From the Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom
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ABSTRACT |
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Serum stimulation of cultured Xenopus
kidney cells results in enhanced phosphorylation of the translational
initiation factor (eIF) 4E and promotes a 2.8-fold increase in the
binding of the adapter protein eIF4G to eIF4E, to form the functional
initiation factor complex eIF4F. Here we demonstrate the
serum-stimulated co-isolation of the poly(A)-binding protein (PABP)
with the eIF4F complex. This apparent interaction of PABP with eIF4F
suggests that a mechanism shown to be important in the control of
translation in the yeast Saccharomyces cerevisiae also
operates in vertebrate cells. We also present evidence that the
signaling pathways modulating eIF4E phosphorylation and function in
Xenopus kidney cells differ from those in several mammalian
cell types studied previously. Experiments with the immunosuppressant
rapamycin suggest that the mTOR signaling pathway is involved in
serum-promoted eIF4E phosphorylation and association with eIF4G.
Moreover, we could find little evidence for regulation of eIF4E
function via interaction with the specific binding proteins 4E-BP1 or
4E-BP2 in these cells. Although rapamycin abrogated serum-enhanced
rates of protein synthesis and the interaction of eIF4G with eIF4E, it
did not prevent the increase in association of eIF4G with PABP. This
suggests that serum stimulates the interaction between eIF4G and PABP
by a distinct mechanism that is independent of both the mTOR pathway
and the enhanced association of eIF4G with eIF4E.
Control of polypeptide synthesis plays an important role in cell
proliferation. The physiological regulation of protein synthesis is
almost always exerted at the level of polypeptide chain initiation (reviewed in Refs. 1 and 2), influenced by elements in the 5'- and
3'-untranslated regions of the mRNA (3). The initiation phase is
regulated, in part, by the phosphorylation and association of
initiation factors involved in binding mRNA to the 40S ribosomal subunit (reviewed in Refs. 1 and 4-8). The cap structure present at
the 5'-end of mRNA facilitates its binding to the ribosome, a
process mediated by at least three initiation factors (eIF4A, eIF4B,
and eIF4F)1 and ATP
hydrolysis. eIF4F is a cap-binding
protein complex composed of three polypeptides; eIF4E, which
specifically recognizes the cap structure, eIF4A, a single strand
RNA-binding protein with helicase activity (9), and eIF4G, which acts
as a bridging molecule between eIF4E and the 40S ribosome via eIF3 (8,
10-14). It is believed that eIF4F functions to unwind secondary
structure in the mRNA 5'-untranslated region to facilitate binding
to the 40 S ribosomal subunit (4). Recent studies in the yeast
Saccharomyces cerevisiae have indicated that a further
association occurs between eIF4G and poly(A)-binding protein (Pab1p),
which binds to the 3' poly(A) tail of mRNA. This interaction allows
functional interaction of the 5'- and 3'-ends of the mRNAs that is
essential for transmitting the stimulatory signal of the poly(A) tail
on translation to the cap structure (8, 15-21).
eIF4E activity can be regulated by both its phosphorylation and by its
availability to participate in the initiation process. Increased levels
of eIF4E phosphorylation have been directly correlated with the
enhancement of translation that follows mitogenic stimulation of
mammalian cells (1, 7, 22). Parallel increases in eIF4E phosphorylation
and interaction of the factor with eIF4G have been observed in a number
of cellular systems (23-27), and the phosphorylated form of eIF4E is
reported to exhibit increased affinity for the cap structure in
vitro (28). Another important mechanism regulating the interaction
between eIF4E and eIF4G is exerted by the eIF4E-binding proteins 4E-BP1
and 4E-BP2 (PHAS-I and PHAS-II). In resting cells, 4E-BP1 and 4E-BP2
are hypophosphorylated and bound to eIF4E (7, 29-32). Stimulation of
cells with growth factors or hormones increases the phosphorylation of
these eIF4E-binding proteins to disrupt their association with eIF4E,
liberating eIF4E to interact with a conserved hydrophobic region of
eIF4G. A similar sequence found in 4E-BP1 is involved in binding to
eIF4E and competes with eIF4G for eIF4E binding (33). Insight into the
signaling pathways regulating the phosphorylation of 4E-BP1/BP2 has
been provided by the use of the immunosuppressant, rapamycin (34, 35).
This drug binds to its cytosolic receptor (FK-506-binding protein),
which then interacts with and inhibits the activity of a protein known
variously as RAFT-1/FRAP/RAPT-1 or mTOR (36). As a consequence of this,
rapamycin causes the selective inhibition of two parallel signaling
pathways downstream of mTOR; the p70 S6 kinase (p70S6K)
signaling pathway and the phosphorylation of 4E-BP1 (36-38). Thus in
mammalian cells rapamycin is thought to impair the enhancement of
protein synthesis initiation in response to serum stimulation by
stabilizing the interaction between eIF4E and 4E-BP1 (1, 2, 7, 32). In
contrast, rapamycin does not prevent the phosphorylation of eIF4E
during the early phases of T cell activation (26), hormone-induced
maturation of Xenopus oocytes (25), or the responses of
Chinese hamster ovary cells to insulin (39) or NIH 3T3 cells to serum
(27, 40).
Using Xenopus kidney cells in culture, we have utilized
rapamycin to examine the signal transduction pathways involved in the
enhanced phosphorylation of eIF4E, its recruitment into eIF4F complexes, and its association with other initiation factors. In
contrast to the situation with the mammalian cells described above,
rapamycin prevented the serum-stimulated increase in eIF4E phosphorylation in these cells. Although rapamycin inhibited eIF4F complex formation, this did not appear to be due to stabilization of
the interaction of eIF4E with 4E-BP1 or 4E-BP2. In addition, we show
that serum stimulated the association between eIF4G and PABP via a
rapamycin-insensitive pathway. These data indicate that multiple
signaling pathways converge at the level of eIF4F complex formation to
influence the interactions between eIF4E, eIF4G, and PABP during the
stimulation of cell growth.
Chemicals and Biochemicals--
Materials for tissue culture
were from Life Technologies; [35S]methionine and
[32P]orthophosphate were from ICN; Immobilon
polyvinylidine difluoride was from Millipore;
m7GTP-Sepharose was from Amersham Pharmacia Biotech;
Microcystin was from Calbiochem; and rapamycin was a kind gift from Dr.
J. Kay (University of Sussex, United Kingdom). Unless otherwise stated, all other chemicals were from Sigma.
Cell Culture--
Xenopus laevis kidney B 3.2 cells
(41) were grown in 10-cm dishes at room temperature (20-24 °C),
containing 60% Leibovitz L-15 medium, 30%
H2O, 10% fetal calf serum supplemented with 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml
streptomycin. For serum starvation, cultures were incubated with 0.5%
fetal calf serum for 24 h prior to treatment with serum (10%), as
described in individual figure legends.
In Vivo [32P]Orthophosphate Labeling--
Cells
were serum-starved for 24 h in phosphate-free Leibovitz
L-15 medium and incubated with
[32P]orthophosphate (200 µCi/ml) for 2 h prior to
treatment with serum (10%). Extracts were prepared as described below.
Preparation of Cell Extracts--
Cultures were placed on ice,
and cells were scraped into 1 ml of Buffer A (80 mM
Measurement of Protein Synthesis--
Serum-starved cells were
incubated in the presence of 10 µCi/ml [35S]methionine
and 10% fetal calf serum in complete medium for the times indicated.
Cells were recovered and washed twice in Buffer A, prior to lysis in
0.3 M NaOH. Incorporation of radioactivity into protein was
determined by precipitation with trichloroacetic acid.
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Vertical Slab
Isoelectric Focusing (VSIEF), and Immunoblotting--
One-dimensional
polyacrylamide gels and vertical slab isoelectric focusing gel
electrophoresis were carried out as described previously (27, 42, 43).
Following the transfer of proteins to polyvinylidine difluoride
membrane, the presence of eIF4E, eIF4G, eIF4A, PABP, eIF2 m7GTP-Sepharose, Poly(A)-Sepharose Chromatography,
and Immunoprecipitation of eIF4G--
For the isolation of eIF4E and
associated proteins, cell extracts of equal protein concentration were
subjected to m7GTP-Sepharose chromatography, the resin was
washed three times with Buffer C (50 mM Mops/KOH, pH 7.4, 100 mM NaCl, 0.5 mM EDTA, 0.5 mM
EGTA, 1 µM microcystin, 50 mM
Antisera to eIF4 Proteins--
Antiserum specific to eIF4E
(peptide sequence, TATKSGSTTKNRFVV) and eIF4A (peptide sequence,
DLPANRENYIHRTGRGGRFGRK) were prepared as described previously (25, 27,
43); rabbit antiserum to the C terminus of eIF4G was generated
following expression of eIF4G920-1396 in bacteria, as
described (44); antiserum specific to the phosphorylated form of eIF4E
was generated in rabbits using the peptide TATKSGS(P)TTKNRFVV (prepared
by Dr. G. Bloomberg, Department of Biochemistry, University of Bristol, United Kingdom) and affinity-purified on the same peptide. In all
cases, detection was within the linear response of the antiserum to the protein.
Rapamycin Prevents the Serum-induced Phosphorylation of eIF4E in
Xenopus Kidney Cells--
In several mammalian cell systems, it has
been possible to show that physiological stimulation of protein
synthesis and cell growth results in parallel enhancement of the
phosphorylation of eIF4E and eIF4G (23-27) and the association of
these polypeptides to form the eIF4F complex (45, 46). To examine this
relationship further, we have analyzed the activation of protein
synthesis in Xenopus kidney cells in culture, using a cell
line well established for studies of serum stimulation of both general
translation and of specific translation of mRNAs encoding ribosomal
proteins (41). Relative to control cells, addition of serum resulted in
a 20% increase in the rate of total protein synthesis within 30 min, reaching a 40% increase by 3 h (Fig.
1A). Consistent with previous studies with this cell line (41), the early activation of protein synthesis was accompanied by a significant increase in the proportion of ribosomes in polysomes (data not shown). This modest effect on the
translation rate at early times after stimulation is similar in extent
to that reported for insulin-stimulated (39, 46-48) and
serum-stimulated mammalian cells in culture (49). To determine the
effect of serum on the phosphorylation of eIF4E, extracts were prepared
from control or stimulated cells, and the level of phosphorylation was
directly monitored by immunoblotting using a phosphospecific antiserum;
the recovery of eIF4E was monitored using antiserum that recognizes
eIF4E irrespective of its phosphorylation status (24, 25, 27, 42). As
shown in Fig. 1B, serum stimulation resulted in a 40%
increase in the phosphorylation of total eIF4E, which was maximal
within 30 min. Because Xenopus cells contain two distinct
forms of eIF4E (25, 50), we have analyzed the phosphorylation of these
independently following m7GTP-Sepharose affinity
purification, VSIEF, and immunoblotting (Fig. 1C). With this
system, for each form of the protein, the more phosphorylated eIF4E
variant is the upper immunoreactive band (25, 27, 42, 43). These data
show that the addition of serum resulted in a 50% increase in the
total phosphorylation of eIF4E within 30 min; the proportion of eIF4E
in the phosphorylated form did not increase any further upon prolonged
incubation of the cells (data not shown). In most experiments, the
enhancement of phosphorylation was similar for both forms of eIF4E, but
in some cases the effect on phosphorylation of the upper form (resolved by VSIEF) was greater (see Fig.
2A). The data on overall eIF4E phosphorylation were confirmed by prelabeling of cells with
[32P]orthophosphate; extracts were prepared from starved
and serum-stimulated cells, and eIF4E and associated proteins were
isolated by affinity chromatography. Serum induced greater than a 50%
increase in the phosphate labeling of total eIF4E, without affecting
the amount of eIF4E protein present in the cell (Fig. 1D).
By virtue of its association with eIF4E, eIF4G was also recovered
following m7GTP-Sepharose chromatography. Following this
step, an 80% increase in the phosphate labeling of eIF4G recovered in
association with eIF4E was observed (Fig. 1D). Although we
have observed modest increases in the phosphorylation of total cellular
eIF4G following serum stimulation (data not shown), the results
presented here mainly reflect the enhanced recovery of eIF4G protein
associated with eIF4E following serum activation of cells (see Fig.
3A). Conversely, by comparing
the signals on Western immunoblots probed with antisera recognizing
either total or phosphorylated eIF2
By using the immunosuppressant rapamycin in conjunction with VSIEF and
immunoblotting, we have also begun to examine the intracellular signaling pathways modulating the enhanced phosphorylation of eIF4E and
the activation of protein synthesis in response to serum. It is now
well established that in a number of mammalian cell types, rapamycin
prevents the activation by serum or insulin of the p70S6K
signaling pathway (36) and the phosphorylation of eIF4E-binding proteins (4E-BP1 and 4E-BP2) (7, 27, 29-32, 51, 52). These effects are
thought to explain the partial abrogation by this agent of the
stimulation of translation (27, 40, 49, 53, 54). As described above,
serum stimulation resulted in an increase in total eIF4E
phosphorylation from 16 to 42% within 30 min of addition (Fig.
2A, top panel, compare lanes 2 and 1;
quantified in the bottom panel), a finding confirmed by
in vivo labeling of eIF4E with
[32P]orthophosphate prior to isolation (middle
panel). Preincubation of Xenopus kidney cells with
rapamycin strongly inhibited the activation of p70S6K (data
not shown), and surprisingly, the serum-stimulated phosphorylation of
eIF4E monitored by immunoblotting or phosphate labeling in vivo (Fig. 2A, lane 1 versus lane 3). Under these
conditions, rapamycin had no obvious effect on the phosphate labeling
of total eIF4G (data not shown). The effect of rapamycin on the
serum-induced activation of protein synthesis was monitored by pulse
labeling of cells with [35S]methionine prior to harvest.
Fig. 2B shows that rapamycin abrogated the early
serum-induced increase in the rate of protein synthesis. Similar
effects on translation rates and eIF4E phosphorylation were observed
with wortmannin and LY294002 (data not shown). These data are in
contrast to investigations with mammalian cells, in which the
enhancement of phosphorylation of eIF4E has been found to be resistant
to attenuation by rapamycin (25-27, 39, 40, 45, 49).
Serum Stimulation Promotes the Co-isolation of Poly(A)-binding
Protein with the eIF4F Complex--
mRNA is posttranscriptionally
modified with a cap structure at the 5'-end and a poly(A) tail at the
3'-end, both of which, individually and in concert, play essential
roles in the regulation of translation (reviewed in Refs. 8, 12, 21,
55, and 56). Cap structure-dependent initiation of
translation involves the assembly of the eIF4F complex
(eIF4E/eIF4A/eIF4G) at the 5'-end of mRNA. Recent studies in the
yeast S. cerevisiae have indicated a further interaction
between eIF4G and poly(A)-binding protein (Pab1p), which results in
circularization of the mRNA (17-20). Evidence has also been
presented for interactions between poly(A) or poly(A)-binding protein
and polypeptides of the eIF4F and eIF (iso)4F complexes in
the wheat germ system (57, 58). Furthermore, synergistic effects of the
cap and poly(A) tail on translational efficiency have been demonstrated
in mammalian cells as well as in yeast and plant systems (3, 21, 55).
Recently, Craig et al. (74) demonstrated the interaction of
poly(A)-binding protein with a homologue of eIF4G, PAIP-1, and showed
that such interaction enhanced translation when over-expressed in COS-7 cells (74). However, there has been as yet no clear evidence from
vertebrate systems on potential interactions between poly(A)-binding protein and eIF4G or on the regulation of such interactions by physiological stimuli.
To address this, we have analyzed eIF4E/eIF4G complex formation and the
association of PABP with eIF4G in serum-stimulated Xenopus
kidney cells. Extracts were prepared from cells, and eIF4E and
associated proteins were isolated by m7GTP-Sepharose
chromatography (in the presence of GTP to reduce nonspecific
interactions between proteins and resin) and visualized by
immunoblotting. Fig. 3A, lane 1, shows that even
in the starved state, there is some association of eIF4E with eIF4A,
eIF4G, and PABP. The specificity of this isolation procedure for eIF4E
and associated proteins was demonstrated by the finding that under these assay conditions, none of these proteins were retained by Sepharose lacking the m7GTP moiety (Fig. 3A,
lane 4) and inclusion of m7GTP in the extraction
buffer prevents recovery of eIF4E and associated proteins (data not
shown). This association of eIF4G, eIF4A, and PABP with eIF4E was
enhanced by at least 2-fold in extracts derived from cells treated with
serum for 30 min (Fig. 3A, lane 2; quantified in
Fig. 3B), and was unaffected by ribonuclease treatment of
the extract prior to affinity chromatography (data not shown).
Preincubation of the cells with rapamycin (Fig. 3A, lane 3)
prevented the serum-induced enhancement of association between eIF4E
and eIF4G, indicating a role for the p70S6K signaling
pathway in modulating eIF4F complex formation. The recovery of eIF4A
and PABP on the m7GTP-Sepharose affinity matrix essentially
paralleled that of eIF4G. To examine more closely the association
between eIF4G and PABP, eIF4G was immunoprecipitated from extracts, and
the level of associated eIF4E and PABP was visualized by immunoblotting
(Fig. 3C). Extracts from serum-stimulated cells showed
approximately a 2-fold increase in recovery of both eIF4E and PABP
associated with eIF4G (Fig. 3C, compare lanes 1 and 2); in the absence of antiserum, little eIF4G, PABP, and
eIF4E was recovered with the protein A-Sepharose (lane 4).
As the antiserum to eIF4G did not react with purified eIF4E or PABP
(data not shown), these data suggest that serum treatment of cells
promoted the interaction of eIF4G with eIF4E and PABP. In agreement
with the data where eIF4G was recovered via eIF4E (Fig. 3B),
pretreatment of cells with rapamycin (lane 3) and wortmannin
or LY294002 (data not shown) abrogated the serum-stimulated interaction
of eIF4E with eIF4G. However, in contrast, the enhanced co-isolation of
PABP in eIF4G immunoprecipitates was still apparent in extracts from
cells stimulated in the presence of either agent (Fig. 3C;
quantified in bottom panel), indicating that serum
stimulates the interaction between eIF4G and PABP by a distinct
mechanism that is independent of both the mTOR signaling pathway and
the association of eIF4G with eIF4E. To confirm these results, we also
examined the recovery of associated proteins with PABP selected from
extracts using the poly(A)-Sepharose affinity matrix (Fig. 3D). Again, these data indicated that serum stimulated the
co-isolation of eIF4G (and associated eIF4E) with PABP by approximately
2-fold (Fig. 3D, lane 1 versus lane 2), whereas eIF4G, PABP,
and eIF4E do not associate with the Sepharose resin lacking the
selection moiety (Fig. 3D, lane 4). The serum-stimulated
increase in the association between eIF4G and PABP was largely
unaffected by rapamycin (Fig. 3D, lane 3), whereas the
association between eIF4G and eIF4E was reduced by inhibition of mTOR
signaling (quantified in Fig. 3D, bottom panel). The
quantitative differences in recovery of PABP associated with eIF4G
using the different resins may reflect, in part, the enhanced stability
of the eIF4F complex when recovered via eIF4E (28, 59, 60) or that the
association of PABP with eIF4G is not as stable as that for eIF4E with eIF4G.
Increased eIF4F Complex Formation Is Not Mediated by Dissociation
of 4E-BP1 from eIF4E--
In addition to phosphorylation, the ability
of eIF4E to participate in the initiation process can also be modulated
by its availability, mediated by its interaction with specific binding proteins, 4E-BP1 and 4E-BP2, identified as downstream signaling targets
of rapamycin-sensitive pathways (7, 37, 61). Following our finding that
serum enhanced the association of eIF4E with eIF4G in
Xenopus kidney cells (Fig. 3), we examined the effects of
serum on the phosphorylation of 4E-BP1 and BP2 and their association with eIF4E, using separate independent sources of anti-4E-BP antisera. In several cell types, the phosphorylation of either 4E-binding protein
has been shown to result in a characteristic mobility shift on SDS-PAGE
analysis and their total release from eIF4E (27, 29, 30, 39, 40, 51,
61-63). We were therefore surprised to find that addition of serum to
starved Xenopus kidney cells did not appear to influence the
distribution of either total 4E-BP1 (Fig.
4A, top panel) or total 4E-BP2
(bottom panel) between phosphorylated and non-phosphorylated
forms. This was further illustrated by the lack of effect of rapamycin
on the mobility of these proteins on SDS-PAGE (Fig.
4A, lane 3). To further confirm this finding, the
migration on SDS-PAGE of 4E-BP1 isolated from starved (Fig. 4B,
lane 1) or serum-stimulated (lane 2) cells was compared
with that of 4E-BP1 isolated from NIH 3T3 cells following incubation in
the absence (lane 3) or presence (lane 4) of
serum, as described previously (27). These data show that although serum induced a characteristic mobility shift of 4E-BP1 to the more
phosphorylated form (
These data suggest that in serum-stimulated Xenopus kidney
cells, the increase in association of eIF4G and PABP with eIF4E does
not correlate well with control at the level of dissociation of
4E-binding proteins from eIF4E. These data are distinct from those
obtained with mammalian cells, but the reasons for such a difference
are unclear. At this time, we cannot discount some regulation at the
level of 4E-BP-2, but this does not appear to involve its
phosphorylation. Also, there is the possibility that as yet unknown
eIF4E interacting proteins may play a role in the effects we observe.
Another binding protein, 4E-BP3, has been identified (75), but our
attempts to analyze the interaction between eIF4E and this protein have
been inconclusive as the rabbit antiserum reacted poorly with the
Xenopus protein (data not shown). One possibility is that
the availability of eIF4E may not be limiting in these cells; attempts
to quantify the relative levels of eIF4E and 4E-BPs have proven
difficult due to the existence of multiple forms of these proteins in
Xenopus cells. On the other hand, studies with recombinant
proteins have indicated that both forms of eIF4E found in
Xenopus cells interact to the same extent with 4E-BP1 or
4E-BP2, suggesting that inhibition of phosphorylation of eIF4E does not
involve the selective interaction of one variant of eIF4E with 4E-BPs
(data not shown). As a consequence, the mechanism by which rapamycin
inhibits eIF4F complex formation in the Xenopus kidney cells
remains unclear. It is clear, however, from work using mammalian cells
that the phosphorylation of eIF4E and 4E-BPs can be regulated
independently and that each may play a distinct role in translation
initiation in vivo (27, 64).
The stimulation of protein synthesis plays a central role in the
activation of cell growth (1, 2, 7). Studies using rapamycin as a
pharmacological probe have implicated the signaling pathway governed by
mTOR in the parallel activation of p70S6K and the increase
in cap structure-dependent translation in mitogen-activated cells (7, 31, 37, 38, 40, 51, 52, 62, 65). Rapamycin attenuates the
stimulation of cap structure-dependent translation in
response to serum re-feeding, such that the overall protein synthesis
rate is decreased by 15-40% (27, 40, 49, 53, 54, 66, 67). Current
models suggest that rapamycin stabilizes the eIF4E/4E-BP interaction
and thus prevents the mitogen-induced liberation of eIF4E and
consequent increase in the availability of eIF4F complexes (1, 27, 32).
However, even in mammalian systems, this may be a simplistic view, as
studies have shown a poor temporal correlation between the inhibition
of phosphorylation of 4E-BP1, the inhibition of translation, and eIF4F
complex formation (27, 40, 68). We now show that Xenopus
kidney cells in culture do not seem to exhibit regulation of eIF4E by
4E-BP-1.
Here we have characterized the effect of rapamycin on the
phosphorylation of eIF4E and its interaction with eIF4G following serum
stimulation in the Xenopus kidney cell line B3.2. In these cells, the activation of protein synthesis is associated with enhanced
phosphorylation of eIF4E and eIF4G and decreased phosphorylation of
eIF2 Unlike several other systems examined to date (25-27, 40), inhibition
of mTOR or PI3-kinase signaling blocked the enhancement of protein
synthesis and the phosphorylation of both forms of eIF4E in
serum-stimulated Xenopus kidney cells (Fig. 2). A similar observation has been reported for IL-3-starved, insulin receptor substrate-1-transfected cells treated with insulin (67). Together, these data indicate that eIF4E phosphorylation can occur downstream of
mTOR in some cell types and may stabilize the eIF4F complex (23-28,
59). One explanation for this is that activation of mTOR causes the
dissociation of 4E-BPs from eIF4E, making the latter available as a
kinase substrate. Indeed, the association of eIF4E with 4E-BP1
abrogates its phosphorylation by protein kinase C in vitro
(70). However, this is an unlikely explanation for the data presented
here, because we find no evidence for the enhanced phosphorylation of
4E-BP1 or BP2 and only small effects on the dissociation of the latter
from eIF4E following serum addition to Xenopus kidney cells
(Fig. 4). These data are in agreement with studies in Chinese hamster
ovary cells, in which it was found that eIF4E associated with 4E-BP1
was phosphorylated to the same extent as total eIF4E (39).
During these studies, we have demonstrated an increase in the
co-isolation of eIF4G with PABP following the activation of protein
synthesis (Fig. 3). PABP is found in all eukaryotes and mediates the
stimulatory effects of the poly(A) tail on translation (reviewed in
Refs. 12, 21, 55, and 56). In S. cerevisiae, Pab1p has been
shown to interact directly (via its RNA recognition motif 2) (71) with
eIF4G through a specific sequence N-terminal to the eIF4E binding site
(17, 20). This association is believed to mediate the circularization
of mRNA and promote the poly(A) and PABP-dependent
stimulation of mRNA translation (12, 21, 55, 56, 72). The
synergistic effects of the poly(A) tail and the mRNA cap structure
on translational efficiency and its hormonal stimulation have been
demonstrated in mammalian cells (3). Our data show that in conjunction
with the phosphorylation of eIF4E and eIF4G, there is a reproducible
increase in the recovery of PABP and eIF4G associated with eIF4E
following serum stimulation of Xenopus kidney cells. The
enhanced association of PABP with eIF4G was confirmed by
co-precipitation with antiserum specific to eIF4G and following
isolation of PABP with poly(A)-Sepharose (Fig. 3C). Whereas
inhibition of mTOR signaling prevented the activation of
p70S6K, the phosphorylation of eIF4E, the association
between eIF4E and eIFG, and the activation of protein synthesis, the
binding of PABP to eIF4G was less sensitive to this inhibition. To our knowledge, this represents the first demonstration of physiological regulation of the interaction between PABP and proteins of the eIF4F
complex. At this time, we do not know whether the interaction between
eIF4G and PABP is direct or indirect. However, work completed after our
study has now demonstrated that PABP can interact directly with eIF4G
via a newly discovered N-terminal motif in mammalian systems, although
the strength of this interaction remains to be
determined.2 Our previous
attempts to delineate the site of interaction between eIF4G and PABP
with bacterially expressed proteins were
unsuccessful,3 as the widely
used eIF4G clone (10, 11) lacks the N-terminal motif required for this
interaction (73).
The data presented here indicate that more than one signaling pathway
is involved in regulating the assembly of the eIF4F/PABP complex during
the activation of cell growth in Xenopus kidney cells:
primarily a rapamycin-sensitive pathway that modulates the
phosphorylation of eIF4E and eIF4F complex formation, and also a
rapamycin-resistant pathway, which may not be rate-limiting for
translation, that involves the association of PABP with eIF4G.
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EXPERIMENTAL PROCEDURES
-glycerophosphate, pH 7.2, 2 mM benzamidine), isolated
by centrifugation, washed in the same buffer, and lysed by the addition
of 200 µl Buffer B (10 mM NaCl, 10 mM
MgCl2, 1 µM microcystin, 10 mM
Tris-HCl, pH 7.5, 1% (by volume) Triton X-100, 1% (by volume) sodium
deoxycholate, 50 mM
-glycerophosphate, 50 mM
NaF, 2 mM EGTA, 2 mM EDTA, 2 mM
benzamidine, 7 mM 2-mercaptoethanol). Following a 5-min
incubation on ice with occasional vortexing, extracts were centrifuged
for 5 min at 15,000 rpm in a cooled microcentrifuge. The supernatant
was frozen in liquid nitrogen and stored at
70 °C.
, 4E-BP1,
and 4E-BP2 was detected with specific antisera, as described in the
individual figure legends. For specific analysis of 4E-BP1 and 4E-BP2,
total cell extract was boiled for 8 min, the precipitated protein was
removed by centrifugation, and the supernatant was subjected to
SDS-PAGE and immunoblotting (29, 30).
-glycerophosphate, 50 mM NaF, 2 mM
benzamidine, 7 mM 2-mercaptoethanol, 0.1 mM
GTP), and recovered protein was eluted directly into sample buffer for
either SDS-PAGE or VSIEF, prior to analysis (25, 27). In a similar manner, to directly isolate PABP and associated proteins, cell extracts
were subjected to affinity chromatography using poly(A)-Sepharose, and
the resin washed in Buffer C prior to elution with SDS-PAGE sample
buffer. Immunoprecipitation of eIF4G and associated proteins from cell
extracts was as described previously, except the resin was washed five
times (with 1 ml of buffer each time) (27, 43).
RESULTS
, we have been able to
demonstrate that serum stimulation caused a reduction in the
phosphorylation of this factor (Fig. 1E).
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Fig. 1.
Serum stimulation of Xenopus
kidney cells enhances the phosphorylation of eIF4E and eIF4F
complex formation. A, Xenopus kidney B3.2
cells were serum-starved for 24 h prior to the addition of serum
(10%) and 10 µCi/ml [35S]methionine. At the times
indicated, cells were harvested, and the labeling of total protein was
determined as described under "Experimental Procedures." The effect
of serum on translation is expressed as percentage of change relative
to the serum-starved cells. The experiment was carried out three times,
each in triplicate, and the error bars indicate the S.E.
B, cells were incubated in the absence (lane 1)
or presence (lane 2) of 10% (v/v) serum for 30 min prior to
the preparation of extracts, as described. Aliquots of extracts
containing equal amounts of protein were resolved directly by SDS-PAGE,
followed by immunoblotting with antiserum specific to total eIF4E (27)
(top panel) or with antiserum that specifically recognizes
the phosphorylated form of eIF4E (bottom panel; see under
"Experimental Procedures"). C, aliquots of extracts
containing equal amounts of protein were subjected to
m7GTP-Sepharose affinity chromatography to isolate total
eIF4E. The two forms of eIF4E present in Xenopus cells were
resolved by VSIEF and visualized by immunoblotting with antiserum
specific to total eIF4E. The migration of the more phosphorylated
variant of each form of eIF4E resolved by VSIEF is indicated
(eIF4E (P)). D, serum-starved cells were
incubated with 200 µCi/ml [32P]orthophosphate in
phosphate-free medium for 2 h prior to further incubation in the
absence (lane 1) or presence (lane 2) of serum
(10%) for 30 min. Extracts were prepared, and eIF4E and associated
proteins were isolated by m7GTP-Sepharose chromatography
and resolved by SDS-PAGE. The resulting autoradiograph is presented,
with the bottom panel showing the phosphorylation of eIF4E
and the top panel showing the labeling of associated eIF4G.
E, in parallel cultures to those described in D,
extracts were prepared, but without [32P]orthophosphate
labeling. Unfractionated cell extracts containing equal amounts of
protein were resolved by SDS-PAGE and immunoblotted with antiserum to
either total eIF2 or with antiserum specific to the phosphorylated
form of eIF2
(bottom panel). In all cases, these data are
representative of those obtained in at least three separate
experiments.
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Fig. 2.
Rapamycin abrogates the serum-induced
phosphorylation of eIF4E in Xenopus kidney cells.
A, serum-starved B3.2 cells were incubated in the absence or
presence of rapamycin (50 nM) for 1 h, prior to the
addition of serum (10%). Following a 30-min incubation, extracts were
prepared and adjusted to equal protein concentration, and total eIF4E
was isolated by m7GTP-Sepharose chromatography prior to
analysis by VSIEF and immunoblotting with anti-eIF4E antiserum. In a
parallel culture, cells were prelabeled with
[32P]orthophosphate as described in Fig. 1D
prior to incubation in the absence or presence of serum (10%) and
rapamycin (50 nM). Total eIF4E was isolated as described
and resolved by SDS-PAGE. The resulting autoradiograph is presented
(middle panel). Using densitometric scanning, the proportion
of total eIF4E in the phosphorylated form was quantified from analysis
of cell extracts by VSIEF and immunoblotting (bottom panel).
Lane 1, control cells; lane 2, serum-stimulated
cells; lane 3, serum-stimulated in the presence of
rapamycin. The experiment was carried out four times, and the
error bars indicate S.E. B, cells were
serum-starved for 24 h prior to the addition of serum (10%) and
10 µCi/ml [35S]methionine for 30 min. The level of
total protein synthesis was determined as described under
"Experimental Procedures," and the effect of serum and rapamycin on
early translation rates is expressed as percentage of change relative
to the serum-starved cells. These data are representative of those
obtained in at least three separate experiments.
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Fig. 3.
Serum promotes the co-isolation of
poly(A)-binding protein with eIF4F. A, cell extracts
were prepared as in Fig. 2A and adjusted to equal protein
concentration, and eIF4E and associated proteins were isolated by
m7GTP-Sepharose chromatography (lanes 1-3) or
Sepharose alone (lane 4). Recovered proteins were resolved
by SDS-PAGE and visualized by immunoblotting using the specific
antiserum indicated. The data presented are from a single experiment but are representative of
those obtained in 10 separate experiments. B, quantification
(by densitometric scanning) of the amount of eIF4G, PABP, and eIF4A
recovered with eIF4E. For each, the amount of protein was normalized
for the recovery of eIF4E and is expressed as percentage of control.
Lane 1, serum-starved cells; lane 2, addition of
serum; lane 3, serum following preincubation with rapamycin.
The experiment was carried out 10 times, and the error bars
indicate S.E. C, extracts containing equal amounts of
protein were subjected to immunoprecipitation with (lanes
1-3) or without (lane 4) anti-eIF4G antiserum, as
described under "Experimental Procedures." Recovered proteins were
resolved by SDS-PAGE and visualized by immunoblotting using the
specific antiserum indicated. The bottom panel shows
quantification of the amount of eIF4G, PABP, and eIF4A recovered in the
assay. For each, the amount of protein was normalized for the recovery
of eIF4G and is expressed as percentage of control. The experiment was
carried out nine times, and the error bars indicate the S.E.
D, PABP was directly isolated from extracts by
poly(A)-Sepharose affinity chromatography (lanes 1-3) or
Sepharose alone (lane 4), and recovered proteins were
resolved by SDS-PAGE and visualized by immunoblotting using the
specific antiserum indicated. The bottom panel shows
quantification of the amount of eIF4G, PABP and eIF4A recovered in the
assay. For each, the amount of protein was normalized for the recovery
of PABP and is expressed as percentage of control. The experiment was
carried out six times, and the error bars indicate the
S.E.
) in the NIH3T3 cell system (27) (Fig.
4B, lane 4 versus lane 3), there was no such mobility shift in the Xenopus cell system (lane 2 versus lane
1). Similar results were found when antiserum specific to 4E-BP2
was used (data not shown). In addition, although
[32P]phosphate labeling of 4E-BP1 and 4E-BP2 was
increased by serum in vivo (Fig. 4B, bottom
panel, lane 2 versus lane 1), again there was no evidence for a
phosphorylation-induced mobility shift of these proteins, and the
increased phosphorylation of 4E-BP1 and BP2 was unaffected by
rapamycin, suggesting phosphorylation on rapamycin-insensitive sites
(Ref. 61 and data not shown). Fig. 4C shows that the
addition of serum had little or no influence on the association of
4E-BP1 with eIF4E, although the amount of associated 4E-BP-2 did
decrease by up to 30% in some experiments (compare lanes 2 and 1; quantified in panel D). In general, the latter effect was more variable between experiments and was often accompanied by decreased recovery of 4E-BP-2 in the cell extracts following serum stimulation (data not shown). The reasons for this are
unclear. Direct isolation of 4E-BP1 by immunoprecipitation confirmed
that serum had little or no effect on the association of this protein
with eIF4E (data not shown). Preincubation of cells with rapamycin
(Fig. 4C, lane 3), which is known to increase the
level of the eIF4E/BP complex above basal levels in mammalian cells (7,
29-32), did not significantly influence the recovery of 4E-BP1
associated with total eIF4E (quantified in Fig. 4D). However, although rapamycin promoted the interaction between eIF4E and
4E-BP-2, inhibition of mTOR signaling did not increase the level of
this complex above basal levels.
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Fig. 4.
Serum-stimulated eIF4F complex formation
occurs without concomitant dissociation of 4E-BP1 from eIF4E.
A, serum-starved B3.2 cells were incubated in the absence
(lanes 1 and 2) or presence (lane 3)
of rapamycin (50 nM) for 1 h prior to the addition of
serum (10%) for 30 min (lanes 2 and 3). Extracts
were prepared and adjusted to equal protein concentration, and 4E-BPs
were isolated as described under "Experimental Procedures." Total
4E-BP1 (top panel) and 4E-BP2 (bottom panel) was
visualized by SDS-PAGE and immunoblotting; for 4E-BP1, the antiserum
(from A. A. Thomas, Utrecht, The Netherlands) was specific to this
protein, whereas the antiserum to 4E-BP-2 (from N. Sonenberg, Montreal,
Canada), also cross-reacted with 4E-BP-1. B, top panel,
extracts prepared from Xenopus kidney cells as above
(lanes 1 and 2) or from serum-starved (lane
3) or stimulated (lane 4) NIH 3T3 cells, as described
(27), were resolved by SDS-PAGE, and 4E-BP1 was visualized by
immunoblotting as in A. In addition, cells were incubated
with [32P]orthophosphate as described in Fig. 1, prior to
incubation in the absence (lane 1) or presence (lane
2) of serum. Extracts were prepared and adjusted to equal protein
concentration, 4E-BPs were isolated as described under "Experimental
Procedures," and the resulting autoradiograph is shown. The identity
of 4E-BP1 and 4E-BP2 was confirmed by immunoblotting (data not shown).
C, extracts were prepared as in A, but eIF4E and
associated proteins were isolated by m7GTP-Sepharose
chromatography prior to resolution by SDS-PAGE. Recovered proteins were
visualized by immunoblotting using the specific antisera described in
A. D, the amount of 4E-BP recovered in
association with eIF4E (C) was quantified by scanning
densitometry and normalized for recovery of eIF4E, and it is expressed
as a percentage of control. The experiment was carried out six times,
and the error bars indicate the S.E.
DISCUSSION
(Fig. 1). In mammalian tumor cells, decreased phosphorylation of eIF2
, with concomitant enhancement of eIF2B function, has been
reported to play a role in serum stimulation of translation (69).
However, to date, our attempts to investigate changes in the activity
of eIF2B in extracts from Xenopus kidney cells have been
unsuccessful. Our data on the phosphorylation of eIF4E and eIF4G are
consistent with earlier studies using Xenopus oocytes and
primary T cells (25, 26), in which the recovery of phosphorylated eIF4G
associated with eIF4E was increased in response to hormones or the
activation of cell growth. As phosphorylation of eIF4E (28) and eIF4G
(59) has been shown to increase the ability of these proteins to
interact with the cap structure, and the association of eIF4E with
eIF4G enhances the binding of eIF4E to the cap structure (60), these
data indicate that eIF4F phosphorylation and complex formation are
important in the regulation of translation in these cells.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Fabrizio Loreni
(Department of Biology, Universitat di Roma "Tor Vergata," Rome,
Italy) for provision of the Xenopus kidney cell line B3.2
and to Dr. N. Sonenberg for communicating data prior to publication.
Various antisera to 4E-BP1 (PHAS-I) were kindly provided by Dr. J. Lawrence, Jr. (Washington University School of Medicine, St. Louis,
MO), Prof. R. M. Denton (Department of Biochemistry, University of
Bristol, United Kingdom), and Dr. A. A. M. Thomas (Molecular
Cell Biology, University of Utrecht, The Netherlands);
antiserum to 4E-BP2 was kindly provided by Dr. N. Sonenberg (Department
of Biochemistry, McGill University, Montreal, Canada); antiserum to
PABP was kindly provided by Dr. S. Arrigo (Medical University of South
Carolina, Charleston, SC), and Dr. D. Schoenberg (Department of
Pharmacology, Ohio State University, Columbus, OH); the monoclonal
antibody recognizing eIF2 was developed in the laboratory of the
late Dr. E. Henshaw, and phosphospecific antiserum was from Dr. G. Krause (Wayne State University School of Medicine, Detroit, MI).
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Note Added in Proof |
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Piron et al. (76) have recently demonstrated that eIF4GI interacts directly with eIF4GI and that PABP is a part of the eIF4F complex.
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FOOTNOTES |
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* This work was supported by Grants 040800/DG/CS, 045109/Z/, 050703/Z/, and 045619/Z/ from The Wellcome Trust and Grant G06562 from the Biotechnology and Biological Sciences Research Council (United Kindom).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.
Supported by a Wellcome Trust Prize Studentship.
§ A Senior Research Fellow of The Wellcome Trust. To whom correspondence should be addressed. Tel.: 01273-678544; Fax: 01273-678433; E-mail s.j.morley{at}sussex.ac.uk.
2 N. Sonenberg, personal communication.
3 L. McKendrick and S. J. Morley, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: eIF, eukaryotic initiation factor; m7GTP, 7-methyl guanosine triphosphate; Mops, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; VSIEF, vertical slab isoelectric focusing; PABP, poly(A)-binding protein..
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REFERENCES |
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