| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 36, 32855-32859, September 6, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Biochemistry Laboratory, School of Biological Sciences,
University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom
Received for publication, June 25, 2002, and in revised form, July 19, 2002
Previous work has suggested that increased
phosphorylation of eukaryotic initiation factor (eIF) 4E at
Ser-209 in the C-terminal loop of the protein often correlates with
increased translation rates. However, the functional consequences of
phosphorylation have remained contentious with our understanding of the
role of eIF4E phosphorylation in translational control far from
complete. To investigate the role for eIF4E phosphorylation in de
novo translation, we studied the recovery of human kidney cells
from hypertonic stress. Results show that hypertonic shock caused a
rapid inhibition of protein synthesis and the disaggregation of
polysomes. These changes were associated with the dephosphorylation of
eIF4G, eIF4E, 4E-binding protein 1 (4E-BP1), and ribosomal protein S6.
In addition, decreased levels of the eIF4F complex and increased
association of 4E-BP1 with eIF4E were observed over a similar time
course. The return of cells to isotonic medium rapidly promoted the
phosphorylation of these initiation factors, increased levels of eIF4F
complexes, promoted polysome assembly, and increased rates of
translation. However, by using a cell-permeable, specific inhibitor of
eIF4E kinase, Mnk1 (CGP57380), we show that de novo
initiation of translation and eIF4F complex assembly during this
recovery phase did not require eIF4E phosphorylation.
Nuclear-encoded mRNAs have a unique cap structure at their 5'
terminus with general composition m7G(5')ppp(5')N (where N
is any nucleotide). The presence of this cap structure has a strong
stimulatory effect on the translation of mRNA, facilitating the
recruitment of translation initiation factors
(eIFs)1 to allow ribosome
binding and initiation at the correct start site (for review, see Refs.
1-5). eIF4E, a protein whose three-dimensional structure resembles a
cupped hand, specifically interacts directly with the cap via its
concave surface (6, 7). It also forms a complex with the scaffold
protein eIF4G (8) on its convex surface. eIF4G in turn recruits other
initiation factors, such as eIF3, eIF4A, and poly(A)-binding protein
(PABP) to the 5' end of the mRNA, allowing for efficient unwinding
of secondary structure in the 5' untranslated region (9) and the
functional circularization of mRNA believed to be necessary to
promote efficient translation (2, 4, 5, 10, 11). In addition, eIF4E
binds a family of regulatory proteins, 4E-binding proteins (4E-BPs),
which compete with eIF4G for sites on the convex surface of eIF4E (12,
13) and inhibit formation of initiation complexes (14). Association of
4E-BPs with eIF4E is modulated by phosphorylation events controlled via
the mammalian target of rapamycin signaling pathway (3, 5, 15,
16).
Human eIF4E undergoes regulated phosphorylation on Ser-209 (17, 18),
and while correlations exist between its enhanced phosphorylation and
increased rates of protein synthesis (for review, see Refs. 1, 5, 19,
and 20), the functional consequences of this modification have remained
contentious. We (20-22) and others (2, 23-25) have shown that
phosphorylation of eIF4E occurs via multiple signaling pathways. Two
protein kinases, Mnk1 and Mnk2, which act at the convergence point of
extracellular-signal regulated kinase and stress-activated p38
mitogen-activated protein (MAP) kinase (25-28), phosphorylate eIF4E at
the physiological site in vitro and in vivo (27,
29, 30). Mnk1/Mnk2 interact directly with eIF4G (30-33), bringing them
in close proximity to eIF4E within the eIF4F complex (2, 5).
While phosphorylation of eIF4E has been reported to increase its
interaction with the cap structure (34), recent studies have suggested
that our understanding of the role of eIF4E phosphorylation in
translational control is far from complete (5). First, increased phosphorylation of eIF4E observed during cell stress is sometimes associated with a global inhibition of protein synthesis (5, 21, 22,
25). In addition, phosphorylation of eIF4E is not required for
restoration of translation in an eIF4E-dependent system
in vitro, and both wild-type and phosphorylation site
variants are able to rescue the lethal phenotype of eIF4E deletion in
Saccharomyces cerevisiae (35). Also it appears that
phosphorylation of eIF4E is not a prerequisite for mitogen-induced
cap-dependent translation in cultured cells (28).
Furthermore, in adult cardiocytes, eIF4E phosphorylation had no effect
on eIF4F complex formation or total rates of protein synthesis (36).
However, eIF4E phosphorylation does appear to be important for cell
growth in Drosophila; transgenic Drosophila
expressing a non-phosphorylatable form of eIF4E in a null background
show reduced viability, smaller cell size, and developmental delays
(37). Despite this, no strong phenotype was observed by expressing a
form of eIF4E that mimicked eIF4E phosphorylation (37). Biophysical
studies by Scheper et al. (33) have demonstrated that
phosphorylation of eIF4E actually reduces its binding to mRNA caps
by promoting its rate of dissociation. These data are consistent with
an earlier prediction (19) that, with analogy to the transcriptional
machinery, phosphorylation may be required for the release of eIF4F
from the cap complex during the initiation process (33). Alternatively,
phosphorylation of eIF4E may be involved in reprogramming (de
novo) translation by promoting the rate of release of factors from
existing initiation complexes to give under-represented mRNAs a
chance to compete for ribosome binding (5, 19, 33).
Exposure of mammalian cells to hypertonic stress is a very effective
means of inducing a transient fall in protein synthesis, which is
rapidly reversible upon restoration of isotonic medium (38-40). This
system has been used as a tool to study the function of the eIF4F
complex during de novo recruitment of mRNA in intact cells (41). In this study we used hypertonic stress to investigate the
role for eIF4E phosphorylation in de novo translation using a cell-permeable inhibitor of Mnk, CGP57380 (28), to prevent the
rephosphorylation of eIF4E during recovery from such stress.
Chemicals and Biochemicals--
Materials for tissue culture
were from Invitrogen, and fetal calf serum was from Labtech
International. [35S]Methionine was from ICN, and
phosphospecific antisera to eIF4E (Ser-209, catalog no. 9741),
eIF4G (Ser-1108, catalog no. 2441), p38 MAP kinase
(Thr-180/Tyr-182, catalog no. 9211), 4E-BP1 (Ser-65, catalog no. 9451),
and ribosomal protein S6 (Thr-421/Ser-424, catalog no. 9204) were from
Cell Signaling Technology. Immobilon polyvinylidene difluoride was from
Millipore, and m7GTP-Sepharose was from Amersham
Biosciences. CGP57380, a specific, cell-permeable inhibitor of Mnk1
(28), was a kind gift from Dr. Hermann Gram, Novartis Pharma AG, Basel.
Unless otherwise stated, all other chemicals were from Sigma.
Cell Culture--
Human 293 kidney cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. When used, CGP57380 or Me2SO alone was added
to cultures and was present in all subsequent incubations as indicated.
Protein Synthesis Measurements--
Cells were cultured in
six-well plates, and [35S]methionine was added to the
complete growth medium at the specific activity and for the times
indicated in the individual figure legends. Cells were harvested, and
the incorporation of methionine into protein was determined by
trichloroacetic acid precipitation as described previously (22).
Preparation of Cell Extracts--
Following treatment, cells
were scraped into PBS containing 40 mM SDS-PAGE, Isoelectric Focusing, and Immunoblotting--
Samples
containing equal amounts of protein were resolved by SDS-PAGE and
processed as described previously (21, 22). Antiserum specific for the
C-terminal domain of eIF4GI and those specific to eIF4E, eIF2 Isolation of eIF4E and Associated Factors by
m7GTP-Sepharose Chromatography--
eIF4E and
associated proteins were isolated by m7GTP-Sepharose
chromatography as described previously (21, 22), and recovered proteins
were visualized by immunoblotting.
Polysome Analysis--
Cells were isolated as described above
and lysed by vortexing in 200 µl of Buffer B (200 mM
Tris-HCl, pH 8.5, 40 mM sodium fluoride, 80 mM
KCl, 7 mM 2-mercaptoethanol, 2 mM benzamidine, 2 mM MgCl2, 200 µg/ml cycloheximide, 0.5%
(by volume) Igepal and deoxycholate). Extracts were fractionated on
15-50% (w/v) sucrose gradients in 20 mM MOPS-KOH, pH 7.2, 100 mM KCl, 2 mM MgCl2 for 75 min
at 42,000 rpm in a Beckman SW50.1 rotor at 4 °C and visualized with
an ISCO UA6 fractionator.
CGP57380 Prevents the Serum-stimulated Phosphorylation of eIF4E
without Affecting eIF4G Phosphorylation or Levels of the eIF4F
Complex--
In general, previous work has suggested that increased
phosphorylation of eIF4E at Ser-209 in the C-terminal loop of the
protein correlates with increased translation rates (1, 5, 19, 23, 42).
These studies have prompted the view that eIF4E phosphorylation may
have a role in modulating the cap binding activity of the protein,
invoking elaborate models based on biochemical studies and
three-dimensional structure analysis (2, 6, 7). However, the functional
consequences of phosphorylation have remained elusive, and several
exceptions have called into question the correlation with global rates
of protein synthesis (for review, see Ref. 5). It has been
suggested that phosphorylation of eIF4E, rather than modulating global protein synthesis, may be involved in reprogramming translation (5, 19), possibly by promoting the rate of release of
factors from existing initiation complexes to give under-represented mRNAs a chance to compete for the 40 S ribosome (33). Such a mechanism could resolve apparently conflicting observations of increased phosphorylation of eIF4E following either mitogenic stimulation or the application of cell stress.
Recently a novel, non-toxic, specific inhibitor of Mnk1 (CGP57380) has
been described that prevents eIF4E phosphorylation in response to
mitogens (28). In our study we have used CGP57380 to investigate the
role of eIF4E phosphorylation in de novo initiation of
translation and eIF4F complex assembly. Fig.
1A shows that CGP57380 had no
significant effect on the serum-stimulated up-regulation of translation
rates in starved 293 cells. In addition, SDS-PAGE analysis of
initiation factors shows that CGP57380 did not affect the
serum-stimulated phosphorylation of eIF4G (Fig. 1B,
lanes 1 and 6 versus lanes 5 and
10; quantified in Fig. 1C). However, although
eIF4E phosphorylation was normally increased in response to serum (Fig.
1B, lane 1 versus 6),
consistent with published data (28), both the basal level and the
serum-stimulated phosphorylation of eIF4E were reduced by CGP57380 at
concentrations greater than 10-15 µM. Isoelectric
focusing and immunoblotting for eIF4E confirmed the dephosphorylation
of eIF4E in response to CGP57380 (Fig. 1B); quantification
of these data is shown in Fig. 1C. Fig. 1D
shows that although CGP57380 could reduce eIF4F levels to a small
extent in starved cells (lane 4 versus lane 1),
treatment of cells with up to 20 µM CGP57380 had no
effect on the serum-stimulated recovery of eIF4G, PABP, or eIF4A
associated with eIF4E (lane 8 versus lane 4).
This finding is consistent with the observation that association of
4E-BP1 with eIF4E was unaffected by CGP57380 treatment alone (data not
shown).
Exposure of Human Kidney Cells to Hypertonic Medium Results in the
Dephosphorylation of Key Translation Initiation Factors--
To
determine conditions for the investigation of the role of eIF4E
phosphorylation in de novo initiation events, human kidney cells were incubated in the presence of NaCl in excess to that already
present in the growth medium. Fig. 2
shows that 50-200 mM added NaCl resulted in a
concentration- (A) and time-dependent (B) inhibition of protein synthesis. To address whether
hypertonic shock is associated with changes in the phosphorylation
status of key initiation factors, extracts were prepared, and proteins were visualized by immunoblotting with phosphospecific antisera. Fig.
2C shows that dephosphorylation of a key phosphorylation site in 4E-BP1 (Ser-65) was complete within 15 min, while that of eIF4G
(Ser-1108, Refs. 3 and 43) and S6 (Thr-421/Ser-424) occurred at a
slower rate with no change in overall levels of initiation factors
recovered in the extracts. eIF4E dephosphorylation (Ser-209) was
evident at 15 min using a phosphospecific antibody for detection
although hypertonic stress activates p38 MAP kinase, an upstream
regulator of the physiological eIF4E kinase, Mnk1 (Refs. 26 and 29, and
see Fig. 2D). However, isoelectric focusing of eIF4E showed
that salt treatment for 60 min was required to reduce levels of the
phosphorylated protein below 5% (Fig. 2B). In agreement
with published data for HeLa cells (40), over the times used in this
study there was little change in the phosphorylation status of eIF2 De Novo Initiation of Protein Synthesis Does Not Require Increases
in the Level of eIF4E Phosphorylation--
Using the conditions
determined above, we investigated the requirement for eIF4E
phosphorylation in de novo protein synthesis following
recovery from hypertonic shock, a process requiring the integrity of
the eIF4F complex (41). Incubation of cells with 200 mM
NaCl for 60 min resulted in an inhibition of the rate of protein
synthesis (Fig. 3A) and
disaggregation of polysomes (Fig. 3B). Together these events
are characteristic of a defect in the initiation stage of translation,
a finding confirmed by the observation that polysomes were stabilized
by the inclusion of cycloheximide in the medium during salt treatment
(data not shown). On return to isotonic medium, protein synthesis rates recovered to control levels within 30-45 min (Fig. 3A) and
polysomes reformed (Fig. 3B). In the presence of 20 µM CGP57380, polysomes reformed on return to isotonic
conditions, but there was a distinct shift toward smaller polysomes,
suggesting that eIF4E phosphorylation may have a subtle role in the
initiation of translation. Further work will be required to address
this possibility.
Consistent with the data presented in Fig. 2, hypertonic shock for 60 min caused a decrease in the phosphorylation of eIF4G, eIF4E, 4E-BP1,
and S6, an increase in the phosphorylation of p38 MAP kinase (Fig.
2C, lane 2 versus lane 1), and a
decrease in the recovery of eIF4G associated with eIF4E. Conversely,
there was an increase in association of 4E-BP1 with eIF4E following salt stress (Fig. 2D, lane 2 versus lane
1). Return of the cells to isotonic medium promoted a
time-dependent phosphorylation of total eIF4G, which
occurred with similar kinetics in the absence or presence of CGP57380
(Fig. 3C). Similarly, recovery was also associated with
phosphorylation of 4E-BP1 on Ser-65 as visualized with the
phosphospecific antiserum and a characteristic mobility shift of the
total 4E-BP1 on SDS-PAGE using antiserum that recognizes the protein
irrespective of its phosphorylation status (Fig. 3C). Furthermore, these events and the dephosphorylation (and inactivation) of p38 MAP kinase were seen to occur with essentially the same kinetics
in the absence (Fig. 3C, lanes 3-6) or presence
of CGP57380 (lanes 9-12). In contrast, although the
phosphorylation of total eIF4E increased from very low levels in the
absence of inhibitor on return to isotonic conditions (Fig.
3C, lanes 3-6), CGP57380 completely prevented
this increase (lanes 9-12). These data were confirmed with
isoelectric focusing/immunoblotting analysis of eIF4E (data not shown)
and were not due to changes in the levels of eIF4E recovered in the
extracts (Figs. 3C). The phosphorylation of S6 lagged behind
that of eIF4E and 4E-BP1 and the increase in the rate of protein
synthesis during the recovery period. These data are in agreement with
previous findings with MPC11 cells in showing that changes in S6
phosphorylation are too slow to account for rapid changes in protein
synthesis following tonicity shifts (39).
We also examined the phosphorylation status of the eIF4F complex during
recovery from salt stress. Following the return of cells to isotonic
conditions, the assembly of the eIF4F complex occurred rapidly both in
the absence and presence of CGP57380. In each instance, this was
associated with the release of 4E-BP1 from eIF4E and enhanced binding
of eIF4G to eIF4E (Fig. 3D). The increase in levels of eIF4F
complex was confirmed by the finding that there was enhanced
association of PABP and eIF4A with eIF4E under such conditions (data
not shown). The phosphorylation of the population of eIF4G
recovered in the eIF4F complex also increased with similar kinetics to
that observed for the total eIF4G in the absence or presence of
CGP57380 (Fig. 3D). In contrast, the phosphorylation of
eIF4E was completely prevented by the presence of CGP57380 during the
recovery period (Fig. 3D, lanes 9-12). Therefore, although the integrity of eIF4G is critical for the reprogramming (de novo) translation during recovery of cells
from hypertonic stress (41), eIF4E phosphorylation was not a
prerequisite for increased rates of de novo translation or
enhanced levels of eIF4F complexes. Further work will be needed to
resolve the role of eIF4E phosphorylation in growth control in cultured cells.
CGP57380, a specific, cell-permeable inhibitor
of Mnk1, was a kind gift from Dr. Hermann Gram, Novartis Pharma AG, Basel.
*
This research was supported by Grants 040800 and 050703 from
The Wellcome Trust.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.
Published, JBC Papers in Press, July 22, 2002, DOI 10.1074/jbc.C200376200
The abbreviations used are:
eIF, eukaryotic
initiation factor;
4E-BP, 4E-binding protein;
m7GTP, 7-methylguanosine triphosphate;
MOPS, 3-(N-morpholino)propanesulfonic acid;
PABP, poly(A)-binding
protein;
MAP, mitogen-activated protein;
PBS, phosphate-buffered
saline.
Phosphorylation of Eukaryotic Initiation Factor (eIF) 4E
Is Not Required for de Novo Protein Synthesis following
Recovery from Hypertonic Stress in Human Kidney Cells*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-glycerophosphate
and 2 mM benzamidine and isolated in a cooled microfuge.
Cell pellets were resuspended in 200 µl of ice-cold Buffer A (50 mM Mops-KOH, pH 7.2, 2.5 mM EGTA, 1 mM EDTA, 40 mM sodium fluoride, 80 mM KCl, 7 mM 2-mercaptoethanol, 2 mM benzamidine, 0.1 mM GTP, 2 mM
Na3VO4) and lysed by the addition of 0.5% (by
volume) each of Igepal and deoxycholate and vortexing. Cell debris and
nuclei were removed by centrifugation in a microcentrifuge for 5 min at
4 °C, and the resultant supernatants were frozen in liquid
N2.
, and
phospho-eIF2 were as described previously (22); total levels of
eIF4G, eIF4E, eIF2, PABP, eIF4A, and 4E-BP1 were visualized with
alkaline phosphatase-conjugated secondary antibodies, while the
determinations using commercial phosphospecific antisera and those for
p38 MAP kinase used enhanced chemiluminescence for detection. In all
cases, care was taken to ensure that detection was within the linear
range of the response. One-dimensional vertical isoelectric focusing
gels used for the analysis of eIF4E phosphorylation were performed as
described previously (21, 22).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (25K):
[in a new window]
Fig. 1.
Inhibition of eIF4E phosphorylation with
CGP57380 does not prevent the activation of protein synthesis in
response to serum. A, cells (in 10-cm plates) at
approximately 50% confluence were serum-starved in 0.1% fetal calf
serum for 48 h and then incubated for 60 min in CGP57380 (or
Me2SO alone) at the concentrations shown. PBS (solid
bars) or fetal calf serum (gray bars) was added to 10%
(by volume), and the incubation continued for 60 min. For the final 30 min of this period, [35S]methionine (50 µCi/ml) was
added, extracts were prepared, and the incorporation of methionine into
protein was determined by trichloroacetic acid precipitation. The
presented data are the means ± S.D. (bars) of
two separate experiments, each performed in triplicate. B,
cells were serum-starved for 48 h and then incubated for 60 min in
the absence or presence of the final concentrations of CGP57380
indicated before the addition of PBS (lanes 1-5) or 10%
fetal calf serum (lanes 6-10) for 60 min. Extracts were
prepared and resolved by SDS-PAGE with the phosphorylation status of
eIF4G and eIF4E visualized by immunoblotting. Results are
representative of those obtained in three separate experiments.
C, quantification of data for the phosphorylation of eIF4G
and eIF4E expressed as percentage of un- stimulated control levels (set at 100%). The presented data are
the means ± S.D. (bars) of three separate experiments.
D, cells were serum-starved for 48 h and then incubated
in the absence (lanes 1 and 5) or presence of 5 µM (lanes 2 and 6), 10 µM (lanes 3 and 7), or 20 µM (lanes 4 and 8) CGP57380 for 60 min before the addition of PBS (lanes 1-4) or 10% fetal
calf serum (lanes 5-8) for 60 min. Extracts were subjected
to m7GTP-Sepharose chromatography, and eIF4E and associated
factors were resolved by SDS-PAGE and visualized by immunoblotting.
Results are representative of those obtained in three separate
experiments. FCS, fetal calf serum.
in response to hypertonic stress (Fig. 2C). We also
investigated the effects of hypertonic shock on the eIF4F complex.
Isolation of eIF4E and associated factors with
m7GTP-Sepharose (Fig. 2E) indicated that
hypertonic shock caused a dissociation of eIF4G, PABP, and eIF4A from
eIF4E, indicative of a decrease in eIF4F complex levels. As predicted
from accepted models (2, 3, 5), the dissociation of eIF4G from eIF4E occurred concomitantly with an increase in binding of the less phosphorylated forms of 4E-BP1 to eIF4E.
View larger version (21K):
[in a new window]
Fig. 2.
Exposure of 293 cells to hypertonic medium
causes an inhibition of protein synthesis. A, cells
were incubated for 30 min in complete growth medium supplemented with
NaCl at the concentrations indicated in addition to what was present in
Dulbecco's modified Eagle's medium. For the last 15 min of this
period, [35S]methionine (5 µCi/ml) was added, and
samples were processed as described in Fig. 1. The presented
data are the means ± S.D. (bars) of two separate
experiments, each performed in triplicate. B, cells were
incubated for the times indicated without or following addition of the
indicated concentrations of NaCl in addition to that present in the
complete growth medium. For the last 10 min of this period,
[35S]methionine (5 µCi/ml) was added, and samples were
processed as described in Fig. 1. Results are expressed as the
percentage of the rate of protein synthesis in untreated cells (set at
100%), and the presented data are the means ± S.D.
(bars) of two separate experiments, each performed in
triplicate. C, cells were incubated in the presence of 200 mM added NaCl, and extracts were prepared at the times
indicated. Equal amounts of protein were resolved by SDS-PAGE, and the
phosphorylation status of eIF4G, eIF4E, eIF2 , 4E-BP1, and ribosomal
protein S6 was visualized by immunoblotting. Results are representative
of those obtained in three separate experiments.
D, cells were incubated in the absence or presence of 200 mM added NaCl for 30 min, and extracts were prepared as
described. Equal amounts of protein were resolved by SDS-PAGE,
and the phosphorylation status of p38 MAP kinase was visualized by
immunoblotting. E, extracts described in C were
subjected to m7GTP-Sepharose chromatography, and eIF4E and
associated factors were resolved by SDS-PAGE and visualized by
immunoblotting. Results are representative of those obtained in three
separate experiments. MAPK, MAP kinase.
View larger version (28K):
[in a new window]
Fig. 3.
Recovery of protein synthesis rates following
salt stress does not require the phosphorylation of eIF4E.
A, cells were incubated in complete growth medium for 60 min
without (con) or following addition of 200 mM
NaCl. For the last 10 min of this period, [35S]methionine
(5 µCi/ml) was added, and samples were processed as described in Fig.
1. Parallel cultures were rinsed in fresh complete medium and
incubated in the absence (open bars) or presence of 20 µM CGP57380 (closed bars) for the times
indicated. For the last 10 min of this period, the rate of
protein synthesis was estimated by [35S]methionine
incorporation into protein as described in Fig. 1. The presented data
are the means ± S.D. (bars) of two separate
experiments, each performed in triplicate. B, left panel,
cells were incubated in complete growth medium for 60 min without
(solid line) or following addition of 200 mM
NaCl (dotted line), and extracts were resolved by sucrose
gradient centrifugation. Sedimentation is from left to
right, and the migration of the 80 S ribosome is indicated.
Right panel, parallel cultures from ongoing
cultures (control, upper panel) or those
incubated with 200 mM NaCl for 60 min were rinsed in fresh
complete medium and incubated in the absence (middle panel)
or presence of 20 µM CGP57380 (bottom panel)
for 90 min. Cell extracts were resolved by sucrose gradient
centrifugation, and the migration of the 80 S ribosome is indicated.
C, cells were incubated in complete growth medium for 60 min
without (C) or following addition of 200 mM NaCl. Cultures were rinsed in fresh complete medium and
incubated in the absence or presence of 20 µM CGP57380
for the times indicated. Extracts were resolved by SDS-PAGE, and the
phosphorylation status of eIF4G, eIF4E, 4E-BP1, S6, and p38 MAP kinase
was visualized by immunoblotting. Results are
representative of those obtained in three separate experiments.
D, extracts from panel C were subjected to
m7GTP-Sepharose chromatography, and eIF4E and
associated factors were resolved by SDS-PAGE and visualized
by immunoblotting. Results are representative of those obtained in
three separate experiments.
ACKNOWLEDGEMENT
FOOTNOTES
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@sussex.ac.uk.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1.
Rhoads, R. E.
(1999)
J. Biol. Chem.
274,
30337-30340 2.
Gingras, A-C.,
Raught, B.,
and Sonenberg, N.
(1999)
Annu. Rev. Biochem.
68,
913-963[CrossRef][Medline]
[Order article via Infotrieve]
3.
Raught, B.,
Gingras, A. C.,
and Sonenberg, N.
(2000)
in
Translational Control of Gene Expression
(Sonenberg, N.
, Hershey, J. W. B.
, and Mathews, M. B., eds)
, pp. 245-294, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
4.
Hershey, J. W. B.,
and Merrick, W. C.
(2000)
in
Translational Control of Gene Expression
(Sonenberg, N.
, Hershey, J. W. B.
, and Mathews, M. B., eds)
, pp. 33-88, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
5.
Morley, S. J.
(2001)
in
Signaling Pathways for Translation
(Rhoads, R. E., ed)
, pp. 1-37, Springer-Verlag, Berlin
6.
Marcotrigiano, J.,
Gingras, A. C.,
Sonenberg, N.,
and Burley, S. K.
(1997)
Cell
89,
951-961[CrossRef][Medline]
[Order article via Infotrieve]
7.
Tomoo, K.,
Shen, X.,
Okabe, K.,
Nozoe, Y.,
Fukuhara, S.,
Morino, S.,
Ishida, T.,
Taniguchi, T.,
Hasegawa, H.,
Terashima, A.,
Sasaki, M.,
Katsuya, Y.,
Kitamura, K.,
Miyoshi, H.,
Ishikawa, M.,
and Miura, K.-I.
(2002)
Biochem. J.
362,
539-544[CrossRef][Medline]
[Order article via Infotrieve]
8.
Morley, S. J.,
Curtis, P. S.,
and Pain, V. M.
(1997)
RNA
3,
1085-1104[Medline]
[Order article via Infotrieve]
9.
Merrick, W. C.
(1992)
Microbiol. Rev.
56,
291-315 10.
Gallie, D. R.
(1998)
Gene (Amst.)
17,
1-11
11.
Wells, S. E.,
Hillner, P. E.,
Vale, R. D.,
and Sachs, A. B.
(1998)
Mol. Cell
2,
135-140[CrossRef][Medline]
[Order article via Infotrieve]
12.
Marcotrigiano, J.,
Gingras, A. C.,
Sonenberg, N.,
and Burley, S. K.
(1999)
Mol. Cell
3,
707-716[CrossRef][Medline]
[Order article via Infotrieve]
13.
Ptushkina, M.,
Von der Haar, T.,
Karim, M. M.,
and McCarthy, J. E. G.
(1999)
EMBO J.
18,
4068-4075[CrossRef][Medline]
[Order article via Infotrieve]
14.
Haghighat, A.,
and Sonenberg, N.
(1997)
J. Biol. Chem.
272,
21677-21680 15.
Pause, A.,
Belsham, G. J.,
Gingras, A. C.,
Donze, O.,
Lin, T. A.,
Lawrence, J. C.,
and Sonenberg, N.
(1994)
Nature
371,
762-767[CrossRef][Medline]
[Order article via Infotrieve]
16.
Heesom, K. J.,
and Denton, R. M.
(1999)
FEBS Lett.
457,
489-493[CrossRef][Medline]
[Order article via Infotrieve]
17.
Joshi, B.,
Cai, A.-L.,
Keiper, B. D.,
Minich, W. B.,
Mendez, R.,
Beach, C. M.,
Stepinski, J.,
Stolarski, R.,
Darzynkiewicz, E.,
and Rhoads, R. E.
(1995)
J. Biol. Chem.
270,
14597-14603 18.
Flynn, A.,
and Proud, C. G.
(1995)
J. Biol. Chem.
270,
21684-21688 19.
Morley, S. J.
(1996)
in
Protein Phosphorylation in Cell Growth Regulation
(Clemens, M. J., ed)
, pp. 197-224, Harwood Academic Publishers, Amsterdam
20.
Morley, S. J.
(1997)
FEBS Lett.
418,
327-332[CrossRef][Medline]
[Order article via Infotrieve]
21.
Morley, S. J.,
and McKendrick, L.
(1997)
J. Biol. Chem.
272,
17887-17893 22.
Fraser, C. S.,
Pain, V. M.,
and Morley, S. J.
(1999)
Biochem. J.
342,
519-526[Medline]
[Order article via Infotrieve]
23.
Flynn, A.,
and Proud, C. G.
(1996)
FEBS Lett.
389,
162-166[CrossRef][Medline]
[Order article via Infotrieve]
24.
Mendez, R.,
Kollmorgen, G.,
White, M. F.,
and Rhoads, R. E.
(1997)
Mol. Cell. Biol.
17,
5184-5192[Abstract]
25.
Wang, X. M.,
Flynn, A.,
Waskiewicz, A. J.,
Webb, B. L. J.,
Vries, R. G.,
Baines, I. A,
Cooper, J. A.,
and Proud, C. G.
(1998)
J. Biol. Chem.
273,
9373-9377 26.
Waskiewicz, A. J.,
Flynn, A.,
Proud, C. G.,
and Cooper, J. A.
(1997)
EMBO J.
16,
1909-1920[CrossRef][Medline]
[Order article via Infotrieve]
27.
Scheper, G. C.,
Morrice, N. A.,
Kleijn, M.,
and Proud, C. G.
(2001)
Mol. Cell. Biol.
21,
743-754 28.
Knauf, U.,
Tschopp, C.,
and Gram, H.
(2001)
Mol. Cell. Biol.
21,
5500-5511 29.
Waskiewicz, A. J.,
Johnson, J. C.,
Penn, B.,
Mahalingam, M.,
Kimball, S. R.,
and Cooper, J. A.
(1999)
Mol. Cell. Biol.
19,
1871-1880 30.
Pyronnet, S.,
Imataka, H.,
Gingras, A.-C.,
Fukunaga, R.,
Hunter, T.,
and Sonenberg, N.
(1999)
EMBO J.
18,
270-279[CrossRef][Medline]
[Order article via Infotrieve]
31.
Pyronnet, S.
(2000)
Biochem. Pharmacol.
60,
1237-1243[CrossRef][Medline]
[Order article via Infotrieve]
32.
Morino, S.,
Imataka, H.,
Svitkin, Y. V.,
Pestova, T. V.,
and Sonenberg, N.
(2000)
Mol. Cell. Biol.
20,
468-477 33.
Scheper, G. C.,
van Kollenberg, B., Hu, J.,
Luo, Y.,
Goss, D. J.,
and Proud, C. G.
(2002)
J. Biol. Chem.
277,
3303-3309 34.
Minich, W. B.,
Balasta, M. L.,
Goss, D. J.,
and Rhoads, R. E.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
7668-7672 35.
McKendrick, L.,
Morley, S. J.,
Pain, V. M.,
Jagus, R.,
and Joshi, B.
(2001)
Eur. J. Biochem.
268,
5375-5385[Medline]
[Order article via Infotrieve]
36.
Saghir, A. N.,
Tuxworth, W. J.,
Hagedorn, C. H.,
and McDermott, P. J.
(2001)
Biochem. J.
356,
557-566[CrossRef][Medline]
[Order article via Infotrieve]
37.
Lachance, P. E. D.,
Miron, M.,
Raught, B.,
Sonenberg, N.,
and Lasko, P.
(2002)
Mol. Cell. Biol.
22,
1656-1663 38.
Saborio, J. L.,
Pong, S. S.,
and Koch, G.
(1974)
J. Mol. Biol.
85,
195-211[CrossRef][Medline]
[Order article via Infotrieve]
39.
Kruppa, J.,
and Clemens, M. J.
(1984)
EMBO J.
3,
95-100[Medline]
[Order article via Infotrieve]
40.
Duncan, R. F,
and Hershey, J. W. B.
(1987)
Mol. Cell. Biol.
7,
1293-1295 41.
Novoa, I.,
and Carrasco, L.
(1999)
Mol. Cell. Biol.
19,
2445-2454 42.
Morley, S. J.
(1994)
Mol. Biol. Rep.
19,
221-231[CrossRef][Medline]
[Order article via Infotrieve]
43.
Raught, B.,
Gingras, A. C.,
Gygi, S. P.,
Imataka, H.,
Morino, S.,
Gradi, A.,
Aebersold, R.,
and Sonenberg, N.
(2000)
EMBO J.
19,
434-444[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  X. Wang, P. Yue, C.-B. Chan, K. Ye, T. Ueda, R. Watanabe-Fukunaga, R. Fukunaga, H. Fu, F. R. Khuri, and S.-Y. Sun Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation Mol. Cell. Biol., November 1, 2007; 27(21): 7405 - 7413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Chauvin, S. Salhi, and O. Jean-Jean Human Eukaryotic Release Factor 3a Depletion Causes Cell Cycle Arrest at G1 Phase through Inhibition of the mTOR Pathway Mol. Cell. Biol., August 15, 2007; 27(16): 5619 - 5629. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Araud, R. Genolet, P. Jaquier-Gubler, and J. Curran Alternatively spliced isoforms of the human elk-1 mRNA within the 5' UTR: implications for ELK-1 expression Nucleic Acids Res., July 9, 2007; 35(14): 4649 - 4663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Hinton, M. J. Coldwell, G. A. Carpenter, S. J. Morley, and V. M. Pain Functional Analysis of Individual Binding Activities of the Scaffold Protein eIF4G J. Biol. Chem., January 19, 2007; 282(3): 1695 - 1708. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Gaccioliy, C. C. Huang, C. Wang, E. Bevilacqua, R. Franchi-Gazzola, G. C. Gazzola, O. Bussolati, M. D. Snider, and M. Hatzoglou Amino Acid Starvation Induces the SNAT2 Neutral Amino Acid Transporter by a Mechanism That Involves Eukaryotic Initiation Factor 2{alpha} Phosphorylation and cap-independent Translation J. Biol. Chem., June 30, 2006; 281(26): 17929 - 17940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. V. Slepenkov, E. Darzynkiewicz, and R. E. Rhoads Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides: EVIDENCE FOR A ONE-STEP BINDING MECHANISM J. Biol. Chem., May 26, 2006; 281(21): 14927 - 14938. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-H. Shen, D. W Boyle, P. Wisniowski, A. Bade, and E. A Liechty Insulin and IGF-I stimulate the formation of the eukaryotic initiation factor 4F complex and protein synthesis in C2C12 myotubes independent of availability of external amino acids J. Endocrinol., May 1, 2005; 185(2): 275 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Topisirovic, M. Ruiz-Gutierrez, and K. L. B. Borden Phosphorylation of the Eukaryotic Translation Initiation Factor eIF4E Contributes to Its Transformation and mRNA Transport Activities Cancer Res., December 1, 2004; 64(23): 8639 - 8642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Naegele and S. J. Morley Molecular Cross-talk between MEK1/2 and mTOR Signaling during Recovery of 293 Cells from Hypertonic Stress J. Biol. Chem., October 29, 2004; 279(44): 46023 - 46034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ueda, R. Watanabe-Fukunaga, H. Fukuyama, S. Nagata, and R. Fukunaga Mnk2 and Mnk1 Are Essential for Constitutive and Inducible Phosphorylation of Eukaryotic Initiation Factor 4E but Not for Cell Growth or Development Mol. Cell. Biol., August 1, 2004; 24(15): 6539 - 6549. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Cuesta, Q. Xi, and R. J. Schneider Structural Basis for Competitive Inhibition of eIF4G-Mnk1 Interaction by the Adenovirus 100-Kilodalton Protein J. Virol., July 15, 2004; 78(14): 7707 - 7716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Caron, M. Charon, E. Cramer, N. Sonenberg, and I. Dusanter-Fourt Selective Modification of Eukaryotic Initiation Factor 4F (eIF4F) at the Onset of Cell Differentiation: Recruitment of eIF4GII and Long-Lasting Phosphorylation of eIF4E Mol. Cell. Biol., June 1, 2004; 24(11): 4920 - 4928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Walsh and I. Mohr Phosphorylation of eIF4E by Mnk-1 enhances HSV-1 translation and replication in quiescent cells Genes & Dev., March 15, 2004; 18(6): 660 - 672. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. H. Faour, A. Mancini, Q. W. He, and J. A. Di Battista T-cell-derived Interleukin-17 Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of the p38 Mitogen-activated Protein Kinase Cascade: ROLE OF DISTAL SEQUENCES IN THE 3'-UNTRANSLATED REGION OF COX-2 mRNA J. Biol. Chem., July 11, 2003; 278(29): 26897 - 26907. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
