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Molecular and Cellular Biology, September 1999, p. 6041-6047, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cell Cycle Progression and Proliferation Despite
4BP-1 Dephosphorylation
Steven O.
Marx1 and
Andrew R.
Marks1,2,*
Molecular Cardiology Program, Divisions of
Cardiology and Circulatory Physiology, Department of
Medicine,1 and Department of
Pharmacology,2 Columbia University College of
Physicians and Surgeons, New York, New York 10032
Received 7 January 1999/Returned for modification 17 March
1999/Accepted 26 May 1999
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ABSTRACT |
Proliferation and cell cycle progression in response to growth
factors require de novo protein synthesis. It has been proposed that
binding of the eukaryotic translation initiation factor 4E (eIF-4E) to
the inhibitory protein 4BP-1 blocks translation by preventing access of
eIF-4G to the 5' cap of the mRNA. The signal for translation initiation
is thought to involve phosphorylation of 4BP-1, which causes it to
dissociate from eIF-4E and allows eIF-4G to localize to the 5' cap. It
has been suggested that the ability of the macrolide antibiotic
rapamycin to inhibit 4BP-1 phosphorylation is responsible for the
potent antiproliferative property of this drug. We now show that
rapamycin-resistant cells exhibited normal proliferation despite
dephosphorylation of 4BP-1 that allows it to bind to eIF-4E. Moreover,
despite rapamycin-induced dephosphorylation of 4BP-1, eIF-4E-eIF-4G
complexes (eIF-4F) were still detected. In contrast, amino acid
withdrawal, which caused a similar degree of 4BP-1 dephosphorylation,
resulted in dissociation of the eIF-4E-eIF-4G complex. Thus, 4BP-1
dephosphorylation is not equivalent to eIF-4E inactivation and does not
explain the antiproliferative property of rapamycin.
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INTRODUCTION |
Cellular proliferation involves
translation of specific mRNAs encoding proteins required for
transition from the G1 phase to the S phase of the cell
cycle (6, 30). Both proliferative and antiproliferative
stimuli (26, 35) can regulate translation initiation, which
is the rate-limiting step in de novo protein synthesis. Eukaryotic
initiation factor 4E (eIF-4E) has been proposed as the critical
regulator of translation (26). eIF-4E binds to the 7-methyl
GTP (m7-GTP) cap on the 5' untranslated region of all
cytoplasmic eukaryotic mRNAs and recruits the 40S ribosomal complex.
This complex comprises eight proteins, including eIF-4A (RNA helicase),
eIF-4B (RNA binding protein), and eIF-4G, a scaffolding protein that
directly interacts with eIF-4E and is believed to unwind the secondary
structure of the 5' untranslated region, allowing efficient translation initiation (26, 33).
4BP-1 (or PHAS-I) has been identified as an important inhibitor of
eIF-4E (23, 33). 4BP-1 is thought to inhibit translation initiation by binding to eIF-4E (which is continuously bound to the 5'
cap) and preventing its association with eIF-4G (14). Phosphorylation of 4BP-1 causes it to dissociate from eIF-4E, thereby
allowing translation initiation to proceed (7, 11, 23).
Overexpression of 4BPs (4BP-1 and 4BP-2) in cells transformed by either
eIF-4E or v-src causes significant reversion of the transformed phenotype, suggesting that members of the 4BP family are
negative regulators of cell growth (43).
The mTOR/FRAP/RAFT1 (4, 17, 44) protein has been shown to
regulate phosphorylation of 4BP-1 (7, 15, 23, 33) and
p70s6k (5). Rapamycin bound to its cytosolic
receptor, the FK506 binding protein (FKBP12) (45), inhibits
the kinase activity of mTOR/FRAP/RAFT1, resulting in dephosphorylation
of 4BP-1, increased 4BP-1-eIF-4E complex formation, and, presumably,
inhibition of translation initiation (7, 11, 13, 23, 28, 33,
46). It has been proposed that inactivation of eIF-4E via 4BP-1
is the mechanism whereby rapamycin inhibits G1-to-S-phase
progression (7). However, disruption of the gene encoding
4BP-1 (PHAS-I) in mice does not cause rapamycin resistance, and
fibroblasts derived from these mice exhibit normal protein synthesis
and growth (2). Furthermore, 4BP-1 may not play a
significant role in rapamycin's antiproliferative effects, as
suggested by the findings that rapamycin does not prevent the early
effects of serum-induced protein translation, polysome formation
(34), eIF-4E phosphorylation, or the recruitment of eIF-4E
into the eIF-4F complex (29).
mTOR has also been implicated in the pathway(s) mediating nutrient
sensing through dephosphorylation of both p70s6k (3,
16, 48) and 4BP-1 (16, 48). For example, amino acid
withdrawal in Chinese Hamster Ovary (CHO) cells causes
p70s6k dephosphorylation and kinase inhibition, 4BP-1
dephosphorylation, increased 4BP-1-eIF-4E association, and reduced
eIF-4E-eIF-4G complex formation (16, 48). Rapamycin
inhibits the ability of amino acids to induce the release of 4BP-1 from
eIF-4E and inhibits the complex formation of eIF-4E-eIF-4G
(48). Therefore, the effect of rapamycin on eIF-4E-eIF-4G
complex formation appears to be related to the method of stimulation;
rapamycin does not inhibit serum-induced eIF-4E-eIF-4G complex
formation (29), whereas it does inhibit amino acid-induced
complex formation (48).
In the present study, we examined the effects of rapamycin on
modulators of protein translation in four different cell lines. We
found that in CHO cells, BC3H1 cells, and two rapamycin-resistant (RR)
cell lines, i.e., (i) RR cells generated from BC3H1 cells and (ii)
murine erythroleukemia cells (MELC), rapamycin caused dephosphorylation
of 4BP-1 and increased association of 4BP-1 and eIF-4E without causing
eIF-4E-eIF-4G dissociation. These results suggest that rapamycin does
not cause cell cycle arrest through inhibition of the eIF-4E-eIF-4G
complex formation.
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MATERIALS AND METHODS |
Cell culture.
BC3H1 and RR cells were grown in Dulbecco's
modified essential medium (DMEM) with the addition of 20% fetal bovine
serum (FBS) and 1% penicillin-streptomycin. The medium was changed
every 48 to 72 h. Rapamycin was added directly to the medium. The
cells were cultured for multiple passages in the presence of high
concentrations of rapamycin (0.1 to 1 µM) followed by dilutional
cloning. Two independent clones were utilized; RR-1 clones were grown
in 1 µM rapamycin, and RR-3 clones were grown in 0.1 µM rapamycin. In all studies, rapamycin was removed 1 week prior to the experiment. MELC were grown in minimal essential medium-
(MEM-) supplemented with 10% FBS (inactivated) and 1% penicillin-streptomycin
(38). CHO cells (obtained from the American Type Culture
Collection) were grown in Ham's F-12 medium supplemented with 10% FBS
and 1% penicillin-streptomycin. Deprivation and restoration of amino acids were performed as previously described (48).
Cell proliferation assays.
BC3H1, RR-1, and RR-3 cells
(25 × 104) were grown in DMEM plus 20% FBS (in
triplicate). Rapamycin (100 nM) was added directly to the medium. After
48 h, the cell number was determined with a Coulter Counter. MELC
(10 × 104) were grown in MEM plus 10% FBS; rapamycin
(0.2 to 1 µM) or a vehicle was added directly to the medium. After 4 days, the cells were counted with a Coulter Counter.
[3H]leucine incorporation.
BC3H1 and RR-1
cells (104) were plated in triplicate in 12-well dishes.
After 48 h, rapamycin (1 µM) was added to the appropriate wells,
and cells were pulsed with 1 µCi of [3H]leucine per
well. [3H]leucine incorporation into protein was
determined by precipitation with trichloroacetic acid. Experiments were
repeated three times.
Fluorescence-activated cell sorter analysis.
BC3H1 and RR-1
cells were placed in DMEM plus 1% FBS for 24 h. The cells were
then stimulated with DMEM plus 20% FBS and treated with either
rapamycin (100 nM) or the vehicle. The cells were washed and harvested
after 24 h and labeled with propidium iodide solution-RNase for
1 h. The cells were analyzed on a fluorescence-activated cell
sorter, with a minimum of 15,000 cells counted.
Western blot analyses.
To detect 4BP-1, eIF-4E, and eIF-4G,
lysates were prepared as previously described (23). Parental
BC3H1 cells and RR cells, MELC, and CHO cells were grown in DMEM plus
20% FBS, MEM plus 10% FBS (inactivated), and Ham's F-12 medium plus
10% FBS, respectively. Asynchronously dividing cells were treated with
either the vehicle or rapamycin for 30 min, and cellular lysates were
prepared. Protein was measured with the Bradford reagent, size
fractionated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), and transferred to nitrocellulose.
Membranes were incubated as previously described (23) and
visualized by enhanced chemiluminescence. For 4BP-1-eIF-4E complex
experiments, cell extracts (130 µg) were incubated with
m7-GTP-Sepharose in duplicate for 1 h at 4°C. The
resin was washed and samples were size fractionated on SDS-8%
(eIF-4G) or -13% (eIF-4E and 4BP-1) polyacrylamide gels, transferred
to nitrocellulose, and blotted with eIF-4G (29), eIF-4E
(Transduction Laboratories), or 4BP-1 (PHAS-I) (23)
antibodies. For immunoprecipitation, cell extracts (100 µg) were
incubated with 2 µl of anti-eIF-4G antibody (49) in 500 µl of lysis buffer, captured with protein A-Sepharose, washed and
eluted with Laemmli buffer, and size fractionated on SDS-11.5%
polyacrylamide gels.
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RESULTS AND DISCUSSION |
Proliferation and cell cycle progression in RR cells.
RR and
BC3H1 cells were selected on the basis of normal growth following
multiple passages in the presence of high concentrations of rapamycin
(either 100 or 1,000 nM). Proliferating RR and parental cells exhibited
similar morphologies (Fig. 1A). Two
independent clonal RR cell lines (RR-1 and RR-3) exhibited no growth
arrest (Fig. 1B) and no decrease in viability (as assessed by trypan blue exclusion) in the presence of rapamycin. Rapamycin induced a delay
in transition from G1 to S phase in the parental cells but
not in the RR cells (Fig. 1C).
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FIG. 1.
Growth characteristics of RR cells. (A) The morphologies
and sizes of proliferating parental (BC3H1) and RR-1 cells are similar.
(B) Rapamycin inhibited cell growth in the parental cells but not in
two RR cell lines, RR-1 and RR-3. Hatched bars show the numbers of
cells that were plated; cell number was determined after 48 h.
Growth was significantly inhibited for rapamycin-treated cells (100 nM,
black bars) compared to untreated BC3H1 cells (white bar). Data are
averages of triplicate samples + standard deviations. (C) Flow
cytometry of parental and RR-1 cells. Cells were serum starved in DMEM
plus 1% FBS for 24 h, followed by stimulation with 20% FBS.
Cells were harvested after 24 h and stained with propidium iodide.
Analysis was based upon results from a minimum of 15,000 cells.
Parental cells treated with rapamycin (100 nM) arrested in
G1/S; RR-1 cells showed no rapamycin-induced inhibition of
cell cycle progression.
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Rapamycin inhibits activation of cyclin-dependent kinases (CDK) by
preventing mitogen-induced down-regulation of the CDK inhibitor p27kip1 (31).
p27kip1 protein regulation has been shown to be
controlled at the translational level (18) and via
ubiquitin-dependent degradation (32). Rapamycin resistance
in RR cells results from a deficiency in p27kip1
due to increased degradation of the protein via a ubiquitin-independent pathway (25). RR cells have constitutively low
p27kip1 levels, and unlike the case with
parental cells, p27kip1 levels are not regulated
in response to mitogens or rapamycin (25). In response to
serum withdrawal, RR cells undergo apoptosis because of their inability
to extinguish hyperphosphorylation of retinoblastoma protein (pRb)
(25).
Inhibition of 4BP-1 phosphorylation.
The ability of RR cells
to proliferate in the presence of high concentrations of rapamycin
provided the opportunity to determine whether inactivation of
proteins thought to be required for translation interferes with cell
cycle progression. Rapamycin has been previously demonstrated to
minimally inhibit protein synthesis, if at all (11, 36). In
both proliferating BC3H1 and RR-1 cells, rapamycin (1 µM)
demonstrated no significant effect on [3H]leucine
incorporation (Fig. 2A).
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FIG. 2.
Generalized protein synthesis is unaffected by
rapamycin, but 4BP-1 is inhibited in parental and RR cells. (A)
Parental BC3H1 and RR-1 cells (104) were cultured in DMEM
plus 20% FBS for 48 h in 12-well dishes; 1 µM rapamycin (black
bars) or vehicle (white bars) was added to the appropriate wells when
the cells were pulsed with [3H]leucine (1 µCi).
Incorporation of [3H]leucine was measured by
precipitation with trichloroacetic acid. Rapamycin had no significant
effect on protein synthesis. Data are representative of three
experiments. (B) Parental BC3H1, RR-1, and RR-3 cells were cultured in
20% FBS; 100 nM rapamycin or vehicle was added to the cultures, and
lysates were prepared after 45 min. Cellular lysates (100 µg) were
analyzed by immunoblotting with an anti-4BP-1 (anti-PHAS-I) antiserum.
,  , and  are arbitrary designations of bands representing the
phosphorylated forms of 4BP-1 (23).
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Rapamycin inhibited phosphorylation of both p70
s6k (data
not shown and reference
25) and 4BP-1 (Fig.
2B) in
proliferating RR
cells. The inhibition of both p70
s6k and
4BP-1 phosphorylation in wild-type and RR cells was observed
more than
48 h after removal of rapamycin from the culture medium
(data not
shown). Rapamycin is a potent inhibitor of cell growth;
therefore, it
has been difficult to determine whether the drug
specifically inhibits
translation or, conversely, whether the
primary effect of rapamycin is
to inhibit cell cycle progression
(e.g., by up-regulating
p27
kip1 [
25,
31]). However, the
ability of rapamycin to inhibit 4BP-1
(PHAS-1) phosphorylation and
eIF-4E function has been interpreted
as indicating that inhibition of
translation is the mechanism
underlying the growth-inhibitory
properties of rapamycin (
7).
The rapamycin-induced
inhibition of 4BP-1 in proliferating RR
cells uniquely demonstrates
that proliferation can proceed despite
dephosphorylation of 4BP-1.
4BP-1-eIF-4E interaction.
Overexpression of eIF-4E increases
the translation of mRNAs containing extensive secondary structures,
including cyclin D1 (40, 41), ornithine decarboxylase
(42), and c-myc (9), and causes
transformation of fibroblasts (10, 22, 39). Immunoblot analyses showed that eIF-4E protein levels were equivalent in the
parental and RR cells (Fig. 3A),
indicating that the stoichiometry between 4BP-1 (Fig. 2B) and eIF-4E
was unchanged in the RR cells. eIF-4E is constitutively bound to the 5'
cap, and its activity is regulated by the binding of the inhibitory
protein 4BP-1 (23, 33). To determine whether 4BP-1 was bound
to eIF-4E in RR cells, an affinity resin containing the 5' cap homolog
m7-GTP was used as previously described (23). In
these experiments dephosphorylation of 4BP-1 caused 4BP-1 to bind to
eIF-4E, which in turn was bound to the m7-GTP resin. RR
cells cultured with rapamycin were able to proliferate despite
persistent inactivation of eIF-4E by 4BP-1 (Fig. 3B). No significant
differences in the amount of 4BP-1 binding to eIF-4E were seen in BC3H1
and RR cells treated with rapamycin.
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FIG. 3.
Rapamycin increases complex formation between 4BP-1 and
eIF-4E in parental and RR cells. (A) Cell extracts (100 µg) were
analyzed by immunoblotting with anti-eIF-4E antibody. eIF-4E protein
levels were equivalent in parental BC3H1 and RR cells. (B) Binding of
4BP-1 to eIF-4E (already bound to m7-GTP-Sepharose resin)
was determined. m7-GTP-Sepharose was incubated with 130 µg of cellular extract for 1 h at 4°C. The resin was washed,
size fractionated by SDS-PAGE, transferred to nitrocellulose, and
blotted with anti-4BP-1 or anti-eIF-4E antibody.
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Like RR cells, MELC exhibited no growth arrest after 4 days of
rapamycin treatment (Fig.
4A). Rapamycin
also inhibits p70
s6k in MELC, as previously reported
(
8). In proliferating MELC,
rapamycin (0.2 µM) treatment
for 1 h inhibited 4BP-1 phosphorylation
(Fig.
4B) and increased
the complex formation between 4BP-1 and
eIF-4E as assessed by
m
7-GTP binding (Fig.
4C). Rapamycin had no effect on eIF-4E
protein
levels in MELC (data not shown).
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FIG. 4.
MELC are rapamycin resistant and demonstrate
rapamycin-induced 4BP-1 dephosphorylation and increased 4BP-1-eIF-4E
complex formation. (A) MELC (10 × 104 cells; hatched
bar) were treated with either a vehicle (white bar) or increasing
concentrations of rapamycin (black bars [numbers are micromolar
concentrations]). Cells were counted at 4 days. These data are
representative of two experiments. (B) Cell extracts were analyzed by
immunoblotting with an anti-4BP-1 antibody. Rapamycin (0.2 µM)
treatment for 1 h induced dephosphorylation of 4BP-1. , ,
and are arbitrary designations of bands representing the
phosphorylated forms of 4BP-1 (23). Data are representative
of three experiments. (C) Binding of 4BP-1 to eIF-4E (bound to
m7-GTP resin) was determined in the presence or absence of
rapamycin (0.2 µM). Cellular extracts were incubated with
m7-GTP resin, size fractionated by SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with anti-4BP-1 and anti-eIF-4E
antibodies. Rapamycin increased the binding of 4BP-1 to eIF-4E.
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Rapamycin does not affect eIF-4E-eIF-4G complex formation.
The binding of eIF-4G or 4BP-1 to eIF-4E is believed to be mutually
exclusive (14, 36). Therefore, the increased association of
eIF-4E and 4BP-1 would theoretically reduce the association of eIF-4E
and eIF-4G (7, 14, 24, 47). Several groups have shown that
increased eIF-4E-eIF-4G association was correlated with decreased
4BP-1-eIF-4E complex formation (12, 21). Moreover, rapamycin prevented amino acid-induced eIF-4E-eIF-4G complex formation in amino acid-depleted CHO cells (48). However, conflicting data have also been reported with regard to the effects of rapamycin on
eIF-4E-eIF-4G interactions (20, 29). Morley and McKendrick demonstrated with NIH 3T3 cells that rapamycin increased the
association of 4BP-1 and eIF-4E on m7-GTP resin but that
rapamycin did not prevent serum-induced eIF-4E-eIF-4G interaction as
assessed by immunoprecipitation assays (29). Moreover, they
demonstrated that rapamycin does not inhibit the recruitment of eIF-4E
to the ribosome and that prolonged incubation (for 20 h) causes
only a 30 to 40% reduction in the amount of eIF-4E associated with
eIF-4G (29). Furthermore, Beretta et al. (1) have
shown a lack of temporal correlation between 4BP-1 dephosphorylation
and inhibition of in vitro cap-dependent translation. Morley and
McKendrick (29) and others (1) have suggested that these data may reflect a low rate of release of 4BP-1 from eIF-4E,
such that eIF-4E is able to associate with 4BP-1 only after release
from eIF-4G.
In CHO cells, rapamycin did not prevent the association of eIF-4E and
eIF-4G as assessed by immunoprecipitation and binding
to
m
7-GTP resin (Fig.
5A).
However, as previously described (
48),
amino acid
deprivation for 1 h did significantly reduce the association
of
eIF-4E and eIF-4G. Rapamycin (1 µM) did not prevent serum-induced
association of eIF-4E and eIF-4G. Morley and McKendrick (
29)
suggested that there might be a population of eIF-4E that is
inaccessible
to dephosphorylated 4BP-1 and thus dissociation from
eIF-4G. However,
our findings that amino acid withdrawal rapidly
induced dissociation
of the eIF-4E-eIF-4G complex in cells in which
serum withdrawal
did not induce the same dissociation suggest that
compartmentalization
may not explain the lack of effect of rapamycin on
this complex
in both CHO cells and BC3H1 or RR cells.
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FIG. 5.
Rapamycin does not prevent serum-induced association of
eIF-4E-eIF-4G in CHO, BC3H1, and RR cells. (A) CHO cells were grown in
DMEM plus 10% FBS and treated with either a vehicle or rapamycin for
24 h. In parallel experiments, CHO cells were placed in Ham's
F-12 medium without serum for 16 h. Cells were washed and amino
acid deprived in Earle's salt solution for 1 h. Cells were
stimulated with Ham's F-12 medium plus 10% FBS following pretreatment
with either a vehicle or rapamycin (1 µM). Cell lysates were prepared
as described in Materials and Methods. Cellular extracts (100 µg)
were incubated with m7-GTP resin at 4°C in duplicate,
washed, size fractionated on an SDS-8% polyacrylamide gel,
transferred to nitrocellulose, and immunoblotted with anti-eIF-4G
antibody (the nitrocellulose was also incubated with anti-eIF-4E
antibody to demonstrate equal uptake on the resin [data not shown]).
Cellular extracts (100 µg) were immunoprecipitated with anti-eIF-4G
antibody, bound with protein A-Sepharose beads, size fractionated on an
SDS-11.5% polyacrylamide gel, transferred to nitrocellulose, and
immunoblotted with either anti-eIF-4E or anti-eIF-4G antibodies. Amino
acid (AA) withdrawal significantly inhibited eIF-4E-eIF-4G complex
formation. Rapamycin had no effect on serum-stimulated eIF-4E-eIF-4G
association. Data are representative of three similar experiments. (B)
BC3H1 and RR-1 cells were grown in DMEM plus 20% FBS. Experiments were
performed as described above (in duplicate) to determine the binding of
eIF-4G and 4BP-1 to eIF-4E bound to m7-GTP-Sepharose
resin. Size fractionation was performed by SDS-PAGE with either 8%
(eIF-4G) or 13% (4BP-1) polyacrylamide. Rapamycin (1 µM) was added
to the medium 24 h prior to cell lysis. Rapamycin and serum
withdrawal (cells maintained in DMEM alone) for 1 h caused
increased association of 4BP-1 with the m7-GTP resin in
BC3H1 and RR cells. However, complex formation between eIF-4E and
eIF-4G persisted despite rapamycin (1 µM) treatment for 24 h.
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In BC3H1 and RR cells, rapamycin (1 µM for 24 h) caused
increased association of 4BP-1 with the m
7-GTP resin but
failed to inhibit the serum-induced association
of eIF-4E and eIF-4G as
assessed by binding to the m
7-GTP (Fig.
5B) and by
coimmunoprecipitation (Fig.
6). In RR
cells
exposed to rapamycin for more than 1 week, eIF-4G-eIF-4E
association
persisted despite increased 4BP-1 association with eIF-4E
(data
not shown), indicating that even prolonged exposure to rapamycin
and 4BP-1 dephosphorylation does not induce eIF-4E-eIF-4G
dissociation.
Amino acid withdrawal in BC3H1 and RR cells caused
increased association
of 4BP-1 and eIF-4E on m
7-GTP resin
and markedly reduced eIF-4E-eIF-4G complex formation
(Fig.
6).
Rapamycin inhibited the amino acid-induced eIF-4E-eIF-4G
complex
formation in nutrient-deprived BC3H1 cells; however, rapamycin
did not
inhibit the serum-induced eIF-4E-eIF-4G complex formation
in BC3H1 and
RR cells (Fig.
6). Therefore, although growth factor
(serum) withdrawal
can lead to dephosphorylation of 4BP-1 and
increased 4BP-1-eIF-4E
association, in the absence of amino acid
withdrawal, it is
insufficient to cause eIF-4E-eIF-4G complex
dissociation.
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FIG. 6.
Differential regulation of the eIF-4E-eIF-4G complex by
rapamycin and amino acid withdrawal in BC3H1 and RR-1 cells. BC3H1 and
RR-1 cells were cultured in DMEM plus 20% FBS and treated with
rapamycin (1 µM). In amino acid (AA) withdrawal experiments, cells
were placed in Earle's balanced salt solution for 1 h. Cells were
pretreated with rapamycin (1 µM) for 15 minutes and then stimulated
for 30 min either with amino acids or with 20% FBS plus amino acids.
Cellular extracts were prepared. eIF-4G and eIF-4E were
coimmunoprecipitated with an anti-eIF-4G antibody and immunoblotted
with either anti-eIF-4G or anti-eIF-4E antibody. In addition, the same
extracts were used in experiments performed as described above (in
duplicate) to determine the binding of 4BP-1 to eIF-4E (already bound
to m7-GTP-Sepharose resin).
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Conclusions.
The mechanism(s) by which rapamycin inhibits
proliferation and induces G1/S arrest have not been fully
elucidated. Rapamycin induces a G1-to-S-phase transition
arrest and inhibits a mitogen-activated signaling pathway that involves
mTOR, p70s6k, p27kip1, 4BP-1,
eIF-4E, cell cycle kinases, and retinoblastoma protein (5, 7, 11,
23, 28, 33). However, the immediate downstream targets of mTOR
and the significance of rapamycin's inhibition of p70s6k,
4BP-1, and eIF-4E remain uncertain. The observations by several groups
that the inhibition of mTOR, p70s6k, and 4BP-1
phosphorylation by rapamycin were coupled to growth arrest and to
inactivation of eIF-4E led to the hypothesis that rapamycin's
antiproliferative properties are mediated via translational control
(7, 13, 19, 37). In the present study, we used two RR muscle
cell lines (RR-1 and RR-3) and a third hematopoietic cell line (MELC)
that is also resistant to rapamycin and confirmed our findings with
rapamycin-sensitive CHO cells. This strategy allowed us to address the
question of whether cell cycle progression and translation can proceed
despite dephosphorylation of 4BP-1 and inactivation of
p70s6k by rapamycin. We showed that rapamycin-mediated
dephosphorylation and increased binding of eIF-4E to 4BP-1 are not
required for rapamycin's antiproliferative effects. Rapamycin does not
cause the dissociation of the eIF-4E-eIF-4G complex in
serum-stimulated cells; therefore, inhibition of protein translation
does not appear to be the mechanism through which rapamycin exerts its
antiproliferative effects. Rapamycin caused no significant reduction in
protein synthesis (Fig. 2A) (11, 36), in contrast to amino
acid withdrawal, which has a more profound effect on generalized
protein synthesis (16). Although amino acid withdrawal and
rapamycin cause similar degrees of dephosphorylation of
p70s6k and 4BP-1, presumably through inhibition of mTOR
activity, they do not have similar effects on the association of eIF-4E
and eIF-4G. This suggests that the mTOR pathway, which is regulated by
amino acids and rapamycin, may play a greater role in
p70s6k inhibition and 4BP-1 phosphorylation than in
eIF-4E-eIF-4G complex regulation.
The ability of rapamycin to inhibit cellular proliferation may have
important applications in the treatment of disorders such
as
accelerated arteriopathy that occurs in transplanted hearts
and
restenosis following the placement of coronary stents (
27).
The present study indicates that the antiproliferative properties
of
rapamycin are probably not mediated by inhibition of protein
translation via inactivation of p70
s6k or eIF-4E. While it
appears that rapamycin does not inhibit cell
growth via translational
control, the CDK inhibitors, in particular
p27
kip1, remain attractive candidates for
mediators of the drug's important
antiproliferative
properties.
| |
ACKNOWLEDGMENTS |
We thank J. Blenis for anti-p70s6k antibody, J. Lawrence for anti-PHAS-I (anti-4BP-1) and anti-eIF-4E antibodies, S. Morley and R. Rhoads for anti-eIF-4G antibodies, V. Richon for MELC and
for data on the effects of rapamycin on MELC growth, and J. Hurwitz and
D. Cobrinik for critical reading of the manuscript.
This work was supported by the NIH, MDA, and AHA (A.R.M.) and the
Richard and Lynne Kaiser Family Foundation and by a grant from the
Johnson and Johnson Focused Giving Program. S.O.M. is a recipient of an
American Heart Association Clinician Scientist Award and the NY Academy
of Medicine Glorney-Raisbeck Fellowship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Molecular
Cardiology Program, Box 65, Columbia University College of Physicians & Surgeons, Rm 9-401, 630 West 168th St., New York, NY 10032. Phone:
(212) 305-0270. Fax: (212) 305-3690. E-mail:
arm42{at}columbia.edu.
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
