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Molecular and Cellular Biology, June 1999, p. 4302-4310, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Translational Homeostasis: Eukaryotic Translation
Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6
Kinase Activities
Kianoush
Khaleghpour,
Stéphane
Pyronnet,
Anne-Claude
Gingras, and
Nahum
Sonenberg*
Department of Biochemistry and McGill Cancer
Center, McGill University, Montreal, Quebec, Canada H3G 1Y6
Received 7 December 1998/Returned for modification 27 January
1999/Accepted 15 March 1999
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ABSTRACT |
Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA 5'
cap binding protein, which plays an important role in the control of
translation. The activity of eIF4E is regulated by a family of
repressor proteins, the 4E-binding proteins (4E-BPs), whose binding to
eIF4E is determined by their phosphorylation state. When
hyperphosphorylated, 4E-BPs do not bind to eIF4E. Phosphorylation of
the 4E-BPs is effected by the phosphatidylinositol (PI) 3-kinase signal
transduction pathway and is inhibited by rapamycin through its binding
to FRAP/mTOR (FK506 binding protein-rapamycin-associated protein or
mammalian target of rapamycin). Phosphorylation of 4E-BPs can also be
induced by protein synthesis inhibitors. These observations led to the
proposal that FRAP/mTOR functions as a "sensor" of the
translational apparatus (E. J. Brown and S. L. Schreiber,
Cell 86:517-520, 1996). To test this model, we have employed the
tetracycline-inducible system to increase eIF4E expression. Removal of
tetracycline induced eIF4E expression up to fivefold over endogenous
levels. Strikingly, upon induction of eIF4E, 4E-BP1 became
dephosphorylated and the extent of dephosphorylation was proportional
to the expression level of eIF4E. Dephosphorylation of
p70S6k also occurred upon eIF4E induction. In contrast, the
phosphorylation of Akt, an upstream effector of both p70S6k
and 4E-BP phosphorylation, was not affected by eIF4E induction. We
conclude that eIF4E engenders a negative feedback loop that targets a
component of the PI 3-kinase signalling pathway which lies downstream
of PI 3-kinase.
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INTRODUCTION |
A key step in ribosome binding to
the eukaryotic mRNA is the recognition of the cap structure
(m7GpppX, where X is any nucleotide) by the cap binding
protein complex, eukaryotic translation initiation factor 4F (eIF4F).
eIF4F is composed of three subunits: eIF4E, a 24-kDa polypeptide which interacts directly with the cap structure; eIF4A, a 50-kDa polypeptide that exhibits RNA helicase activity; and eIF4G, which serves as a
scaffolding protein for the binding of eIF4E, eIF4A, and eIF3, an
initiation factor associated with the 40S ribosomal subunit (reviewed
in references 62 and 66). eIF4F
is an important target for translational control by extracellular
stimuli, stress, and viral infections (30).
eIF4F function is regulated through multiple mechanisms, including
transcriptional activation of eIF4E (39), changes in eIF4E
phosphorylation state (reviewed in reference 67),
and cleavage of eIF4G upon infection with some viruses, causing the shutoff of host protein synthesis (reviewed in references
7 and 23). In addition, members
of a family of proteins, termed eIF4E-binding proteins (4E-BPs)
(53), also called PHAS, for phosphorylated heat- and
acid-stable protein (47), bind to eIF4E and prevent its
interaction with eIF4G and thus the formation of the eIF4F complex
(32, 49). Three mammalian members of the 4E-BP family have
been cloned (53, 56). One functional analog of the 4E-BPs,
p20, has been identified in Saccharomyces cerevisiae,
although there is no sequence homology between the yeast protein and
the mammalian 4E-BPs, other than the eIF4E binding site (3).
The interaction of mammalian 4E-BPs with eIF4E is modulated by the
extent of 4E-BP phosphorylation. The 4E-BPs strongly interact with
eIF4E when in their hypophosphorylated state and dissociate from eIF4E
upon hyperphosphorylation (47, 53).
Phosphorylation of 4E-BP is induced by growth factors, hormones,
mitogens, G protein-coupled receptor agonists, and cytokines (for
reviews see references 30, 45, and
67). A large body of evidence documents the
involvement of the phosphatidylinositol (PI) 3-kinase signalling
pathway in the phosphorylation of 4E-BP1 and a serine/threonine kinase,
p70S6k, which phosphorylates ribosomal S6 protein
(37). For example, the phosphorylation of both 4E-BP1 and
p70S6k is strongly diminished by wortmannin, an inhibitor
of PI 3-kinase (14, 17, 72). In addition, a
dominant-negative mutant bearing a mutation of the 85-kDa regulatory
subunit of the PI 3-kinase inhibits the phosphorylation and activity of
p70S6k (14), and expression of an activated
110-kDa catalytic subunit of PI 3-kinase stimulates the phosphorylation
of 4E-BP1 (30). Signalling from PI 3-kinase to 4E-BP1 and
p70S6k is mediated at least in part through Akt (30,
40, 71).
Another kinase that modulates the activity of p70S6k and
4E-BP1 is FRAP/mTOR (FK506 binding protein
[FKBP]-rapamycin-associated protein or mammalian target of
rapamycin) (10, 12, 13, 15, 30, 34). FRAP/mTOR belongs to a
growing family of kinases (PIK, PI 3-kinase related) which have
homology in their catalytic domains with PI 3-kinase (reviewed in
reference 36). The immunosuppressant rapamycin, in a
complex with the immunophilin FKBP12, binds to a specific site in
FRAP/mTOR and inhibits its activity (70). FRAP/mTOR
phosphorylates 4E-BP1 and p70S6k in vitro (12, 13,
15). Furthermore, the signalling pathway which leads to S6 and
4E-BP1 phosphorylation bifurcates at a rapamycin-sensitive point
immediately upstream of p70S6k (73). This
explains the findings that treatment of cells with rapamycin decreases
phosphorylation of 4E-BP1 and p70S6k and that addition of
the FKBP12-rapamycin complex prevents phosphorylation of 4E-BP1 by
FRAP/mTOR in an immune complex kinase assay (13).
In addition to extracellular stimuli, the rapamycin-sensitive
phosphorylation of p70S6k and 4E-BP1 can be induced by
inhibitors of protein synthesis, such as cycloheximide and anisomycin
(51, 72). These and other observations led Brown and
Schreiber to propose that FRAP/mTOR may be a component of a
"sensor" apparatus for the activity of the translational machinery
(11). Thus, there might exist a cellular feedback mechanism
to regulate translational homeostasis. Were such a model correct, it
might be expected that overexpression of eIF4E, which results in
translation upregulation, would downregulate the FRAP/mTOR pathway. To
address this idea, we established an eIF4E tetracycline-regulatable
expression system to modulate cellular eIF4E levels.
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MATERIALS AND METHODS |
Vectors.
For construction of pRep9-CMVt-eIF4E,
the coding sequence of eIF4E was cloned downstream of the
tetracycline-responsive promoter CMVt, which consists of a
minimal cytomegalovirus early promoter sequence fused to seven copies
of a tet operator sequence. Murine eIF4E cDNA was excised
from pBluescript KS-4E by digestion with HindIII/BamHI and ligated directionally into
pRep9-CMVt (5).
Cell lines.
An NIH 3T3-derived cell line, S2-6, expressing
Tetr-VP16 transactivator (tTA) (65), was
transfected with 10 µg of pRep9-CMVt-eIF4E by calcium
phosphate precipitation. G418-resistant colonies were isolated after 14 days of selection in medium containing 0.7 mg of G418/ml. Cells were
maintained in medium containing 1 µg of tetracycline hydrochloride
(Sigma) per ml. To induce expression of eIF4E, cells were rinsed three
times in 1× phosphate-buffered saline (PBS) and grown in the absence
of tetracycline for the times indicated in the figure legends.
Western blotting.
Cells were rinsed twice with ice-cold PBS
and incubated with the appropriate lysis buffer. For analysis of eIF4E
and 4E-BP1, the lysis buffer contained 10 mM Tris-HCl, pH 7.5; 1%
Triton X-100; 50 mM KCl; 1 mM dithiothreitol (DTT); 2 mM
MgCl2; 0.2 mM phenylmethylsulfonyl fluoride (PMSF). For
analysis of p70S6k and Akt, the lysis buffer contained 50 mM sodium phosphate, pH 7.2; 2 mM EGTA; 25 mM NaF; 25 mM
-glycerophosphate; 0.5% Triton X-100; 100 µM
Na3VO4; 1 mM PMSF; 1 µg of leupeptin/ml; 1 µg of pepstatin/ml; 1 mM benzamidine; 2 mM DTT. Lysates were
centrifuged at 14,000 rpm for 10 min to remove cellular debris, and
total protein concentration was determined in duplicate by using the bicinchoninic acid protein assay (Pierce) and quantitated against a
standard curve of bovine serum albumin protein concentrations. For each
experiment, an equal amount of total protein (25 to 50 µg) was
electrophoresed on a sodium dodecyl sulfate (SDS)-8 to 15%
polyacrylamide gel and electroblotted onto nitrocellulose membranes
(Millipore) in 25 mM Tris-HCl, pH 7.5; 190 mM glycine; and 20%
(vol/vol) methanol, at 75 V for 2 h. Filters were then blocked in
Tris-buffered saline containing 0.2% Tween 20 (TBST) and 5% (wt/vol)
dry milk at 25°C for 1 h. Membranes were incubated overnight at
4°C with one of the following antibodies at the indicated dilution:
mouse monoclonal antiactin at 1:500 (ICN Biomedicals), rabbit
polyclonal anti-eIF4E at 1:1,000 (26), rabbit polyclonal anti-4E-BP1 at 1:1,000 (31), rabbit polyclonal
anti-p70S6k at 1:300 (Santa Cruz Biotechnology), rabbit
polyclonal anti-phosphospecific p70S6k (Thr421/Ser424) at
1:1,000 (New England Biolabs), rabbit polyclonal anti-phosphospecific
Akt (Ser473) at 1:1,000 (New England Biolabs), and goat polyclonal
anti-Akt (C-20) at 1:1,000 (Santa Cruz Biotechnology). After they were
washed with TBST, the membranes were incubated as follows: for rabbit
polyclonal anti-4E-BP1, anti-p70S6k, anti-phosphospecific
p70S6k, and anti-phosphospecific Akt antibodies, membranes
were incubated with donkey anti-rabbit horseradish
peroxidase-conjugated immunoglobulin G (IgG) (Amersham); and for goat
anti-Akt (C-20), membranes were incubated with rabbit anti-goat
horseradish peroxidase-conjugated IgG at 1:5,000 (Jackson Laboratories)
for 30 min. Membranes were then washed with TBST three times, and the
signals were detected by using an enhanced chemiluminescence (ECL) kit
(Amersham) after exposure to an X-ray film (Du Pont). Membranes probed
with the monoclonal antiactin antibody were incubated for 1 h with
0.1 µCi of 125I-labelled rabbit anti-mouse IgG (NEN) per
ml, and for polyclonal anti-eIF4E, membranes were incubated for 1 h with 0.1 µCi of 125I-labelled protein A (Amersham) per
ml. The membranes were washed three times in TBST and exposed to an
X-ray film. Signals were quantified by using the Fuji Bas 2000 phosphorimager.
Far-Western analysis.
Heart muscle kinase (HMK)-eIF4E fusion
protein was purified as described previously (53). HMK-eIF4E
(3 µg) was incubated with HMK (20 U) (Sigma) in the presence of
[
-32P]-ATP (50 µCi) for 1 h at 37°C and
purified by using a Sephadex G-50 column (Pharmacia Biotech). Total
cellular protein (50 µg) was subjected to electrophoresis on an
SDS-15% polyacrylamide gel and electroblotted onto a
0.2-µm-pore-size nitrocellulose membrane as described above for
Western blotting. The membrane was blocked in 5% milk in HBB (25 mM
HEPES-KOH, pH 7.7; 25 mM NaCl; 5 mM MgCl2; 0.1 mM EDTA; 1 mM DTT; 0.05% Nonidet P-40) for 2 h and then incubated overnight
at 4°C in hybridization buffer (20 mM HEPES-KOH, pH 7.7; 75 mM KCl;
2.5 mM MgCl2; 0.1 mM EDTA; 1 mM DTT; 0.05% Nonidet P-40;
1% milk) containing 32P-labelled HMK-eIF4E (250,000 cpm/ml
[9]). The membrane was washed four times with
hybridization buffer and exposed to an X-ray film (Du Pont).
ODC assay.
Cells were rinsed twice in ice-cold PBS, scraped
in lysis buffer (0.25 M Tris-HCl, pH 7.4; 1 mM EDTA; 1 mM DTT), and
subjected to two freeze-thaw cycles. The lysate was centrifuged at
14,000 rpm for 10 min to remove cellular debris. Protein content of the lysate was determined in duplicate by using the bicinchoninic acid
protein assay (Pierce) and quantitated against a standard curve of
bovine serum albumin protein concentrations. Equal amounts of total
protein (50 µg) were incubated with 2.5 µCi of
14C-labelled ornithine (Amersham) and 50 µM pyridoxal
5-phosphate for 1 h at 37°C. Incubations were performed in
96-well microtiter plates. Liberated 14CO2 was
trapped in a covering 3MM paper saturated with a solution of barium
hydroxide. The 3MM paper was rinsed with acetone, dried, and exposed to
an X-ray film. Spots were cut from the paper, and the radioactivity was
determined by using a beta-scintillation counter.
p70 S6 kinase assay.
Cell extract (20 µg) was diluted with
extraction buffer (50 mM Tris-HCl, pH 8.0; 120 mM NaCl; 20 mM NaF; 1 mM
benzamidine; 1 mM EDTA; 6 mM EGTA, 1% [vol/vol] Nonidet P-40; 0.1 mM
PMSF) and incubated with rabbit polyclonal anti-p70S6k
antibody (Santa Cruz Biotechnology) on ice for 2 h. A 37.5%
suspension of protein A-Sepharose (Repligen) resin was added and
incubated end over end at 4°C for 1 h. The resin was washed
twice with extraction buffer and once with dilution buffer (50 mM
morpholinepropanesulfonic acid [MOPS], pH 7.2; 5 mM
MgCl2; 10 mM NaF; 30 mM -glycerophosphate). p70S6k activity was assayed with 40S ribosomal subunits in
a mixture (10 µl) containing 50 mM MOPS, pH 7.2; 1 mM DTT; 5 mM
MgCl2; 5 mM p-nitrophenyl phosphate; 100 µM
ATP; 0.5 µM protein kinase inhibitor (Sigma); 6 µCi of
[32P]-ATP; 20 µg of 40S ribosomal subunits
(44). The 40S ribosomes were a generous gift from G. Thomas.
After incubation at 37°C for 30 min, reactions were terminated by
addition of Laemmli buffer. Samples were resolved by SDS-12%
polyacrylamide gel electrophoresis (PAGE), and S6 phosphorylation was
visualized by autoradiography.
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RESULTS |
4E-BP1 dephosphorylation in eIF4E-transformed cells.
In an
initial attempt to determine the effect of eIF4E overexpression on
4E-BP1 phosphorylation, we used an NIH 3T3-transformed cell line
overexpressing eIF4E (46). We also used NIH 3T3 cell lines
transformed with a mutant of another initiation factor, eIF2
(Ser51Ala, which cannot be phosphorylated [22]), as
well as a mutant of its kinase (a dead-kinase mutant), the
interferon-inducible PKR (protein kinase, double-stranded RNA dependent
[42]). Overexpression of eIF2ala and a
dominant-negative mutant of PKR, but not wild-type eIF2 (eIF2wt), results in malignant transformation of NIH 3T3
cells (22, 42).
Murine 4E-BP1 can be resolved by SDS-PAGE into three bands as detected
by Western blotting: the slowest-migrating, hyperphosphorylated isoform
() does not interact with eIF4E. The middle form () is less
phosphorylated and binds to eIF4E with moderate affinity, while the
fastest-migrating form () is hypophosphorylated and interacts
strongly with eIF4E (8, 31, 48). In parental NIH 3T3 cells,
4E-BP1 was detected exclusively in its phosphorylated ( and )
isoforms, while the isoform was absent (Fig.
1, lane 1). However, in contrast to the
parental cells, only the two faster-migrating isoforms ( and )
were detected (lane 2) in NIH 3T3 cells overexpressing eIF4E
(46). This effect is similar to that seen in the presence of
rapamycin (8). Overexpression of eIF2ala
(lane 3) or eIF2wt (lane 4) did not affect the state of
4E-BP1 phosphorylation in NIH 3T3 cells, as 4E-BP1 was present in the
and isoforms. The status of 4E-BP1 phosphorylation in NIH 3T3
cells expressing a dominant-negative mutant of PKR was also unchanged
relative to that in parental NIH 3T3 cells (compare lanes 1 and 5). In addition, in NIH 3T3 cells which are transformed by overexpressing eIF4G (27), 4E-BP1 is not dephosphorylated (data not shown). Therefore, it appears that 4E-BP1 phosphorylation is altered in cells
transformed with eIF4E but not in cells transformed by other initiation
factors (this is further addressed in the Discussion section).
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FIG. 1.
4E-BP1 phosphorylation state in transformed NIH 3T3
cells. Immunoblot analysis of total cell extract (50 µg) obtained
from parental NIH 3T3 cells or NIH 3T3 cells overexpressing eIF4E
(eIF4E), eIF2ala mutant [2 (Ala)] and wild type
[2 (wt)], and mutant PKR 6. The blot was probed with a rabbit
polyclonal anti-4E-BP1 antibody, as described in Materials and Methods.
The three isoforms of 4E-BP1 are indicated.
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Tetracycline-inducible eIF4E-expressing cell lines.
To
demonstrate that the dephosphorylation of 4E-BP1 is a direct effect of
eIF4E overexpression rather than the result of a secondary effect, such
as an autocrine feedback loop or some aberration in the cell lines
tested, we generated eIF4E inducible cell lines. We chose to use the
tetracycline-inducible system rather than transient transfections, as
the effects can be measured in shorter periods, thus minimizing
possible autocrine effects. We utilized a tetracycline-inducible system
in which both the eIF4E and the tTA genes are under the control of a
tetracycline-inducible promoter, which is activated upon the removal of
tetracycline (65).
An NIH 3T3-derived stable cell line containing the autoregulatory tTA
expression plasmid, S2-6 (
65), was transfected with
a
CMV-eIF4E plasmid. G418-resistant cells were isolated and expanded,
and
the amount of eIF4E protein expressed was determined by Western
blotting. Cell extract from each clone was assayed for eIF4E expression
in cells grown in the presence (uninduced state) or absence
(eIF4E-induced
state) of tetracycline for 24 h. eIF4E expression
was compared
to that of control S2-6 cells cultured in the presence of
tetracycline,
and eIF4E protein levels were quantified and normalized
against
actin. The average (for three experiments) maximal increases in
the amount of eIF4E after induction were 3.4-fold in clone 1 (Fig.
2 presents a representative experiment;
compare lanes 4 and 1),
2.2-fold in clone 2 (compare lanes 6 and 1),
and 1.6-fold in clone
3 (compare lanes 8 and 1) as compared to the
amount in S2-6 cells.
The amount of eIF4E in the stably transfected
clones in the uninduced
state was somewhat less (0.6- to 0.8-fold) than
that found in
the S2-6 cells (compare lane 1 to lanes 3, 5, and 7).
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FIG. 2.
Expression of eIF4E in stably transfected
tetracycline-inducible NIH 3T3 cells. (Upper panel) Immunoblot analysis
was performed on total protein (25 µg) from cells cultured for
24 h in the presence or absence of tetracycline. S2-6 cells are
the parental cells, which express tTA upon induction. C1, C2, and C3
are stably transfected cell lines expressing eIF4E. The blot was probed
first with a rabbit polyclonal anti-eIF4E antibody, followed by a mouse
monoclonal anti-actin antibody. After probing with
125I-labelled anti-rabbit and anti-mouse secondary
antibodies, the amount of eIF4E expressed was quantitated with a Bas
2000 phosphorimager. Relative molecular masses for protein standards
are indicated in kilodaltons to the right. (Lower panel) Quantitation
of the results shown in the upper panel. eIF4E expression was
normalized against actin levels and expressed as the ratio relative to
the amount of eIF4E in S2-6 cells in the presence of tetracycline,
which was set at 1. Values are the averages of three experiments, and
the standard errors are shown.
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The expression kinetics of eIF4E were analyzed by Western blotting for
clone 1, the clone expressing the highest level of
eIF4E. Cells were
cultured in the absence of tetracycline for
72 h, and the levels
of eIF4E at several time points were compared
to that in the cells in
the uninduced state (tetracycline present,
time zero) (Fig.
3, lane 1). eIF4E expression reached a
maximal
level (5.8-fold induction) at 24 h after removal of
tetracycline
(compare lanes 3 and 1), then declined to a 3.2-fold
increase
at 48 h, and remained stable over the remainder of the
time course
(lanes 5 to 7).
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FIG. 3.
Time course of induction of eIF4E. (Upper panel) Total
cell extract (25 µg) was prepared from C1 cells cultured in the
absence of tetracycline for the indicated times. Extracts were
immunoblotted and probed with a rabbit polyclonal anti-eIF4E antibody.
The blot was subsequently probed with a mouse monoclonal anti-actin
antibody to normalize for protein loading. After probing with
125I-labelled anti-rabbit and anti-mouse secondary
antibodies, the amount of eIF4E expressed was quantitated with a Bas
2000 phosphorimager. (Lower panel) Quantitative analysis of the results
presented in the upper panel. eIF4E expression levels were normalized
against actin levels and are expressed as the ratios relative to the
amount of eIF4E in C1 cells at time 0, which was set at 1. Results are
representative of two experiments for which the values differed by less
than 14%.
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eIF4E expression increases ODC activity.
It was important to
determine whether the overexpressed eIF4E is functional. We therefore
examined ornithine decarboxylase (ODC) activity, which was previously
shown to correlate with the amount of the protein (55, 59,
69). ODC is a downstream target of eIF4E, as its amount is
increased in cells constitutively overexpressing eIF4E (61,
64). Within the first 4 h of eIF4E induction in clone 1 cells, ODC activity did not change significantly as compared to the
activity measured in extracts prepared from cells cultured in the
presence of tetracycline (Fig. 4, lower panel). However, ODC activity increased as early as 6 h
post-induction (Fig. 4, upper panel; the activity was 141% of that at
time zero), concomitant with the time line required for a detectable
increase in the level of eIF4E. Maximal enzymatic activities were
reached at 12 and 24 h (the activities were 370 and 350% as
compared to that at time zero). Note that ODC activity starts to
decline after 12 h, which could be due to the synthesis of
polyamines that induce the degradation of ODC (50) or the
downregulation of translation by 4E-BPs (see below). These results
indicate that the induced eIF4E protein is active and bolster the
original conclusion (61, 64) that the increase in ODC
translation observed in cells constitutively overexpressing eIF4E is a
direct result of eIF4E overexpression.
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FIG. 4.
ODC activity is stimulated by eIF4E overexpression. C1
cells were maintained in the absence or presence of tetracycline for
the indicated times. Total cell extract (50 µg) was assayed for ODC
activity as described in Materials and Methods. ODC activity is
expressed as the percentage of the value obtained at time 0. ODC
activity at time 0 was 3.1 (without tetracycline) and 2.7 (in the
presence of tetracycline) nmol of CO2/h/mg of protein.
Results are representative of two experiments for which the activities
differed by less than 26%.
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4E-BP1 is dephosphorylated upon eIF4E induction.
The inducible
eIF4E-expressing cell lines were used to correlate 4E-BP1
phosphorylation status with eIF4E levels. A Western blot analysis of
eIF4E induction for clone 1 cells was performed after removal of
tetracycline. Overexpression of eIF4E was detected as early as 6 h
postinduction (Fig. 5A, lane 6), and
maximal eIF4E expression was detected after 24 h (lane 8). Western
blot analysis of 4E-BP1 revealed a gradual dephosphorylation that
coincided with the induction of eIF4E. Under uninduced conditions, only the slower-migrating phosphorylated isoforms ( and ) of 4E-BP1 were detected (Fig. 5B, lane 1). However, 4E-BP1 was found only in its
faster-migrating isoforms ( and ) at 12 and 24 h
postinduction of eIF4E (lanes 7 and 8). Dephosphorylation of 4E-BP1 was
detected as early as 4 and 6 h after induction of eIF4E (lanes 5 and 6), when eIF4E levels were also increased. At this time, the amount of the hyperphosphorylated () isoform of 4E-BP1 began to decrease, while the isoform began to predominate. It appears that the amount
of 4E-BP1 is increased following eIF4E induction; however, because we
are not completely sure whether our anti-4E-BP1 antibody has equal
affinity for the various phosphorylation forms of 4E-BP1, we cannot
conclude with certainty that there is increased synthesis or stability
of 4E-BP1 upon induction of eIF4E.
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FIG. 5.
eIF4E induction results in dephosphorylation of 4E-BP1.
Cell extracts were prepared from C1 cells in the absence of
tetracycline for the indicated times. (A) Total cell extract (25 µg)
was resolved on an SDS-12.5% polyacrylamide gel, electroblotted onto
a 0.45-µm-pore-size nitrocellulose membrane, and probed with rabbit
polyclonal anti-eIF4E and mouse monoclonal anti-actin antibodies. (B)
Total cell extract (50 µg) was resolved on an SDS-15%
polyacrylamide gel, electroblotted onto a 0.2-µm-pore-size
nitrocellulose membrane, and probed with a rabbit polyclonal
anti-4E-BP1 antibody. (C) Far-Western analysis of cell extract (50 µg) by using 32P-labelled HMK-eIF4E as a probe was
performed as described in Materials and Methods. In panels B and C, the
different phosphorylation isoforms of 4E-BP1 are indicated with
arrows.
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To measure the binding capacity of 4E-BP1 to eIF4E over the time course
of eIF4E induction, a far-Western analysis was performed,
using a
32P-labelled recombinant eIF4E protein (
53).
Since phosphorylation
of 4E-BP1 prevents its binding to eIF4E, only the
and
isoforms
are detected in this assay (
8,
31,
47,
53). The results
of the far-Western analysis are consistent with
those of the Western
blot analysis of 4E-BP1 (compare Fig.
5C with Fig.
5B). The interaction
of eIF4E with 4E-BP1 was first detected at 6 h postinduction,
when a significant shift in 4E-BP1 phosphorylation
occurred (Fig.
5C, lane 6), and this interaction was further
strengthened as
4E-BP1 shifted predominantly to its
and
isoforms at 12 and
24 h postinduction (lanes 7 and 8). Taken
together, these results
show that upon induction of eIF4E, an increase
in the amount of
the hypophosphorylated (active) isoform of 4E-BP1
occurs, leading
to increased interaction of 4E-BP1 with
eIF4E.
The degree of 4E-BP1 dephosphorylation is directly proportional to
eIF4E levels.
To further establish the correlation between eIF4E
induction and 4E-BP1 dephosphorylation, we analyzed the phosphorylation of 4E-BP1 in eIF4E-expressing stably transfected inducible cells, which
express different amounts of eIF4E upon induction. Cells were cultured
in the absence of tetracycline for up to 36 h. Western blot
analysis shows that in the uninduced state (0 h), 4E-BP1 was present in
the phosphorylated state ( and isoforms) both in the parental
S2-6 cells (Fig. 6, lane 1) and in the
eIF4E stable transfectants (lanes 4, 7, and 10). Removal of
tetracycline from S2-6 cells appears to cause a shift of the 4E-BP1 to
the isoform (compare lane 1 to lanes 2 and 3; because the levels of
total 4E-BP1 protein in lanes 2 and 3 were low in this experiment, the shift is not conclusive, but the shift was reproducible in other experiments). This shift is most probably due to activation of Akt as
described below (see Fig. 8). Upon induction of eIF4E in the stable
transfectants, 4E-BP1 was dephosphorylated to varying degrees. For
clone 1, which exhibits the greatest overexpression of eIF4E, the
dephosphorylation of 4E-BP1 was such that only the two faster-migrating
isoforms ( and ) were detected after 24 h (compare lane 4 to
lanes 5 and 6). For clone 2, which overexpresses eIF4E to a lesser
degree than clone 1 (2.2-fold in contrast to 3.4-fold; see Fig. 2),
4E-BP1 dephosphorylation, upon eIF4E induction, was less dramatic than
that for clone 1 (compare lane 7 to lanes 8 and 9). For clone 3, the
cell line which overexpresses the least amount of eIF4E (1.6-fold)
(Fig. 2), phosphorylation of 4E-BP1 was altered to the smallest extent
upon eIF4E induction (compare lane 10 to lanes 11 and 12). In
conclusion, the degree of 4E-BP1 dephosphorylation is directly
proportional to the level of eIF4E expression, in that higher eIF4E
expression is correlated with greater dephosphorylation of 4E-BP1.
Taken together, these results suggest that an upstream element in a
signal transduction pathway that regulates 4E-BP1 phosphorylation is
affected by eIF4E overexpression.
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FIG. 6.
The extent of 4E-BP1 dephosphorylation is proportional
to the level of eIF4E expression. Parental cells (S2-6) and
eIF4E-expressing cell clones C1, C2, and C3 were cultured in the
absence of tetracycline for the indicated times. Total cell extract (50 µg) was immunoblotted and probed with a rabbit polyclonal anti-4E-BP1
antibody. Arrows indicate the different phosphorylation isoforms of
4E-BP1.
|
|
eIF4E overexpression influences p70S6k
phosphorylation.
Since the signalling pathway acting upon 4E-BP1
and p70S6k bifurcates downstream of a common effector in
the PI 3-kinase-FRAP/mTOR signalling pathway (73), we
reasoned that the direct target of the negative feedback loop
engendered by overexpression of eIF4E could be an upstream component of
the shared pathway. Hence, eIF4E overexpression should affect both
4E-BP1 and p70S6k phosphorylation. Consequently, we
examined the state of p70S6k phosphorylation upon induction
of eIF4E. The activity of p70S6k is regulated by
phosphorylation (e.g., see reference 4; see also
reviews in references 37, 57, and
58), which influences the migration of the protein
on SDS-PAGE (18). Decreased migration of p70S6k
was observed upon addition of 10% serum to serum-starved NIH 3T3 cells
(Fig. 7A; compare lane 9 to lane 7). A
small shift to a slower-migrating form occurs upon removal of
tetracycline in the parental cell line (compare lane 1 to lane 2). When
eIF4E was induced, p70S6k migrated more rapidly, indicating
hypophosphorylation of the protein. The shift was most evident in clone
1 cells, where the level of eIF4E is the highest (compare lane 4 to
lane 3). In comparison to clone 1, a much less pronounced shift was
noted for clone 3 (compare lane 6 to lane 5). These data show that the
extent of this shift was largely proportional to the amount of eIF4E
synthesized in the two clones tested.
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FIG. 7.
Effect of eIF4E induction on p70S6k
phosphorylation. eIF4E-expressing cells were maintained in the presence
or absence of tetracycline for 36 h (lanes 1 through 6); NIH 3T3
cells were grown in Dulbecco's minimal essential medium containing
0.5% fetal bovine serum (FBS) for 16 h (lanes 7 to 9), after
which cells were either mock treated (lane 7), incubated for 30 min in
the presence of 20 ng/ml of rapamycin prior to stimulation with 10%
FBS for 1 h (lane 8), or stimulated with 10% FBS alone (lane 9).
Total cell extract (50 µg) was electrophoresed on two separate
SDS-8% polyacrylamide gels and electroblotted onto a
0.45-µm-pore-size nitrocellulose membrane. One membrane was probed
with a rabbit polyclonal anti-p70S6k antibody which
recognizes p70S6k irrespective of its phosphorylation state
(A), while the other was probed with a rabbit polyclonal
anti-phosphopeptide antibody which is specific for phospho-Thr421 and
phospho-Ser424 in p70S6k (B). (C) Total cell extract (20 µg) was immunoprecipitated with rabbit polyclonal
anti-p70S6k antibody. The immunoprecipitate was assayed for
p70S6k activity by using 40S ribosomal subunits as a
substrate as described in Materials and Methods. The figure is a
representative of three independent experiments.
|
|
To substantiate these conclusions, Western blot analysis using a
phosphospecific p70
S6k antibody which recognizes
phospho-Thr421 and phospho-Ser424 was
performed (
35,
75).
These sites are contained in the autoinhibitory
domain of the kinase
and become phosphorylated upon activation
of the PI 3-kinase pathway
(
58). To demonstrate that the phosphospecific
antibody binds
only to the phosphorylated form of p70
S6k, serum-deprived
NIH 3T3 cells were stimulated with serum to induce
phosphorylation. The
antibody failed to interact with p70
S6k from serum-deprived
or rapamycin-treated cells, but it bound
to p70
S6k from
serum-fed cells (Fig.
7B, compare lanes 7 and 8 to lane
9). The
phosphorylation of p70
S6k at Thr421 and Ser424 is
consistent with the shifts in mobility
described above. Thr421 and
Ser424 phosphorylation was increased
in the parental S2-6 cells upon
removal of tetracycline (compare
lane 1 to lane 2), an effect similar
to the observed effect on
4E-BP1 phosphorylation, which was probably
due to phosphorylation
of Akt (see Fig.
8). In the eIF4E-overexpressing
cells, p70
S6k phosphorylation at Thr421 and Ser424
decreased, and this reduction
was proportional to the amount of eIF4E
expressed upon induction
in the two cell clones tested. The decrease in
phosphorylation
was most evident in clone 1 cells, where the level of
eIF4E is
the highest (compare lane 4 to lane 3). In comparison to clone
1, a lesser decrease in phosphorylation of p70
S6k at Thr421
and Ser424 was noted for clone 3 (compare lane 6 to
lane
5).
Finally, it was imperative to show directly that eIF4E overexpression
affects p70
S6k activity. An immune-complex kinase assay was
performed using
extracts from eIF4E-expressing cells as the source of
the kinase
and 40S ribosomal subunits as the source of the substrate.
S6
phosphorylation increased (1.4-fold) upon the removal of
tetracycline
in the parental S2-6 cells, consistent with the increase
in phosphorylation
at Thr421 and Ser424 (Fig.
7C; compare lane 1 to
lane 2). In contrast,
induction of eIF4E resulted in decrease in S6
phosphorylation
in eIF4E-transfected cells (compare lanes 3 and 4 and
lanes 5
and 6). The decrease in S6 phosphorylation was proportional to
the level of eIF4E in the two cell clones tested. The decrease
in
p70
S6k activity was most noticeable in clone 1 cells, where
S6 phosphorylation
decreased 62% (compare lane 4 to lane 3). For clone
3, a lesser
decrease in p70
S6k activity was noted, as S6
phosphorylation decreased only by 35%
(compare lane 6 to lane
5).
eIF4E does not inhibit Akt phosphorylation.
The protein Akt is
a serine/threonine kinase (6), which is activated by PI
3-kinase (20, 25). Phosphorylation of Ser473 and Thr308
activates Akt (2, 68). Akt acts upstream in the 4E-BP1
signalling pathway and stimulates the phosphorylation of 4E-BP1
(30, 40, 71). In addition, Akt stimulates the
phosphorylation of p70S6k (14, 40, 41). The
mechanism by which Akt stimulates the phosphorylation of 4E-BP1 and
p70S6k is not known, although it was recently reported that
Akt activates FRAP/mTOR (63). To determine whether the
target of the negative feedback loop induced by eIF4E overexpression
lies upstream or downstream of Akt, we examined whether Akt
phosphorylation is influenced by eIF4E. The levels of Akt protein did
not change upon eIF4E induction or upon serum withdrawal in the
different cell lines (Fig. 8A; an
anti-actin antibody was used to normalize for loading variability
[Fig. 8B]). We monitored the phosphorylation of Akt at Ser473 during
a 36-h time course following tetracycline removal by using a
phosphospecific antibody against Ser473. The antibody did not detect
Akt in serum-deprived cells, as expected (Fig. 8C, lane 13). Upon
stimulation of NIH 3T3 cells with serum, Akt became phosphorylated and
was detected by the antibody (lane 14). In addition, wortmannin
treatment blocked the phosphorylation of Akt (lane 15). Since rapamycin
exerts its effect downstream of Akt, phosphorylation of Akt in
rapamycin-treated cells, as expected, was not altered (lane 16).
Western blot analysis performed by using the phosphospecific antibody
showed that the phosphorylation of Akt at Ser473 was elevated upon
induction in the eIF4E-transfected cells rather than decreased, as
would be expected were eIF4E to inhibit Akt phosphorylation (compare
lanes 4 to 6, 7 to 9, and 10 to 12). Note, however, that an increase of
Akt phosphorylation was observed in the parental cells upon removal of
tetracycline (compare lanes 1 through 3). This increase can explain the
increase in phosphorylation of 4E-BP1 and p70S6k upon
removal of tetracycline in the parental cells (see Fig. 5, 6, and 7).
Although an explanation for the increase is not immediately clear, it
is possible that the tTA causes Akt phosphorylation and activation. Our
results show that eIF4E does not downregulate Akt phosphorylation.
Furthermore, a decrease in the phosphorylation of Akt was not observed
in a cell line stably overexpressing eIF4E (data not shown). This
excludes the possibility that an upstream component signalling to Akt
is affected by eIF4E. However, an intriguing possibility not addressed
here is that eIF4E inhibits Akt activity through direct binding or
indirectly by another mechanism. In conclusion, the target of the
negative feedback loop established by eIF4E overexpression must lie at
or downstream of Akt.
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FIG. 8.
Akt phosphorylation is not inhibited by eIF4E
overexpression. Tetracycline-inducible cells (S2-6, C1, C2, and C3)
were incubated in the absence of tetracycline for the indicated times
(lanes 1 through 12); NIH 3T3 cells were grown in Dulbecco's minimal
essential medium containing 0.5% fetal bovine serum (FBS) for 16 h (lanes 13 through 16), after which cells were either mock treated
(lane 13), incubated for 30 min in the presence of 100 nM wortmannin
(lane 15) or 20 ng of rapamycin/ml (lane 16) prior to stimulation with
10% FBS for 1 h, or stimulated with 10% FBS alone (lane 14).
Cells were then harvested, and total cell extract (50 µg) was
subjected to electrophoresis on an SDS-8% polyacrylamide gel,
electroblotted, and probed by using the following: (A) goat polyclonal
anti-Akt antibody (C-20), which recognizes Akt irrespective of its
phosphorylation state; (B) mouse monoclonal anti-actin antibody; (C)
rabbit polyclonal anti-phosphospecific (Ser473) Akt antibody. The
result shown is a representative of two independent experiments.
|
|
| |
DISCUSSION |
Translation plays an important role in control of cell growth
(19). Rates of protein synthesis are also coupled to cell cycle progression, since inhibition of translation leads to arrest of
cell growth, generally at the G1 phase (52).
Because the control of the cell cycle is of paramount importance to
cell growth and to the organism's survival, there exist many
checkpoints to ensure its integrity and appropriate functioning. It is
conceivable that checkpoint mechanisms may function to monitor the
activity of the translational machinery and to compensate for changes
in the levels or activities of translation factors. One such checkpoint mechanism has been proposed by Brown and Schreiber (11) to
explain the increase in the phosphorylation of p70S6k
and/or 4E-BP1 observed in cells treated with protein synthesis inhibitors, such as anisomycin and cycloheximide (51, 72). Based on these observations it was postulated that FRAP/mTOR, an
upstream regulator of the phosphorylation of p70S6k and
4E-BP1 (10, 13, 15, 30), is a sensor of translational activity in the cell and a target of a feedback loop. This feedback loop would then act to maintain translational homeostasis in the cell
in order to prevent uncontrolled growth and proliferation, or cell death.
Here we report evidence for the existence of a potential translational
homeostasis mechanism regulated by a negative feedback mechanism in
response to an increase in the concentration of an endogenous
translation factor, eIF4E. A model (depicted in Fig. 9) summarizes the PI 3-kinase signal
transduction pathway which results in the phosphorylation of 4E-BPs and
p70S6k. Both in a cell line stably overexpressing eIF4E and
in cell lines in which eIF4E overexpression is inducible,
dephosphorylation of 4E-BP1 correlates well with eIF4E protein
upregulation. We have also noted dephosphorylation of 4E-BP2 upon
overexpression of eIF4E (39a). Significantly, upregulation
of eIF4E also results in the dephosphorylation of p70S6k.
Akt, which is an upstream effector of 4E-BP1 (30, 40, 71) and might lie upstream of FRAP/mTOR (63; reviewed in
reference 67) in this signalling pathway, is not
dephosphorylated when eIF4E is overexpressed, but it could potentially
be inhibited by direct interaction with eIF4E or by another mechanism,
indicating that the negative feedback target lies at or downstream of
Akt.
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|
FIG. 9.
Proposed model for translational homeostasis in response
to changes in eIF4E concentration. eIF4E incorporation into an eIF4F
complex is regulated by the PI 3-kinase pathway, through Akt and
FRAP/mTOR, which leads to the phosphorylation of 4E-BPs and
p70S6k. Forced expression of eIF4E engenders a negative
feedback loop by downregulation of a component of the Akt-FRAP/mTOR
pathway or by activating a phosphatase (PPase) which dephosphorylates
4E-BP1 and p70S6k. This results in the dephosphorylation of
p70S6k and 4E-BP1.
|
|
We do not know whether the negative feedback effect of eIF4E is by a
direct or indirect stimulation of translation of a negative regulator.
Since the phosphorylation of both 4E-BP1 and p70S6k is
reported to be either directly or indirectly affected by FRAP/mTOR (10, 13, 15), the latter appears to be one intriguing
candidate for the target of eIF4E. However, it is equally possible that eIF4E enhances the activity of 4E-BP1 and p70S6k-directed
phosphatase(s) (Fig. 9). To directly address the question of whether
FRAP/mTOR activity is modulated by eIF4E overexpression, we have
attempted to determine whether in vitro FRAP/mTOR autokinase activity
(10) or FRAP/mTOR-associated 4E-BP1 kinase activity (13) was enhanced upon upregulation of eIF4E expression.
However, we failed to observe any significant changes in these
activities following eIF4E upregulation (1, 28a). This
result is not entirely surprising in view of the findings of Avruch and
collaborators that FRAP/mTOR activity, in response to amino acid
depletion followed by readdition or insulin stimulation, is not changed
when assayed in an in vitro kinase assay (34, 35). Hara et
al. also noted that FRAP/mTOR from rapamycin-treated cells, which is
completely inactive in vivo, showed no decrease in autokinase and
4E-BP1 kinase activity in vitro, and they thus concluded that
inhibition of FRAP/mTOR is apparently readily reversible during cell
extract preparation (35). However, it should be noted that
in other experiments FRAP/mTOR activity on 4E-BP1 in an in vitro kinase assay was shown to be stimulated by insulin by 1.8-fold
(63). Also, we did not detect any changes in the level of
mTOR upon eIF4E induction (39a).
The finding that eIF4E overexpression causes dephosphorylation of
p70S6k at Thr421 and Ser424 is of interest. These Ser/Thr
residues, which are two of the four initially identified sites of
mitogen-induced phosphorylation, are followed by proline residues and
are located in the carboxy terminus of p70S6k (24,
70). The carboxy terminus is thought to act as a pseudosubstrate autoinhibitory domain through interaction with its amino terminus. Phosphorylation of Ser411, Ser418, Ser424, and Thr421 has been postulated to disrupt the interaction between the amino and carboxy termini and allow the phosphorylation of Thr229 and Thr389, resulting in the activation of p70S6k (21). There are
conflicting reports as to the rapamycin sensitivity of the
phosphorylation of Thr421 and Ser424. In metabolic labelling experiments, followed by tryptic map analyses, it was shown that phosphorylation at these residues is rapamycin insensitive (33, 54). However, more recent experiments using a phosphospecific antibody against phosphorylated Thr421 and Ser424 peptides demonstrated that the phosphorylation of these sites in response to insulin is
rapamycin sensitive (35, 75).
The expression of a dominant-negative PKR, a mutant of eIF2, and
even eIF4G did not cause the dephosphorylation of 4E-BP1. Expression of
either of these proteins in NIH 3T3 cells results in cellular
transformation (22, 27, 42), presumably because of an
increase in translation of key growth-promoting proteins. Thus, the
effects reported here cannot be elicited by all translation factors. It
is also noteworthy that several colon tumor cell lines and tumor
tissues which overexpress eIF4E also show an increase in the
hypophosphorylated isoforms of 4E-BP1 (39a).
Some translational inhibitors (such as anisomycin and cycloheximide)
stimulate, in addition to the FRAP/mTOR pathway, the p38
mitogen-activated protein kinase and JNK/SAPK pathways (16, 38,
43). Although anisomycin and cycloheximide exert this effect at
low concentrations which do not inhibit general translation (16), it is possible that translation of sensitive mRNAs is inhibited. A downstream target of the p38 kinase is the
serine/threonine kinase, Mnk1 (28, 60), which phosphorylates
eIF4E on the physiologically relevant Ser 209 (74) and is
thought to be required for optimal eIF4E activity (29).
Thus, a translational feedback mechanism might be achieved by the
integration of signals from several signal transduction pathways, via
the activity of different effector proteins.
| |
ACKNOWLEDGMENTS |
We thank the anonymous reviewers for their insightful comments on
the manuscript. We thank Robert D. Polakiewicz and Michael J. Comb
for phosphospecific antibodies against p70S6k and Akt,
George Thomas for providing 40S ribosomes, and Kazuei Igarashi for
providing NIH 3T3 cells overexpressing eIF4G. We thank Christine
DeMaria and Andrew Craig for valuable comments on the manuscript. We
also thank Colin Lister and Chantal Binda for technical assistance and
Avak Kahvejian for assistance in preparation of the manuscript.
This research was supported by a grant from the National Institute of
Canada and the Howard Hughes Medical Institute International Scholar
Program to N.S. N.S. is a Medical Research Council of Canada
Distinguished Scientist and a Howard Hughes International Scholar. K.K.
is a recipient of a predoctoral studentship from the Medical Research
Council of Canada. S.P. is a recipient of a postdoctoral fellowship
from the Association pour la Recherche sur le Cancer. A.-C.G. is a
recipient of a 1967 Centennial Studentship from the National Science
and Engineering Research Council of Canada.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and McGill Cancer Center, McGill University, 3655 Drummond St., Montreal, Quebec, Canada H3G 1Y6. Phone: (514) 398-7274. Fax:
(514) 398-1287. E-mail: nsonen{at}med.mcgill.ca.
| |
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Abstract
Introduction
