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J. Biol. Chem., Vol. 276, Issue 26, 23922-23928, June 29, 2001
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§,
, and**
From the Department of Biochemistry and Immunology,
Cellular and Molecular Sciences Group, St. George's Hospital Medical
School, Cranmer Terrace, London SW17 0RE, United Kingdom and the
¶ Department of Biochemistry, School of Biological Sciences,
University of Sussex, Brighton BN1 9QG, United Kingdom
Received for publication, January 16, 2001, and in revised form, March 5, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Eukaryotic initiation factor (eIF) 4B interacts
with several components of the initiation pathway and is targeted for
cleavage during apoptosis. In a cell-free system, cleavage of eIF4B by caspase-3 coincides with a general inhibition of protein synthetic activity. Affinity chromatography demonstrates that mammalian eIF4B
interacts with the poly(A)-binding protein and that a region consisting
of the N-terminal 80 amino acids of eIF4B is both necessary and
sufficient for such binding. This interaction is lost when eIF4B is
cleaved by caspase-3, which removes the N-terminal 45 amino acids.
Similarly, the association of eIF4B with the poly(A)-binding protein
in vivo is reduced when cells are induced to undergo
apoptosis. Cleavage of the poly(A)-binding protein itself, using human
rhinovirus 3C protease, also eliminates the interaction with eIF4B.
Thus, disruption of the association between mammalian eIF4B and
the poly(A)-binding protein can occur during both apoptosis and
picornaviral infection and is likely to contribute to the inhibition of
translation observed under these conditions.
Binding of mRNA to eukaryotic ribosomes involves numerous
protein-RNA and protein-protein interactions at both ends of the mRNA (reviewed in Refs. 1-4). The 40 S ribosome and associated initiation factors (5-8) binds to
eIF4G,1 which acts as a
scaffold molecule (1, 3, 9-11). eIF4G associates with the mRNA cap
structure via eIF4E (3, 12, 13), as well as with eIF4A (3) and the
poly(A)-binding protein (PABP) linked to the poly(A) tail (2, 14-18).
This complex facilitates the ability of the 40 S ribosomal subunit to
"scan" along the mRNA until it reaches a suitable initiation
codon, whereupon the 60 S subunit joins to form the 80 S initiation
complex (3, 19). eIF4B, which can also interact with the cap (20, 21),
stimulates the RNA helicase activity of eIF4A (13, 22-25) and is
required to mediate mRNA binding to ribosomes (26-28). It
possesses three potential regulatory domains: an RNA binding domain
(RNA recognition motif), a hydrophilic region (DRYG) that
mediates its binding to eIF3p170 (29), and a serine-rich region at the
C terminus (22).
Both the induction of apoptosis and viral infection result in a rapid
but incomplete inhibition of protein synthesis, accompanied by the
proteolytic cleavage of certain initiation factors (reviewed in Refs.
3, 4, and 30). During apoptosis, degradation of both forms of eIF4G
(eIF4GI and eIF4GII) occurs in a variety of cell types (31-38).
Selective cleavages of eIF4B, eIF3p35, a population of eIF2 In plants, eIF4B can interact with PABP, and this leads to enhancement
both of the RNA binding activity of PABP (42) and of the RNA helicase
activity of the [eIF4A·eIF4B] complex (43). It has remained
unclear whether such an interaction occurs in mammalian cells and, if
so, whether it might be affected by the cleavage of eIF4B that occurs
in apoptotic cells. PABP itself can also be a target for proteolytic
cleavage, notably in picornavirus-infected cells, in which inhibition
of host protein synthesis and the selective translation of viral
mRNAs correlate with the cleavage of both eIF4GII and PABP (30,
44-48). The cleavage of PABP by the picornavirus-encoded 2A or 3C
proteases separates a large N-terminal fragment from a C-terminal
homodimerization domain (47, 48), and this may contribute to the
inhibition of protein synthesis.
Here, we provide evidence for a direct interaction between mammalian
eIF4B and PABP. We demonstrate that recombinant eIF4B can bind to PABP
via the N terminus of eIF4B and that this interaction is ablated when
eIF4B is cleaved by caspase-3 in vitro or when cells are
induced to undergo apoptosis. Furthermore, the cleavage of PABP
with human rhinovirus (HRV) 3C protease eliminates the ability of PABP
to bind to eIF4B. These data suggest that in addition to the cleavage
of eIF4G that occurs during apoptosis and picornavirus infection, the
association between mammalian eIF4B and PABP is targeted under these
conditions by the cleavage of eIF4B and PABP, respectively.
Purification of Recombinant Proteins--
The vector encoding
full-length human His-eIF4B (in pET15b) was a gift from Dr. C. Hellen
(State University of New York Health Center, Brooklyn, NY) and
that encoding human glutathione S-transferase (GST)-PABP (in
pGEX2T) was provided by Dr. J. Hensold (Case Western Reserve
University). Vectors encoding truncated forms of human His-eIF4B were
provided by Dr. J. W. B. Hershey (University of California-Davis). His-tagged forms of full-length and truncated eIF4B,
4E-BP1, and the C-terminal region of eIF4GI were expressed in BL21
cells and purified on nickel-NTA-agarose (Qiagen) (49). GST-PABP was
expressed in baculovirus-infected Sf9 insect cells and purified
on glutathione-Sepharose (Amersham Pharmacia Biotech), as per
the manufacturer's instructions. Purified, recombinant proteins were
dialyzed against Buffer A (20 mM MOPS-K+, pH
7.2, 25 mM NaCl, 10 mM KCl, 7 mM
2-mercaptoethanol, 2 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride) and stored in aliquots at Immunoblotting--
Antisera to eIF4B and PABP were gifts from
Drs. J. W. B. Hershey and D. Schoenberg, respectively.
Antiserum specific for the C-terminal domain of eIF4G was as described
previously (36). Proteins were resolved by SDS-PAGE (50, 51),
transferred to polyvinylidene difluoride membranes, and visualized by
immunoblotting with the antisera described, using
alkaline-phosphatase-coupled secondary antibodies.
Protein Cleavage in Vitro--
Reticulocyte lysate was incubated
for the indicated times at 30 °C with purified caspase-3 (TCS
Biologicals, Buckingham, UK) (5 or 10 µg/ml, as indicated), in
the absence or presence of 50 µM zVAD·FMK
(Alexis, UK). Lysates were also incubated with 200 units/ml recombinant
HRV protease 3C at 5 °C for 2 h prior to translation assays.
Purified His-eIF4B was incubated in phosphate-buffered saline
containing 2 mM benzamidine and 0.5% (v/v) Triton X-100 (PBS-TB) with 0.5 µg/ml purified caspase-3 for 2 h at 37 °C.
Untagged PABP or GST-PABP was incubated with 60 units/ml HRV protease
3C in PBS-TB for 30 min at 37 °C.
In Vitro Translation Assays--
Protein synthesis assays used
non-nuclease-treated reticulocyte lysates essentially as described in
Ref. 52. Incubations were carried out in the absence or presence of 25 µg/ml poly(A)+ or poly(A) Co-isolation of Recombinant Proteins--
Following incubation
together in PBS-TB, His-eIF4B or GST-PABP and their associated proteins
were recovered on nickel-NTA-agarose or glutathione-Sepharose,
respectively. All resins were washed five times each (with 1 ml of
PBS-TB), and the recovered proteins were eluted in SDS-PAGE sample
buffer prior to analysis by SDS-PAGE and immunoblotting.
Induction of Apoptosis and Analysis of Cell Extracts--
Jurkat
cells were incubated in the absence or presence of 250 ng/ml anti-Fas
antiserum for 2 h at 37 °C. Extracts from control and apoptotic
cells were prepared as described previously (33).
eIF4B Cleavage Coincides with Inhibition of Protein Synthesis by
Caspase-3--
Initiation factor eIF4B is cleaved in apoptotic
cells to yield a smaller fragment ( eIF4B Interacts with PABP in a Caspase-sensitive
Manner--
Cleavage of eIF4B by caspase-3 removes an N-terminal
fragment of 45 amino acids (36). As eIF4B interacts with PABP in
plants to modulate the activity of the cap-binding complex (42, 43), we
have examined, using affinity chromatography, whether eIF4B and PABP
interact directly in the mammalian system and whether caspase-3
cleavage of eIF4B affects this binding. Fig.
2A shows that whereas
mammalian His-eIF4B did not itself bind to glutathione-Sepharose (lane 3), there was substantial recovery of the factor on
these beads following incubation with GST-tagged PABP
(lane 5). Conversely, PABP could be isolated from a
reticulocyte lysate by chromatography on nickel-agarose in the presence
but not in the absence of His-eIF4B that was bound to the beads (Fig.
2B). An irrelevant His-tagged protein (the eIF4E-binding
protein 4E-BP1) did not bind PABP in this system, ruling out the
possibility of nonspecific interactions between PABP and nickel-agarose
or the His tag. Similarly, purified His-eIF4B associated with GST-PABP
could be recovered with the latter protein on glutathione-Sepharose,
whereas another His-tagged protein (the C-terminal part of eIF4GI (49))
did not bind under the same conditions (Fig. 2C). This
eliminates the possibility of nonspecific interactions between the His
tag and PABP, GST, or glutathione-Sepharose itself. The interaction
between eIF4B and PABP was not prevented by incubation with RNase A
(Fig. 2A, lane 6), suggesting that it is not mediated by RNA
bridging between the two proteins. However, we cannot entirely discount
a role for a fragment of RNA that is protected from RNase action by one or both proteins.
To delineate the region of eIF4B that interacts with PABP, we incubated
truncated versions of His-eIF4B with untagged PABP. Fig.
3 shows that whereas PABP alone was not
recovered on nickel-agarose (lane 1), the N-terminal region
of eIF4B (amino acids 1-150) permitted the co-isolation of PABP
(lane 5). Further delineation of this region showed that
amino acids 81-180 of eIF4B did not interact with PABP (lane
3), but amino acids 1-80 (lane 4) were sufficient to
allow the interaction.
To determine whether the caspase-mediated truncation of eIF4B at the N
terminus disrupts the interaction of the factor with PABP, we incubated
full-length and caspase-cleaved forms of eIF4B with GST-PABP. Protein
complexes were subsequently isolated on glutathione-Sepharose. Fig.
4A shows that neither
full-length His-eIF4B (lane 2) nor the caspase-truncated
PABP Interacts with eIF4B in a Protease-sensitive
Manner--
Mammalian PABP is cleaved during picornavirus infection by
the virally encoded proteases 2A and 3C, and the sites of cleavage have
been mapped to near the C terminus (47, 48). We have used HRV protease
3C to determine whether such cleavage interferes with the ability to
interact with eIF4B. Protease 3C was able to utilize both GST-PABP
(Fig. 4B, lane 3) and untagged PABP (Fig. 4C) as
substrates but did not degrade eIF4B (Fig. 4C). Following cleavage of GST-PABP, there was a reduction in the co-isolation of
full-length eIF4B with PABP (Fig. 4B, lane 3 versus lane 2). Protease 3C cleavage also
prevented the association of untagged PABP with the His-tagged N
terminus of eIF4B (Fig. 4C, lane 5 versus lane 4). These
data suggest that the highly conserved C-terminal domain of PABP (53)
is required for the interaction with the N-terminal region of eIF4B.
We have investigated the consequences for protein synthesis of the
cleavage of PABP in the reticulocyte lysate. In contrast to the
reported effect of foot-and-mouth disease virus protease 3C in
vivo (54), HRV protease 3C, at the concentration and times used
here, did not degrade eIF4G or eIF4A in vitro but caused cleavage of ~50% of the PABP in the lysate (Fig.
5A). Consequently, it was
possible to examine the effect of this enzyme specifically on PABP
cleavage and protein synthesis in parallel. In a non-nuclease-treated lysate, to which was added either poly(A)+ or
poly(A) The Interaction between PABP and eIF4B Is Diminished in Apoptotic
Cells--
A prediction that can be made on the basis of our results
is that in cells induced to undergo apoptosis, where eIF4B is cleaved (36), the association of PABP with eIF4B should be disrupted. To
address this, extracts were prepared from control and apoptotic Jurkat
cells. The integrity of eIF4G, PABP, eIF4B, and eIF4E and the
association of these proteins with each other were monitored by
immunoblotting. In agreement with published data (31, 36), Fig.
6A shows that eIF4G
(lane 2 versus lane 1) and eIF4B (lane 4 versus lane
3), but not PABP or eIF4E, were cleaved during apoptosis. PABP and
associated proteins were isolated by affinity chromatography on
poly(A)-Sepharose (Fig. 6B). Although similar levels of PABP were recovered from apoptotic and control cell extracts, the amount of
eIF4B associated with PABP was substantially reduced in apoptotic extracts (lane 2 versus lane 1). In addition, the amount of
eIF4E recovered was greatly diminished (lane 2 versus lane
1), as would be predicted from the cleavage of eIF4G, which
results in the separation of the eIF4E binding site from the PABP
binding site (35). We have also used m7GTP-Sepharose
chromatography to investigate the association of PABP with eIF4F (Fig.
6C). Following induction of apoptosis, there was a decrease
in the level of PABP recovered with eIF4E (lane 2 versus lane
1). However, in this instance, it is not possible to discern
whether this effect is attributable to the loss of integrity of eIF4B
or to the caspase-mediated cleavage of eIF4G (31, 35, 37), because both
these proteins bind PABP independently.
The direct interaction between mammalian eIF4B and PABP, in
conjunction with the binding of eIF4G to PABP, may facilitate the
functional association of the 5' and 3' ends of mRNA (1-3, 55-57). In wheat germ, the association of eIF4B with PABP increases the efficiency of reinitiation of protein synthesis (58), a process
that is also enhanced by the interaction between eIF4G and PABP (17,
43). As depicted in Fig. 7A,
the loss of the N terminus of eIF4B (by caspase-mediated cleavage) or
of the C terminus of PABP (by picornavirus protease-mediated cleavage) disrupts complex formation between the two proteins. Such an effect would be predicted to inhibit reinitiation. Indeed, cleavages of eIF4B
(Fig. 1 and Ref. 36) and PABP (Fig. 5 and Refs. 47 and 48) are
associated with inhibition of protein synthesis in vivo and
in vitro.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and the
eIF4E-binding protein 4E-BP1 have also been observed (33, 35, 36,
39-41).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
The baculovirus vector encoding untagged Xenopus PABP was
provided by Dr. D. Schoenberg (Ohio State University). Seventy-two
hours after baculovirus infection, insect cell extracts were prepared
by cell lysis with 1% (v/v) Igepal and stored in aliquots at
70 °C (34).
mRNA encoding
luciferase, which was generated using the Ambion Message Machine
system as per the manufacturer's instructions. Samples were
subsequently resolved by SDS-PAGE. Translation of the luciferase
reporter mRNA was quantified by analysis on a PhosphorImager (Molecular Dynamics Storm 860) using ImageQuant software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
eIF4B). In addition, other
factors, including eIF4GI, eIF4GII, eIF3p35, and eIF2 are
cleaved under the same conditions (31-40). However, the relative
contribution of each of these events to the overall down-regulation of
protein synthesis during apoptosis is not yet known. Using a cell-free
system, we have investigated the time at which cleavage of eIF4B occurs
relative to that of other caspase targets in the protein synthetic
machinery upon addition of purified caspase-3. Fig.
1A (right panel)
shows that in the reticulocyte lysate system, eIF4B is cleaved within 10 min of caspase-3 treatment, whereas eIF4G remains intact (as judged
by the appearance of the specific cleavage products p120 and M-FAG
(35)) until at least 20 min of incubation. At similarly early
times following caspase-3-treatment, eIF3p35 and eIF2 are also not
significantly cleaved (data not shown). The early cleavage of eIF4B
coincides with the start of caspase-induced loss of protein synthetic
activity, which affects both endogenous globin translation (Fig.
1A, left panel, and Fig. 1B, left panel, lanes 2, 5, and 8) and the translation of exogenous capped or
uncapped polyadenylated luciferase mRNA (Fig. 1B, left panel,
lanes 5 and 8, respectively). The caspase-mediated
cleavages are prevented by inclusion of zVAD·FMK in the incubation
(Fig. 1B, right panel), as is the inhibition of protein
synthesis (Fig. 1, A and B).
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Fig. 1.
Cleavage of eIF4B coincides with the
onset of caspase-mediated inhibition of protein synthesis in
vitro. A, reticulocyte lysate was incubated
without (closed squares) or with caspase-3 (5 µg/ml)
(triangles) or caspase-3 and zVAD·FMK (open
squares). Aliquots were assayed for the incorporation of
[35S]methionine into protein (52) (left panel)
and for cleavage of eIF4B and eIF4G by immunoblotting (right
panel). Intact eIF4B and eIF4G and their caspase-generated
cleavage products are indicated. B, lysate was preincubated
without (control (C)) or with recombinant caspase-3 (10 µg/ml) (3), or caspase-3 and zVAD·FMK (3Z)
for 15 min at 30 °C. Further enzyme activity was then blocked by the
addition of zVAD·FMK to the caspase-3 incubation. Aliquots (1 µl)
were taken for analysis of the integrity of eIF4B and eIF4G by
immunoblotting (right panel). Intact eIF4B and eIF4G and
their caspase-generated cleavage products are indicated. Further
aliquots of lysate were assayed for the ability to translate endogenous
globin mRNA and added capped or uncapped polyadenylated luciferase
mRNAs (ClucA+ and UlucA+, respectively) in
the presence of [35S]methionine for 60 min at 30 °C.
The translation products indicated were analyzed by SDS-PAGE and
autoradiography of 2-µl samples (left panel).
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Fig. 2.
Mammalian eIF4B and PABP interact directly
in vitro. A, purified His-eIF4B (4 µg) and GST-PABP (4 µg) were incubated in PBS-TB in the
combinations indicated. Following capture on nickel-NTA-agarose or
glutathione (GSH)-Sepharose beads, the proteins were
analyzed by immunoblotting with antibodies against PABP (top
panel) or eIF4B (bottom panel). Lane 6, proteins were subjected to RNase A treatment (10 µg/ml for 15 min at
37 °C) prior to recovery on glutathione-Sepharose. B,
reticulocyte lysate was passed over nickel-NTA-agarose beads that had
been preincubated with buffer alone (lane 1) or with equal
amounts of His-4E-BP1 (lane 2) or His-eIF4B (lane
3). The beads were washed, and the bound proteins were analyzed as
in A, using antibodies against PABP (top panel)
or eIF4B (bottom panel). C, purified GST-PABP (4 µg) was incubated alone or with His-eIF4B (4 µg) or the His-tagged
C-terminal region of eIF4GI (His-eIF4G(C)) (8 µg), as in
A. Following capture on GSH-Sepharose, the bound proteins
were analyzed by immunoblotting with antibodies against PABP (top
right panel), eIF4B (middle right panel), or eIF4G(C)
(bottom right panel). The left panel shows the
Coomassie-stained proteins (one-fifth of total input) used in the
incubations. The positions of migration of the three proteins on the
gel and the corresponding immunoblots are indicated.
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Fig. 3.
The N-terminal region of eIF4B is necessary
and sufficient for interaction with PABP. Uninfected Sf9
cell extract (lane 2) or extract prepared from cells
infected with baculovirus expressing untagged Xenopus PABP
(lanes 1 and 3-5) was incubated with or without
the following forms of truncated His-eIF4B: lane 3, amino
acids 81-180; lane 4, amino acids 1-80; lane 5,
amino acids 1-150. Following chromatography on nickel-NTA-agarose, the
recovered proteins were analyzed by SDS-PAGE. The top panel
shows an immunoblot for PABP, and the bottom panel shows a
Coomassie-stained gel of the input forms of His-eIF4B. The 1-80 form
of His-eIF4B runs anomalously on this gel system.
eIF4B (lane 3) alone was recovered on the resin. However,
in the presence of GST-PABP, full-length His-eIF4B was co-isolated by
this procedure (lane 5). In contrast, eIF4B did not
associate with GST-PABP (lane 6). Thus, loss of the
N-terminal 45 amino acids, as a result of cleavage of eIF4B by
caspase-3, is sufficient to ablate the interaction with PABP.
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Fig. 4.
Cleavage of eIF4B by caspase-3 or cleavage of
PABP by HRV protease 3C eliminates the interaction between eIF4B and
PABP. A, His-eIF4B, caspase-3-cleaved eIF4B ( eIF4B),
and GST-PABP were incubated in the combinations shown and subjected to
affinity chromatography on nickel-NTA-agarose beads or glutathione
(GSH)-Sepharose beads. The recovered proteins were
visualized by immunoblotting for PABP or eIF4B as indicated (left
panel). The positions of GST-PABP, His-eIF4B, and eIF4B are
shown. Right panel, to demonstrate that eIF4B cleavage was
complete, aliquots of the untreated and caspase-3-cleaved eIF4B were
resolved by SDS-PAGE and visualized by immunoblotting. B,
His-eIF4B was incubated in the presence or absence of GST-PABP and HRV
protease 3C and subjected to chromatography on glutathione-Sepharose.
The bound proteins were eluted and analyzed by immunoblotting using
antibodies against PABP (top panel) or the His tag
(bottom panel). C, untagged PABP and/or His-eIF4B
(amino acids 1-80) were incubated in the presence or absence of
protease 3C as in B and subjected to chromatography on
nickel-NTA-agarose. The bound proteins were analyzed by immunoblotting
using antibodies against PABP (top panel) or the His tag
(bottom panel). The two right lanes of the
top panel show the input of intact PABP and the
protease-cleaved PABP, respectively.
luciferase mRNA, HRV protease 3C had a small
inhibitory effect on the translation of both the endogenous globin
mRNA (data not shown) and the exogenous poly(A)+ RNA
(Fig. 5B, top panel). In contrast, the protease treatment resulted in a substantial increase in the ability to translate the
poly(A) luciferase mRNA (Fig. 5B, bottom
panel). These data suggest that either the PABP cleavage product
shows a gain of function toward translation of poly(A)
mRNA or, more likely, the latter acquires a translational advantage in competition with the endogenous globin mRNA. These results also
indicate that exposure to HRV protease 3C does not impair the function
of other initiation factors required for the translation of both
poly(A)+ and poly(A) mRNAs.
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Fig. 5.
Partial cleavage of PABP by HRV protease 3C
increases the efficiency of translation of non-polyadenylated mRNA
in vitro. A, reticulocyte lysate was
preincubated in the absence (control (C)) or presence
(3C) of recombinant protease 3C. Samples (2 µl) were
analyzed by immunoblotting for PABP, eIF4G, and eIF4A, as indicated.
B, aliquots were assayed for their ability to translate
capped, polyadenylated or capped, non-polyadenylated mRNA encoding
luciferase (ClucA+ and ClucA-, respectively).
The protein was labeled with [35S]methionine (52) and
analyzed by SDS-PAGE followed by phosphorimaging. The result of a
typical experiment is shown in the upper panel, with the luciferase
translation product (luc) indicated. Quantification of the
labeled luciferase is shown in the lower panel as the % (± S.E.) of
the value obtained for each mRNA in the absence of protease 3C in
five separate experiments.
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Fig. 6.
Induction of apoptosis reduces the
association of eIF4B with poly(A)-interacting proteins in
vivo. A, Jurkat cells were incubated for
2 h in the absence (control (C)) (lanes 1 and 3) or presence (Fas) (lanes 2 and
4) of an agonistic antibody to the Fas receptor, and
extracts were prepared. Equal amounts of protein were analyzed by
immunoblotting for eIF4G (and its cleavage product M-FAG), PABP,
eIF4B/ eIF4B, and eIF4E, as indicated. B, PABP and
associated proteins were isolated from control (lane 1) or
apoptotic (lane 2) cell extracts using affinity
chromatography on poly(A)-Sepharose. Nonspecific binding of proteins
was minimized by the inclusion of poly(C) (1 mg/ml) in the binding
buffer. The resin was washed and the bound proteins analyzed as in
A. C, eIF4E and associated proteins were isolated
from control (lane 1) or apoptotic (lane 2) cell
extracts using affinity chromatography on m7GTP-Sepharose.
The samples were processed and analyzed as in B.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
A, schematic representation of the
interaction of eIF4B and PABP. The domain structures of human
PABP and eIF4B and the sites of protein-protein interaction and
cleavage by picornaviral proteases and caspase-3 are shown. For eIF4B,
the RNA recognition motif (RRM), the region containing DRYG
repeats, and the basic domain (BD) are indicated (22, 29).
For PABP, the four RNA recognition motifs and the proline-rich domain
are shown (22, 24, 47, 48). The region of eIF4B required for PABP
binding contains a sequence (residues 6-20) with similarity to amino
acid sequences 133-145 and 135-147 within the PABP-binding domains of
human eIF4GI and eIF4GII, respectively (17, 60). Homologous regions in
the three sequences are boxed, with identical residues
indicated in italics. B, disruption of the
initiation complex by proteolytic cleavages of initiation factors. The
known interactions between eIF3, eIF4A, eIF4B, eIF4E, eIF4G, and PABP
are shown on the left, and the sites at which these factors
are cleaved by caspase-3 and by the picornavirus-encoded 2A, 3C, and L
proteases are indicated. In apoptotic cells, caspase-mediated cleavages
result in the appearance of three fragments of eIF4G and a truncated
form of eIF4B (top right). The cleavages separate the
N-terminal region of eIF4G that binds PABP from the central region that
binds eIF4E, eIF4A, and the [eIF3·eIF4B] complex (4, 35) and also
disrupt the interaction of eIF4B with PABP. In cells infected with
poliovirus, HRV, or foot-and-mouth disease virus, viral
protease-mediated cleavage results in the appearance of two fragments
of eIF4G and a truncated form of PABP (bottom right). The
fragmentation of eIF4G again separates the regions that bind PABP and
the [eIF3·eIF4B] complex (9, 63, 64). In both apoptotic and
infected cells the links between the 5' end of an mRNA (via
recognition of the cap by eIF4E) and the 3' end (via recognition of the
poly(A) tail by PABP) or between the mRNA and the ribosome (via
binding of eIF3 to the latter) are broken, potentially reducing the
efficiency with which reinitiation of protein synthesis on capped
poly(A)+ mRNA can take place.
We have previously pointed out (36) that eIF4B still contains the
DRYG domain required for self-association and binding to eIF3p170 (29)
and also retains the RNA recognition motif domain required for
binding RNA (22, 59). As the region of eIF4B required for the
stimulation of the RNA helicase activity of eIF4A is located in the
C-terminal half of the protein (24, 29), this activity also may not be
directly affected. In contrast, interaction of the extreme N terminus
of eIF4B with PABP provides a potential mechanism by which cleavage of
eIF4B during apoptosis may contribute to the down-regulation of protein
synthesis (4). Because PABP also enhances the RNA helicase activity of
the [eIF4A·eIF4B·eIFiso4F] complex in wheat germ (43), such a
regulatory mechanism would also be impaired when the eIF4B-PABP
interaction is abolished, resulting in less efficient unwinding of
mRNA secondary structure.
The site of binding of PABP on eIF4GI has been assigned to amino acids 132-160 (17), and there is strong conservation of this sequence in eIF4GII (60). Interestingly, a region with significant similarity to parts of these sequences is present near the N terminus of eIF4B (Fig. 7A), and our data are consistent with the possibility that this site is critical for eIF4B-PABP interaction. Further deletion mapping and site-directed mutagenesis studies will be required to test this.
Inhibition of the eIF4B-PABP interaction during infection, as a consequence of the cleavage of PABP by viral proteases (Fig. 4), may be additive or synergistic with that of the cleavages of eIF4GI and eIF4GII in contributing to the shut-off of translation in infected cells (44-48). Only a partial cleavage of PABP in vitro is sufficient to enhance the translation of non-polyadenylated mRNA relative to that of mRNA with a poly(A) tail, perhaps because the latter is translated less efficiently under these conditions and competes less well.
As illustrated in Fig. 7B, disruption of several
protein-protein interactions, as a result of cleavages of initiation
factors, is a feature of both the early stages of apoptosis and of
infection with picornaviruses. The patterns of the cleavages are
different in the two situations, but a degree of evolutionary
convergence has apparently occurred between the mechanisms by which
protein synthesis is targeted for down-regulation. Interestingly, both poliovirus proteases 2A and 3C can themselves induce apoptosis (61,
62), adding weight to the view that initiation factor modifications may
have important functional significance for the development of an
apoptotic response.
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ACKNOWLEDGEMENTS |
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We thank Drs. Dan Gallie, Linda McKendrick, and Ruth Simon for discussions concerning this work.
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FOOTNOTES |
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* Research in our laboratories is supported by grants from the Wellcome Trust (Grants 040800, 058915, 057494, 045619, and 056778), the Leukemia Research Fund, the Cancer Prevention Research Trust, and Glaxo-Wellcome.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.
§ Present address: Dept. of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305.
Senior Research Fellow of the Wellcome Trust.
** To whom correspondence should be addressed. Tel.: 44-020-8725-5762; Fax: 44-020-8725-2992; E-mail: M.Clemens@sghms.ac.uk.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M100384200
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ABBREVIATIONS |
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The abbreviations used are: eIF, eukaryotic initiation factor; GST, glutathione S-transferase; HRV, human rhinovirus; MOPS, 4-morpholinepropanesulfonic acid; PABP, poly(A)-binding protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; zVAD.FMK, z-Val-Ala-DL-Asp-fluoromethylketone.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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