at the BioMolecular
Engineering Research Center at Boston University (Boston, MA) for type
2 analysis for multimeric proteins. The secondary structure prediction
indicates a high probability that the region of eIF2
, including
amino acids 260-285, will form an
-helix. Most of the predicted
-helical structure is removed in the eIF2
deletion mutant
truncated at amino acid 268, and the probability that the remainder of
the domain will form an
-helix is greatly reduced. This result is
noteworthy because of the finding in the present study that binding of
the eIF2
268 mutant to either wild-type eIF2B, eIF2B
, or eIF2B
is reduced by greater than 75% compared with the full-length protein,
suggesting that this putative
-helical domain is important in the
interaction of the two proteins.
A caveat to the above experiments is that they do not directly
demonstrate an interaction between eIF2B and the C terminus of
the -subunit of eIF2. An alternative explanation for the results is
that deletion of the C-terminal portion of the protein interferes with
the correct folding of the remainder. If the correct folding of the
remainder of the protein is prevented, but necessary for binding of
eIF2B, then deletion of the C terminus could indirectly prevent binding
of eIF2B to the
-subunit of eIF2.
To demonstrate that the methods used above could detect a difference in
affinity of eIF2B for eIF2 following phosphorylation of eIF2,
unphosphorylated eIF2 holoprotein and eIF2 holoprotein phosphorylated
with HCR were bound to wells in parallel rows in a microtitre plate.
The wells were then probed with various amounts of eIF2B, and the
binding of eIF2B to eIF2 was quantitated using a monoclonal antibody to
eIF2B
. As shown in Fig. 6, the
titration curve for the binding of eIF2B to eIF2 phosphorylated on the
-subunit was shifted to the left of the curve for binding to
unphosphorylated eIF2. The amount of eIF2B required to obtain
half-maximal binding to unphosphorylated eIF2 was approximately 3-fold
greater than for the phosphorylated protein, suggesting that
phosphorylation of eIF2 by HCR increases the affinity of eIF2B for eIF2
by a factor of ~3.
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Finally, the effect of phosphorylation of the -subunit of eIF2 on
the activity of eIF2B was examined. The
-subunit of eIF2 is a
substrate for at least three different protein kinases. CK-II phosphorylates Ser2 and Ser67, PKC
phosphorylates Ser13, and PKA phosphorylates
Ser218 (8). In these studies, the
eIF2·[3H]GDP complex was formed and then phosphorylated
with nonradiolabeled ATP using either CK-II, PKA, or PKC prior to use
as a substrate for eIF2B. As shown in the top panel of Fig.
7, all three kinases incorporated similar
amounts of radioactivity into the
-subunit of eIF2; no incorporation
of 32P into eIF2
was observed in the absence of added
kinase. Furthermore, no incorporation of 32P into eIF2
was observed (data not shown). Phosphorylation by either CK-II or PKC
had no effect on the activity of eIF2B (Fig. 7, bottom
panel). In contrast, phosphorylation of eIF2
by PKA stimulated
the guanine nucleotide exchange activity of eIF2B by 44%
(p < 0.01). Thus, phosphorylation of eIF2
in the
C-terminal part of the protein, but not in the N-terminal region,
resulted in stimulation of eIF2B activity.
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DISCUSSION |
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In this study we demonstrate for the first time the identification
of the subunits of eIF2B to which eIF2 binds and the subunit of eIF2 to
which eIF2B binds. We were surprised to find that eIF2B exhibits little
or no binding to the -subunit of eIF2 regardless of whether or not
the
-subunit of eIF2 has been phosphorylated by the eIF2
kinase,
HCR. Previous studies have shown that eIF2B has a greater affinity for
eIF2 phosphorylated by HCR than for the unphosphorylated protein,
suggesting that eIF2B might bind to eIF2
(4, 5, 14). In two separate
studies (5, 14), the affinity of eIF2B for the eIF2·GDP binary
complex was determined by kinetic analysis of the guanine nucleotide
exchange reaction. In the reported studies, a comparison of the
Km value obtained for eIF2·GDP with the
KI value for eIF2(
P) suggested that the affinity
of eIF2B for eIF2(
P) was 150-fold higher than that for
unphosphorylated eIF2 (5). A study using fluorescence anisotropy to
determine the dissociation constants (KD) of the
eIF2B·eIF2 and eIF2B·eIF2(
P) complexes also found that eIF2B has
a higher affinity for eIF2(
P) than for unphosphorylated eIF2
although the difference in KD between phosphorylated and unphosphorylated eIF2 was only 2-fold in the presence of GDP (4)
rather than the 150-fold difference noted above (5). Furthermore, it
was reported that the affinity of eIF2B for eIF2 varied 5-fold as GDP
concentration was altered (4). In the present study, it was found that
eIF2B had a 3-fold greater affinity for eIF2(
P) than for
unphosphorylated eIF2, a value closer to that observed using
fluorescence anisotropy (4) than was seen using kinetic measurements
(5). Although GDP was not included in the buffers in the present study,
we have found that as much as 70% of the eIF2 purified from rat liver
has GDP bound to it (unpublished observation), which may explain why
the value obtained herein is closer to the lower of the two values
reported by Goss et al. (4). The simplest explanation for
the above observations is that phosphorylation of eIF2
results in a
conformational change in the eIF2 holoprotein that alters the affinity
of eIF2B for eIF2
. In support of this model, we have found that,
like the eIF2B holoprotein, the
-subunit of eIF2 binds to
eIF2
,2 which could provide
a basis for a conformational change in eIF2
in response to
phosphorylation of eIF2
. A second explanation for the observations
is that, in addition to binding to the
-subunit of eIF2, eIF2B might
bind to an area on eIF2 that is shared between the individual subunits
and would only be detected when the subunits were present in the
holoprotein. Such an interaction would not have been detected by
far-Western analysis.
In the present study we also obtained the novel finding that eIF2 binds
to the - and
-subunits of eIF2B. In related studies in
Saccharomyces cerevisiae, Hinnebusch and co-workers (15) have identified point mutations in the
-,
-, and
-subunits of
eIF2B that result in a phenotype characterized by an apparent insensitivity to phosphorylation of eIF2
. Based on these results, it
was proposed that the
-,
-, and
-subunits of S. cerevisiae eIF2B perform a regulatory role in recognizing that the
-subunit of eIF2 is phosphorylated and that one or more of this
group of subunits binds to eIF2. However, of the three subunits, it is unlikely that the primary interaction between eIF2 and eIF2B occurs through binding of eIF2 to the
-subunit of eIF2B because cells deleted for eIF2B
grow at the same rate as wild-type cells, whereas deletion of any of the other four subunits of eIF2B is lethal (reviewed
in Ref. 16). In the model proposed by Hinnebusch (16), the remaining
two subunits, i.e. the
- and
-subunits, would presumably play a role in catalyzing the guanine nucleotide exchange reaction. The results of the present study confirm the postulate that
eIF2 binds to the
-subunit of eIF2B but do not support a model where
eIF2 binds to either the
- or
-subunits of eIF2B.
In summary, the present study reports the identification of the
subunits of eIF2 and eIF2B that interact with each other and suggest
that eIF2B binds to the C terminus of eIF2. The results presented
herein both cast doubt on the long held belief that eIF2B binds to the
-subunit of eIF2 and require the construction of new models where
the predominant interactions between eIF2B and eIF2 occur through the C
terminus of the
-subunit of eIF2 and the
- and
-subunits of
eIF2B.
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ACKNOWLEDGEMENT |
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We acknowledge the excellent technical assistance of Joan McGwire.
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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 (to L. S. J.) and Research Grant 195058 from the Juvenile Diabetes Foundation International (to S. R. K.).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, The Pennsylvania State University, College of
Medicine, P. O. Box 850, Hershey, PA 17033. Tel.: 717-531-8970; Fax:
717-531-7667; E-mail: scot.kimball{at}hmc.psu.edu.
§ Recipient of a Postdoctoral Fellowship from the Juvenile Diabetes Foundation International and supported in part by Training Grant DK07684 from the National Institutes of Health.
1 The abbreviations used are: Met-tRNAi, methionyl-tRNAi; PKA, protein kinase A; PKC, protein kinase C; PVDF, polyvinylidene difluoride; CK-II, casein kinase II; HCR, heme-controlled repressor; GSK-3, glycogen synthase kinase 3.
2 S. R. Kimball, N. K. Heinzinger, R. L. Horetsky, and L. S. Jefferson, unpublished observations.
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REFERENCES |
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