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INTRODUCTION |
Most of the cells in the adult mammalian liver are in a quiescent
state, but they are induced to re-enter the cell cycle by chemical or
surgical treatments that remove or destroy a portion of the liver. A
commonly used method for inducing liver regeneration in the rat is by
performing a partial hepatectomy in which the two main lobes of the
liver are excised, resulting in 68% tissue loss (1). After partial
hepatectomy, the remaining liver cells rapidly undergo one or two
proliferative cycles so that the mass of the liver remnant nearly
doubles after 2 days and approaches the original liver weight by 7 days. The kinetics of this proliferative response have been well
documented (reviewed in Ref. 2). DNA synthesis in parenchymal cells
(hepatocytes) begins about 12 h after partial hepatectomy and
reaches a peak by 20-24 h, followed by a gradual decline. Mitosis
follows 6-8 h later. Nonparenchymal cells lag 24 h behind
hepatocytes in initiating DNA synthesis and mitosis. DNA synthesis and
restoration of the cell deficit in the liver remnant are mostly
complete by 72 h.
Partial hepatectomy also leads to an increase in hepatic protein
synthesis that is essential for regeneration (3, 4). Superimposed on a
general increase in the rate of protein synthesis are marked increases
in translation of specific mRNAs (5). Experiments using a variety
of model systems have shown that increased translation of these
growth-regulated mRNAs is exerted mainly at the level of
initiation. In translation initiation, the 43 S preinitiation complex
binds to the m7GpppN (where N is any nucleotide) cap on the
5' end of the mRNA, the complex translocates to the initiation
codon, and then the 60 S ribosomal subunit is added to form an active
80 S ribosome (reviewed in Refs. 6 and 7). In general, binding of the 43 S preinitiation complex to mRNA is the rate-limiting step in translation initiation. This step is mediated by eukaryotic initiation factor (eIF)1 4F. One subunit
of the eIF4F complex, eIF4E, binds directly to the m7GpppN
cap on the mRNA, and a second subunit, eIF4G, interacts with the 43 S preinitiation complex. Activation of translation initiation results
in increased loading of ribosomes onto growth-regulated mRNAs.
After partial hepatectomy, a variety of growth factors and hormones
carried in the bloodstream activate signaling pathways that stimulate
proliferation of the remaining liver cells (reviewed in Refs. 2 and 8).
Experiments done mainly in cultured cells have shown that some of these
signaling pathways activate protein synthesis by inducing the
phosphorylation of various components of the translational machinery
(6, 7, 9, 10). For example, many growth factors promote the
phosphorylation of the S6 protein in 40 S ribosomal subunits (9). The
major kinase that phosphorylates S6 is the Mr = 70,000 S6 kinase (p70 S6 kinase) (9, 11). p70 S6 kinase is activated by
phosphorylation of the enzyme at multiple serine and threonine residues
(12, 13). Phosphorylation of the 40 S ribosomal subunit by p70 S6
kinase is thought to selectively increase translation of a class of
growth-regulated mRNAs characterized by the presence of a
polypyrimidine tract adjacent to the m7GpppN cap
(5'-terminal oligopyrimidine mRNAs) (14, 15). A second mechanism
that mediates growth factor-induced activation of protein synthesis is
phosphorylation of the translation repressor eIF4E-binding protein 1 (4E-BP1) (10). In quiescent cells, hypophosphorylated forms of 4E-BP1
bind tightly to eIF4E on the mRNA cap, thus excluding eIF4G from
the eIF4F complex. Treatment of cells with growth factors leads to
phosphorylation of 4E-BP1 on multiple sites and its dissociation from
eIF4E, thereby allowing assembly of a functional eIF4F complex (10,
16). Translation of mRNAs with extensive secondary structure at the
5' end is thought to be particularly sensitive to regulation by
4E-BP1.
Treatment of cultured cells with a variety of stimuli leads to
the simultaneous phosphorylation of both p70 S6 kinase and 4E-BP1. In
addition, growth factor-induced activation of p70 S6 kinase and
disruption of the 4E-BP1·eIF4E complex in vitro is blocked
by nanomolar concentrations of the immunosuppressant rapamycin (17-20). The drug exerts this effect by inhibiting the phosphorylation of specific functionally important sites in both proteins (13, 21).
Rapamycin, when bound to its intracellular receptor FKBP12, inhibits
the function of the mammalian target of rapamycin (mTOR), a protein
kinase whose catalytic domain is structurally related to that of
phosphatidylinositol 3-kinase (22, 23). Although it is well accepted
that mTOR activity is required for p70 S6 kinase activation and 4E-BP1
phosphorylation, the mechanism of action of mTOR is controversial. On
one hand, it has been proposed that mTOR is a growth factor-activated
kinase (24, 25) that directly phosphorylates the two proteins at
functionally important sites (26-28). Alternatively, it has been
suggested that mTOR inhibits a phosphatase that can dephosphorylate p70
S6 kinase and 4E-BP1 (29; see discussion in Ref. 13).
Francavilla et al. (29) showed earlier that injection of
rats with rapamycin before partial hepatectomy strongly inhibited DNA
synthesis and mitosis in the liver remnant. Since rapamycin is
currently being used in liver transplant patients (30), it will be
important to determine whether the drug exerts a similar growth
inhibitory effect in humans and, if so, to elucidate the inhibitory
mechanism. Results from studies in cultured cells would suggest that
the growth inhibitory effect of rapamycin in rat liver is due to a
reduction in p70 S6 kinase activity and/or 4E-BP1 phosphorylation.
However, no in vivo studies have been done to test this
hypothesis. In this study, we asked whether rapamycin targets p70 S6
kinase and 4E-BP1 to inhibit liver regeneration. We first tested if p70
S6 kinase activity and functional phosphorylation of 4E-BP1 are altered
after partial hepatectomy. Then we determined whether p70 S6 kinase
activity and 4E-BP1 phosphorylation are reduced in rapamycin-treated
rats. Our data indicate that rapamycin, at a dose that significantly
attenuates liver regeneration, selectively inhibits p70 S6 kinase
activation but not phosphorylation of 4E-BP1 at sites that regulate
binding to eIF4E. These results suggest that the in vivo
effect of rapamycin on 4E-BP1 function might differ significantly from
that observed in cell culture models, which may have important
therapeutic implications for this clinically useful drug.
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EXPERIMENTAL PROCEDURES |
Animals and Surgical Procedures--
Male Harlan Sprague-Dawley
rats weighing ~250 g were purchased from Harlan Sprague-Dawley, Inc.
(Indianapolis, IN). The rats were housed in a quiet room with a 12/12-h
light/dark cycle. Experimental protocols were approved by the
Institutional Animal Care and Use Committee. All rats undergoing
surgery were anesthetized by intramuscular injection with 0.5 ml of 10 mg/ml nalbuphine plus 0.2 ml of a mixture containing 60% ketamine and
40% Rompun. After adequate anesthesia was attained, animals
were placed in the supine position, intubated, and ventilated with
100% O2 using an SAR 830 pressure-fed small animal
respirator (Stoelting Co., Wood Dale, IL). PH rats were subjected to
partial hepatectomy as described previously (1). SHAM rats were
subjected to a sham operation involving laparotomy and manipulation of
the liver without partial hepatectomy. RAPA rats were injected
intraperitoneally with 0.4 mg/kg rapamycin (Calbiochem) in 250 µl of
dimethyl sulfoxide 2 h before partial hepatectomy, and the same
dose of rapamycin was given daily after surgery. Me2SO rats
were treated the same as the RAPA group except that 250 µl of
dimethyl sulfoxide was injected instead of rapamycin. After surgery on
the above animals, the incisions were closed with sutures in two
layers, and the rats were fed and watered ad libitum until
sacrifice. CONT rats were not subjected to any treatment or handling
before sacrifice. For growth factor treatment, laparotomy was
performed, and 5 µl of 1 mM insulin (Sigma) or 500 µl
of 0.2 mg/ml human recombinant epidermal growth factor (Sigma) was
injected into the portal vein. The rats were sacrificed 10 min
(insulin) or 30 min (epidermal growth factor) after the injection.
p70 S6 Kinase Assay--
At specific times postoperatively,
animals were weighed and sacrificed by decapitation, and the livers
were removed. Livers were weighed, rinsed in cold phosphate-buffered
saline, and homogenized with an Ultra-Turrax (Janke & Kunkel, Staufen,
Germany) in ice-cold lysis buffer (25 mM HEPES, pH 7.5, 1%
Triton X-100, 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of
aprotinin and leupeptin). Homogenates were centrifuged at 15,000 × g for 30 min at 4 °C, and the supernatants were stored
at
80 °C. For kinase assays, 50 µl of liver extract was mixed
with 300 µl of lysis buffer, and 5 µl of antibody to p70 S6 kinase
(Santa Cruz Biotechnology, Santa Cruz, CA) was added. After 3 h on
ice, 12.5 µl of packed protein A-agarose beads were added to each
sample, and the tubes were agitated for 1 h at 4 °C. The beads
were washed twice with lysis buffer and twice with S6 kinase assay
buffer (31) minus dithiothreitol. S6 kinase activity in the
immunoprecipitates was then measured using 40 S ribosomal subunits as
substrate as previously described (31). The 32P
incorporated into S6 was then normalized to equal amounts of extract
protein. Protein concentration of liver extracts was determined with a
Bradford assay (Bio-Rad) using bovine serum albumin as a standard.
Immunoblotting--
Equal amounts of liver extract protein were
subjected to SDS-PAGE, and the proteins were transferred onto
polyvinylidene difluoride membranes. Membranes were blocked in 5%
nonfat milk for 1 h and then incubated with antibody to p70 S6
kinase, the hemagglutinin (HA) epitope tag (Covance, Richmond, CA),
4E-BP1 (Santa Cruz Biotechnology), eIF4E (Transduction Laboratories,
Lexington, KY), or 4E-BP1 phosphorylated at Thr-36, Thr-45, Ser-64, or
Thr-69 (Cell Signaling Technology, Inc., Beverly, MA; numbering is
based on the sequence of the rat protein (32)) overnight at 4 °C.
After extensive washing, membranes were incubated with secondary
antibody coupled to horseradish peroxidase (Amersham Pharmacia Biotech)
for 1 h at room temperature. Signals were visualized with an
enhanced chemiluminescence kit (PerkinElmer Life Sciences). The
integrated density of bands was quantified using NIH Image 1.62.
4E-BP1 Constructs--
The 4E-BP1 cDNA was isolated from
Swiss mouse 3T3 cell total RNA by reverse transcriptase-polymerase
chain reaction (RT-PCR) using Pfu DNA polymerase
(Stratagene, La Jolla, CA) and primers 5'-CGCGAATTCATGTCGGCGGGCAGCAGC
(forward) and 5'-CGCGGATCCTTAAATGTCCATCTCAAATTGTGAC (reverse). The PCR
fragment was digested with EcoRI and BamHI and
subcloned into pBluescript, and both strands of the insert were
sequenced. The insert showed two differences when compared with the
published sequence for 4E-BP1 from 3T3-L1 adipocytes (the T at position
109 was C and the G at position 118 was C; Ref. 19), but neither change
resulted in an amino acid difference. An optimal Kozak sequence (33)
followed by an HA tag was introduced onto the 5' end of the 4E-BP1
cDNA by PCR using primers
5'-CGCGAATTCGCCACCATGGCATACCCCTACGACGTGCCCGACTACGCCTCGGCGGGCAGCAGCTGC (forward) and the reverse primer shown above. The PCR fragment was digested with EcoRI and BamHI and subcloned
into pcDNA3.1(
). HA-4E-BP1 phosphorylation site mutants were made
using the QuikChange site-directed mutagenesis kit (Stratagene) and the
following primers (only the forward primers are shown): for Thr-36 to
Ala, 5'-GACTACAGCACCGCCCCGGGCGG; for Thr-45 to Ala,
5'-CTCTTCAGCACCGCCCCGGGAGG; for Ser-64 to Ala, 5'-GGAGTGTCGGAACGCACCTGTGGCC; and for Thr-69 to Ala,
5'-CACCTGTGGCCAAAGCACCCCCAAAGG. All mutations were confirmed by
sequencing both strands.
To construct the glutathione S-transferase (GST)-4E-BP1
fusion protein, primers 5'-CGCGGATCCTGTCGGCGGGCAGCAGC (forward) and 5'-CGCGAATTCTTAAATGTCCATCTCAAATTGTG (reverse) were used to produce a
PCR fragment using 4E-BP1 in pBluescript as template. The fragment was
digested with EcoRI and BamHI and ligated into
pGEX-5X-3 (Amersham Pharmacia Biotech). Bacteria were transformed with
the expression construct, and the GST-4E-BP1 fusion protein was
purified on glutathione-Sepharose.
Analysis of 4E-BP1 Mutants--
COS7 cells were seeded at 5 × 105 cells/6-cm dish in growth medium (Dulbecco's
modified Eagle's medium plus 10% fetal calf serum). The next day the
cells were transfected with 3 µg of DNA in 3 ml of Opti-MEM plus 7.5 µl of LipofectAMINE (Life Technologies, Inc.). After 5 h, the
transfection solutions were replaced with growth medium, and the cells
were allowed to grow for 2 days. After treatment with insulin or
LY294002, the cells were rinsed twice with cold phosphate-buffered
saline and scraped into cold extraction buffer containing 50 mM Tris, 120 mM NaCl, 20 mM NaF, 1 mM benzamidine, 5 mM EGTA, 30 mM
sodium pyrophosphate, 1% Triton X-100, 30 mM
p-nitrophenylphosphate, 0.5 mM dithiothreitol,
and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.2. Homogenates were centrifuged at 12,000 × g for 10 min
at 4 °C, and the supernatants were retained. Equal amounts of cell
lysate protein were subjected to immunoblotting as described above, and
the blots were probed with various 4E-BP1 antibodies.
Binding of 4E-BP1 to m7GTP-Sepharose--
4E-BP1
binding to m7GTP-Sepharose was performed essentially as
previously described (34). 4E-BP1 protein bound to the beads was eluted
by boiling in SDS-polyacrylamide gel electrophoresis sample buffer. The
samples were subjected to SDS-polyacrylamide gel electrophoresis, and
4E-BP1 was detected by immunoblotting as described above.
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RESULTS |
Rapamycin Inhibits Liver Regeneration--
Francavilla et
al. (29) show that injection of rats with 0.1-1 mg/kg rapamycin
for 4 days before partial hepatectomy inhibited DNA synthesis and
mitosis measured 24-26 h after surgery. A different treatment protocol
was used here to examine the effect of rapamycin on the weight of
regenerating liver over a longer time course. Rats were injected with
vehicle (Me2SO group) or with 0.4 mg of rapamycin/kg of
body weight (RAPA group) 2 h before surgery and on each day after
partial hepatectomy. Rats in the PH group were subjected to partial
hepatectomy only. Livers were removed and weighed 2 days and 4 days
after surgery. As expected, the liver remnant of PH rats nearly doubled
in size after 2 days and grew to about 80% of the original liver
weight by 4 days post-partial hepatectomy (Fig.
1). Liver regeneration was suppressed at
2 days in the Me2SO control group but was equivalent to the
PH animals after 4 days. By contrast, liver weight in RAPA animals was
significantly lower than in the Me2SO controls at both 2 days and 4 days (Fig. 1). Average liver weight in the RAPA group was
less than 60% of that in the Me2SO animals 4 days after
partial hepatectomy. Inhibition of liver regeneration by rapamycin is
probably not due to the immunosuppressive action of the drug because
treatment of partially hepatectomized rats with two other
immunosuppressants, cyclosporin A and FK506, augments liver
regeneration (35). Thus, liver regeneration after partial hepatectomy
is strongly inhibited by rapamycin treatment of rats using this
experimental protocol.

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Fig. 1.
Effect of rapamycin on liver
regeneration. Rats undergoing partial hepatectomy were treated
with or without rapamycin or vehicle as described under "Experimental
Procedures." The excised liver lobes, representing 68% of the
tissue, were weighed immediately after partial hepatectomy, and the
original liver mass was calculated. Liver remnants were removed at the
indicated times after surgery and weighed. Liver remnant weight as a
percentage of the original liver weight was calculated for each animal.
Data were analyzed by one-way analysis of variance, and pairwise
comparisons were obtained using Fisher's post hoc tests.
The single asterisk designates a significant difference
between 2-day Me2SO (DMSO) versus
2-day RAPA (p < 0.05), and the double
asterisk designates a significant difference between 4-day
Me2SO versus 4-day RAPA (p < 0.001). Shown are the means ± S. D.; n = 6 for 4-day RAPA; n = 3 for all others.
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Partial Hepatectomy Activates p70 S6 Kinase--
The inhibitory
effect of rapamycin seen in Fig. 1 suggests that p70 S6 kinase and/or
4E-BP1 might play a role in liver regeneration. Gressner and Wool (36)
reported nearly three decades ago that the phosphate content of
ribosomal protein S6 increases significantly within 24 h after
partial hepatectomy. Two subsequent studies showed that total S6 kinase
activity is elevated in extracts of regenerating rat liver (37, 38),
but reagents were not available at that time to specifically assay p70
S6 kinase. Here we used specific immunocomplex kinase assays to
determine whether p70 S6 kinase is activated by partial hepatectomy.
Fig. 2 shows that p70 S6 kinase was
activated almost 50-fold in the liver of rats 24 h after partial
hepatectomy as compared with unhandled rats in the CONT group. Kinase
activity in PH rats dropped sharply between 24 and 48 h but was
still slightly elevated 96 h after surgery. By contrast, p70 S6
kinase activity in sham-operated animals was not higher than CONT
levels at the 24-h time point (Fig. 2). Kinase activity in the SHAM
group increased ~3-fold between 24 and 48 h to a level
equivalent to that measured in PH animals. Western blot analysis showed
that the amount of p70 S6 kinase protein in SHAM and PH livers at the
24-h time point was similar, but active enzyme from PH animals
exhibited an upward mobility shift on SDS-polyacrylamide gels that
indicates increased phosphorylation of the enzyme (see Fig.
4B).

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Fig. 2.
Effect of partial hepatectomy on p70 S6
kinase activity. p70 S6 kinase activity was assayed in liver
extracts prepared from control rats or at various times following
partial hepatectomy or sham operation. Shown are means ± S.D.;
n = 3 for PH and SHAM; n = 2 for
CONT.
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Partial Hepatectomy Induces Functional Phosphorylation of
4E-BP1--
We next tested whether partial hepatectomy induces
hyperphosphorylation of 4E-BP1 by using gel mobility shift assays.
4E-BP1 migrates in SDS-polyacrylamide gels as three bands (
,
,
and
). The
band is the least phosphorylated form, and the
and
bands are more highly phosphorylated species. 4E-BP1 in the liver of CONT rats appeared on Western blots mainly as the
species, and sham operation did not significantly alter the phosphorylation state of the protein at any time examined (Fig.
3A). By contrast, partial
hepatectomy led to a substantial increase in 4E-BP1 phosphorylation as
judged by the mobility shift of the protein. A significant portion of
4E-BP1 was converted to the highly phosphorylated
species 24 h
post-partial hepatectomy, and this band was still prominent 48 h
after the operation (Fig. 3A). 4E-BP1 in PH rats subsequently underwent some dephosphorylation, as the
band was more
intense than the
band at 96 h (Fig. 3A).

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Fig. 3.
Effect of partial hepatectomy on 4E-BP1
phosphorylation. Liver extracts were prepared from control rats or
at various times after partial hepatectomy or sham operation.
A, equal amounts of protein were subjected to
immunoblotting, and the various phosphorylated forms of 4E-BP1 were
detected. Representative results from one animal in each group are
shown. B, binding of 4E-BP1 to m7GTP-Sepharose
was assayed as described under "Experimental Procedures." Results
from three different animals in the PH 24 h group are shown.
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Because 4E-BP1 can be phosphorylated on multiple sites (21), gel
mobility shift assays reveal little about the functional state of the
protein. To directly assess the effect of partial hepatectomy on
binding of 4E-BP1 to eIF4E, the amount of 4E-BP1 that coprecipitates
with eIF4E was measured using the mRNA cap affinity resin
m7GTP-Sepharose. Liver extracts of CONT, SHAM, and PH
animals were incubated with m7GTP-Sepharose, and 4E-BP1
associated with the beads was visualized on Western blots. A large
amount of 4E-BP1 was recovered from extracts of CONT livers due to
tight binding between eIF4E and hypophosphorylated 4E-BP1 (Fig.
3B). Binding of 4E-BP1 to eIF4E was unchanged relative to
the control 24 h after sham operation (Fig. 3B). By
contrast, the amount of 4E-BP1 precipitated by
m7GTP-Sepharose was greatly reduced in the liver of PH rats
24 h after surgery (Fig. 3B). These results are
consistent with the interpretation that partial hepatectomy promotes
phosphorylation of 4E-BP1 at sites that disrupt the 4E-BP1·eIF4E complex.
Rapamycin Inhibits p70 S6 Kinase Activation in Regenerating
Liver--
Having established that partial hepatectomy induces both
p70 S6 kinase activation and 4E-BP1 phosphorylation, we tested if rapamycin attenuates either of these responses. Animals were injected with increasing dosages of rapamycin 2 h before partial
hepatectomy, and p70 S6 kinase activity was measured in liver extracts
24 h after surgery. As shown in Fig.
4A, rapamycin strongly
inhibited the activation of p70 S6 kinase in the liver of PH rats at
every dose examined. The lowest dose tested (0.1 mg/kg) decreased p70 S6 kinase activity ~80%, whereas 0.4 mg of rapamycin/kg body weight reduced the amount of kinase activity to the basal level seen in
sham-operated animals (Fig. 4A). Western blot analysis
showed that the amount of p70 S6 kinase protein in liver extracts was not decreased by rapamycin treatment, but the drug prevented the appearance of phosphorylated species of the protein (Fig.
4B). In a second experiment, rats were injected with vehicle
or with 0.4 mg/kg rapamycin before surgery and on each day after
partial hepatectomy, according to the protocol used in Fig. 1. p70 S6 kinase activity remained much lower in the RAPA group than in the
Me2SO animals 2 and 4 days after surgery (Fig.
4C). Thus, treatment of rats with rapamycin prevents the
phosphorylation and activation of p70 S6 kinase in response to partial
hepatectomy. It should be noted that p70 S6 kinase activity in
Me2SO animals was higher than in PH animals 2 and 4 days
post-partial hepatectomy (compare Figs. 2 and 4C). Daily
handling and injection of vehicle into the Me2SO rats may
induce a stress response that causes p70 S6 kinase to remain active for
a longer time.

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Fig. 4.
Effect of rapamycin on p70 S6 kinase
activation. A, rats were injected intraperitoneally
with increasing dosages of rapamycin 2 h before partial
hepatectomy. Livers were harvested 24 h after surgery, and p70 S6
kinase activity was measured in extracts. Results from individual
animals are shown. B, equal amounts of liver extract protein
from animals treated as described under A were subjected to
Western blotting, and p70 S6 kinase was detected. C, rats
undergoing partial hepatectomy were treated with rapamycin or vehicle
as described under "Experimental Procedures." Livers were harvested
at the indicated times after surgery, and p70 S6 kinase activity was
measured in extracts. Shown are means ± S.D.; n = 6 for 4-day RAPA and n = 3 for all others.
DMSO, Me2SO.
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Phosphorylation of 4E-BP1 in Regenerating Liver Is
Rapamycin-resistant--
The in vivo effect of rapamycin on
4E-BP1 function was examined in the same liver extracts that were used
to analyze p70 S6 kinase activity. m7GTP-Sepharose binding
assays detected a large amount of 4E-BP1 bound to eIF4E in liver
extracts of SHAM animals, and phosphorylation of 4E-BP1 in response to
partial hepatectomy disrupted the 4E-BP1·eIF4E complex (Fig.
5A, upper panel).
However, to our surprise, 4E-BP1 did not coprecipitate with eIF4E in
liver extracts of rats treated with increasing dosages of rapamycin
before partial hepatectomy (Fig. 5A, upper
panel). The Western blot used for these assays was stripped and
reprobed with an antibody to eIF4E to confirm that the amount of eIF4E
pulled down by m7GTP-Sepharose was not altered by partial
hepatectomy or rapamycin treatment (Fig. 5A, middle
panel). These results suggested that rapamycin does not inhibit
the phosphorylation of 4E-BP1 in response to partial hepatectomy. To
further examine this possibility, the phosphorylation state of 4E-BP1
was examined using gel mobility shift assays. As suspected, 4E-BP1
phosphorylation was not reduced in rapamycin-treated rats as compared
with the PH controls 24 h after partial hepatectomy (Fig.
5A, lowest panel). In fact, the ratio of the
band to the total amount of 4E-BP1 appeared to slightly increase at
higher doses of rapamycin, suggesting that 4E-BP1 might be more
phosphorylated in rapamycin-treated animals than in PH animals (Fig.
5A, lowest panel).

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Fig. 5.
Effect of rapamycin on 4E-BP1
phosphorylation. A, rats were injected
intraperitoneally with increasing dosages of rapamycin 2 h before
partial hepatectomy. Livers were harvested 24 h after surgery, and
4E-BP1 binding to m7GTP-Sepharose was measured in extracts
(upper panel). The blot was stripped and reprobed with
antibody to eIF4E (middle panel). Equal amounts of liver
extract protein were also subjected to immunoblotting, and the various
phosphorylated forms of 4E-BP1 were detected (lowest panel).
Results from individual animals are shown. B, rats
undergoing partial hepatectomy were treated with rapamycin or vehicle
as described under "Experimental Procedures." Liver extracts were
prepared at the indicated times after surgery, and 4E-BP1 binding to
m7GTP-Sepharose was measured in extracts (upper
panel). Equal amounts of protein were also subjected to
immunoblotting, and the various phosphorylated forms of 4E-BP1 were
detected (lower panel). Data from individual animals in each
group are shown. The numerical values represent the fraction
of 4E-BP1 bound to m7GTP-Sepharose (obtained by dividing
the integrated density of bands in the upper panel by that
of bands in the lower panel). DMSO,
Me2SO.
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m7GTP-Sepharose binding assays were also performed on liver
extracts from rats treated for 2 or 4 days with vehicle or rapamycin after partial hepatectomy. At both time points, there was less 4E-BP1
bound to the resin in drug-treated animals than in the controls (Fig.
5B, upper panel). This result could be obtained if (a) 4E-BP1 is more highly phosphorylated at sites that
regulate binding in RAPA rats than in Me2SO animals or
(b) the total amount of 4E-BP1 in RAPA animals is much less
than in Me2SO rats. Quantitation of total 4E-BP1 protein by
densitometric scanning of Western blots (Fig. 5B,
lower panel) showed that RAPA livers contained on average 45% and 25% less 4E-BP1 than Me2SO livers at 2 and 4 days, respectively. To correct for the lower level of 4E-BP1 in
drug-treated animals, the amount of 4E-BP1 bound to
m7GTP-Sepharose was divided by the total amount of 4E-BP1
for each animal. These values (shown above the upper panel
in Fig. 5B) confirm that the fraction of 4E-BP1 bound to
m7GTP-Sepharose is significantly less in RAPA rats than in
the Me2SO controls. Quantitation of individual bands in the
lower panel of Fig. 5B indicated that the amount
of the
isoform in comparison to the
and
species was higher
in the RAPA group than in the Me2SO group at 2 days. 4E-BP1
underwent some dephosphorylation after 4 days of rapamycin treatment,
as the
isoform appeared (Fig. 5B, lower
panel) and an increased signal was detected in the
m7GTP-Sepharose binding assay (Fig. 5B,
upper panel). However, as noted above, 4E-BP1·eIF4E
complex formation was still reduced in 4-day RAPA rats as compared with
the 4-day Me2SO controls (Fig. 5B,
numerical values), and quantitation of the 4E-BP1 isoforms in the lower panel of Fig. 5B indicated that the
highly phosphorylated
species still represented a slightly higher
percentage of the total 4E-BP1 in the 4-day RAPA rats than in the
Me2SO controls. Thus, rapamycin treatment of rats inhibits
the activation of p70 S6 kinase but seems to slightly augment the
functional phosphorylation of 4E-BP1 in response to partial
hepatectomy. Furthermore, liver regeneration after partial hepatectomy
is inhibited by rapamycin at a concentration that completely inhibits
p70 S6 kinase activation but not 4E-BP1 phosphorylation.
Rapamycin Sensitivity of 4E-BP1 Phosphorylation Induced by
Cycloheximide or Growth Factors--
The above result was unexpected,
as growth factor-induced phosphorylation of 4E-BP1 has been shown to be
inhibited by rapamycin in a variety of cultured cells. One possible
explanation for our data is that higher concentrations of rapamycin
might be required to inhibit 4E-BP1 phosphorylation in vivo.
Alternatively, metabolism of rapamycin in the rat might cause
production of a substance that selectively inhibits p70 S6 kinase (39).
If such were the case, one might expect that 4E-BP1 phosphorylation in
rat liver would be rapamycin-resistant regardless of the stimulus used. To test this hypothesis, rats were injected with vehicle or with 0.4 mg/kg rapamycin, and 24 h later were treated with cycloheximide or
growth factors to induce 4E-BP1 phosphorylation. Western blot analysis
showed that exposure of vehicle-treated rats to cycloheximide, insulin,
or epidermal growth factor caused increased phosphorylation of 4E-BP1
(Fig. 6A), and
m7GTP-Sepharose binding assays showed a corresponding
decrease in the amount of 4E-BP1 bound to eIF4E (Fig. 6B).
More important, the phosphorylation of 4E-BP1 in control and
agonist-treated rats was reduced in the presence of rapamycin (Fig.
6A), and increased formation of the 4E-BP1·eIF4E complex
was also observed (Fig. 6B). Of the three agonists tested,
insulin was the most potent in disrupting the binding between 4E-BP1
and eIF4E, and it was also the least sensitive to rapamycin (Fig.
6B). p70 S6 kinase activity measured in the same extracts
was increased by these agonists and strongly inhibited in each case by
rapamycin treatment (data not shown). Together, our results demonstrate
that 4E-BP1 phosphorylation induced in vivo by cycloheximide
or growth factor treatment is relatively rapamycin-sensitive, whereas
the pathway that mediates 4E-BP1 phosphorylation in response to partial
hepatectomy is rapamycin-resistant.

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Fig. 6.
Effect of rapamycin on 4E-BP1 phosphorylation
induced by cycloheximide and growth factors. Rats were injected
intraperitoneally with 0.4 mg/kg rapamycin or vehicle. Twenty-four h
later, animals were treated for 10 min with insulin (INS) or
30 min with epidermal growth factor as described under "Experimental
Procedures." Additional rats were treated for 1 h with 2.5 ml of
5 mg/ml cycloheximide (CHX) injected intraperitoneally.
Control rats were not handled after the initial injection with
rapamycin or vehicle. The animals were sacrificed, and liver extracts
were prepared after the various treatments. A, equal amounts
of protein were subjected to immunoblotting, and the various
phosphorylated forms of 4E-BP1 were detected. Data from individual
animals are shown. B, binding of 4E-BP1 to
m7GTP-Sepharose was assayed as described under
"Experimental Procedures."
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Characterization of 4E-BP1 Phospho-specific Antibodies--
4E-BP1
is phosphorylated on five Ser/Thr-Pro sites in cells (21), and mTOR has
been reported to preferentially phosphorylate two of these sites,
Thr-36 and Thr-45, in vitro (27, 40-42). Paradoxically,
phosphorylation of Thr-36 and Thr-45 is relatively resistant to
rapamycin, whereas phosphorylation of two other sites, Ser-64 and
Thr-69, is rapamycin-sensitive (21, 43). A model has been proposed by
Sonenberg and coworkers (41) proposing that phosphorylation of 4E-BP1
on Thr-36 and Thr-45 by mTOR is required for subsequent phosphorylation
of the other sites by growth factor-activated kinases. To further
elucidate the mechanisms that control 4E-BP1 binding to eIF4E in this
in vivo model system, we decided to examine individual
phosphorylation sites in 4E-BP1 using phospho-specific antibodies.
The specificity of these antibodies was first tested using HA-tagged
4E-BP1 mutants in which Thr-36, Thr-45, Ser-64, or Thr-69 were changed
to Ala (see "Experimental Procedures"). COS7 cells expressing
wild-type or mutant proteins were either stimulated with insulin to
induce 4E-BP1 phosphorylation or treated with LY294002 to reduce
phosphorylation to basal levels. Western blots of cell extracts were
then probed with the phospho-specific antibodies. The patterns obtained
using the phospho-Thr-36 and phospho-Thr-45 antibodies were essentially
the same (Fig. 7A, upper
two panels). Both antibodies are phospho-specific, as treatment of
cells with LY294002 greatly reduced the binding to wild-type 4E-BP1.
Both antibodies also showed a significant reduction in binding to the T36A and T45A mutants, but they still recognized the S64A and T69A
proteins. The sequences surrounding Thr-36 and Thr-45 in 4E-BP1 are
almost identical (19), so we conclude that these antibodies cross-react
with both sites.

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Fig. 7.
Characterization of phospho-specific 4E-BP1
antibodies. A, wild-type HA-tagged 4E-BP1
(WT) and phosphorylation site mutants were
constructed and expressed in COS7 cells as described under
"Experimental Procedures." Cells were treated for 20 min with 1 µM insulin or 50 µM LY294002
(LY) before making extracts. Equal amounts of protein were
subjected to Western blotting, and signals were detected with the
indicated phospho-specific antibodies (upper panels) or HA
antibody (lowest panel). B, extracts of
insulin-treated COS7 cells expressing the indicated 4E-BP1 mutants were
prepared as described under "Experimental Procedures." Equal
amounts of protein were subjected to immunoprecipitation with HA
antibody. For the control, the immunoprecipitation contained cell lysis
buffer instead of cell extract. The immunocomplexes were washed twice
with cell lysis buffer and twice with Erk2 kinase buffer (New England
Biolabs). The immunoprecipitates were incubated for 5 h at
30 °C in 17 µl of Erk2 kinase buffer containing 0.5 mM
ATP and 50 units of recombinant murine Erk2 (New England Biolabs). The
samples were subjected to Western blotting, and the membrane was probed
with phospho-Ser-64 4E-BP1 antibody (upper panel) followed
by a general 4E-BP1 antibody (lower panel). C,
combinations of GST-4E-BP1 (1.6 µg) and human recombinant cdc2/cyclin
B complex (20 units; New England Biolabs) were incubated in 17 µl of
cdc2 kinase buffer containing 0.5 mM ATP. After 5 h at
30 °C, the samples were subjected to Western blotting, and the
membrane was probed with phospho-Thr-69 4E-BP1 antibody (upper
panel) followed by a general 4E-BP1 antibody (lower
panel).
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The phospho-Ser-64 antibody recognized endogenous 4E-BP1 in COS7 cells
transfected with empty vector; the exogenous HA-tagged proteins migrate
above this band (Fig. 7A, middle panel). Loss of
the signal in LY294002-treated cells indicates that this antibody only
recognizes phosphorylated 4E-BP1. As expected, the antibody did not
bind to the S64A mutant, but little or no signal was detected with the
T36A, T45A, and T69A proteins either. A 4E-BP1 Ser-64 phospho-specific
antibody recently characterized by Lawrence and coworkers (44) exhibits
these same characteristics. Substitution of an Ala for Thr-36, Thr-45,
or Thr-69 is thought to prevent the phosphorylation of Ser-64 in intact
cells (44). To circumvent this problem, the T36A, T45A, S64A, and T69A
mutant proteins were immunoprecipitated from COS7 cell extracts and
phosphorylated in vitro with Erk2, which has been shown to
phosphorylate all four of these sites in wild-type 4E-BP1 (21). The
samples were then subjected to Western blotting, and the membrane was
probed with the phospho-Ser-64 antibody. The antibody bound to all of the proteins except S64A, indicating that phosphorylation of Ser-64 is
required for recognition of 4E-BP1 by this antibody (Fig.
7B, upper panel).
Finally, the phospho-Thr-69 antibody recognized wild-type 4E-BP1 and
all of the Ala mutants except T69A, as expected (Fig. 7A,
fourth panel). However, this antibody still recognized
4E-BP1 from cells exposed to LY294002, suggesting either that the
antibody is not phospho-specific or that incubation of cells with
LY294002 does not reduce the phosphorylation of Thr-69. To distinguish between these possibilities, purified recombinant GST-4E-BP1 was incubated in vitro with or without cdc2, a kinase that
phosphorylates Ser/Thr-Pro sites. Western blot analysis showed that the
phospho-Thr-69 antibody reacted only with phosphorylated 4E-BP1 protein
(Fig. 7C).
Effect of Rapamycin on the Phosphorylation of Individual Sites in
Rat Liver 4E-BP1--
The results in Fig. 7 indicated that the
phospho-specific antibodies could be used to examine the
phosphorylation of rat liver 4E-BP1. A low level of phosphorylation was
detected in Thr-36/Thr-45 in unhandled CONT rats (Fig.
8, upper panel). The amount of
phosphate was relatively unchanged after sham operation, but a
substantial increase was detected 24 h after partial hepatectomy.
Rapamycin treatment increased the phosphorylation of Thr-36/45 in
response to partial hepatectomy. The bottom panel in Fig. 8
shows that the total amount of 4E-BP1 in the PH samples with or without
rapamycin is the same, so signals on the phospho-specific antibody
blots for these samples can be directly compared with each other.
Similarly, phosphorylation of Ser-64 was strongly increased in PH rats
relative to CONT animals, and rapamycin slightly increased this
response (Fig. 8, second panel). This stimulatory effect of
rapamycin on 4E-BP1 phosphorylation may be indirect, for example, via
the release of humoral factors from other tissues, since these
experiments are conducted in vivo. Nonetheless, increased
phosphorylation of 4E-BP1 in response to such indirect mechanisms is
still not inhibited by rapamycin. It should be noted that one of the
SHAM animals exhibited elevated phosphorylation of 4E-BP1 that was most
evident on the phospho-Ser-64 blot. Finally, Thr-69 appeared to be
constitutively phosphorylated in CONT rats, and there was little change
in the amount of phosphate after partial hepatectomy or rapamycin
treatment (Fig. 8, third panel). These results indicate that
4E-BP1 is phosphorylated at the expected sites after partial hepatectomy, but none of these phosphorylation events is inhibited by
rapamycin treatment.

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Fig. 8.
Effect of rapamycin on 4E-BP1 phosphorylation
at individual sites. Rats were treated as described under
"Experimental Procedures" and the legend of Fig. 4A.
Rapamycin was used at 0.4 mg/kg. Equal amounts of liver extract protein
were subjected to immunoblotting using phospho-specific antibodies to
4E-BP1. The bottom panel shows total 4E-BP1. Data from
individual animals are shown.
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DISCUSSION |
The results presented here demonstrate that treatment of rats with
rapamycin at a dose that impairs the regenerative response after
partial hepatectomy selectively inhibits p70 S6 kinase activation but
not the functional phosphorylation of 4E-BP1. Other investigators such
as Lawrence and Abraham (45) and Denton and co-workers (46) have
previously reported that under conditions in which p70 S6 kinase
activity is abolished by rapamycin, phosphorylation of 4E-BP1 was only
partly inhibited, suggesting that both rapamycin-sensitive and
-insensitive pathways are involved (reviewed in Refs. 45 and 46).
However, these investigators also found that insulin-induced 4E-BP1·eIF4E complex dissociation was blocked by rapamycin (19, 46),
in contrast to our findings in regenerating liver, where rapamycin was
completely ineffective at blocking this dissociation (Fig.
5A). The reason why this lack of effect of rapamycin on 4E-BP1·eIF4E complex dissociation has not been seen in other studies may be due to differences between cells cultured in vitro
and cells growing in vivo. Rapamycin is not only an
important research tool, but it is also a clinically useful drug, so
elucidating its mechanism of action in vivo will be
important for our understanding of its therapeutic use.
The inhibitory effect of rapamycin on liver regeneration suggested that
signaling molecules downstream of mTOR, such as p70 S6 kinase and
4E-BP1, might be involved in the growth response. We found that p70 S6
kinase is activated 50-fold in regenerating rat liver after partial
hepatectomy (Fig. 2). Partial hepatectomy also leads to phosphorylation
of the translation repressor 4E-BP1 and disruption of the
4E-BP1·eIF4E complex (Fig. 3). The kinetics of these two responses
were similar. The increase in p70 S6 kinase activity appears to be due
to phosphorylation and not to increased synthesis of the protein (Fig.
4B). p70 S6 kinase was most active during the first 2 days
after surgery (Fig. 2); DNA synthesis is also maximal during this
period (2). This result is consistent with our earlier studies in
synchronized Swiss mouse 3T3 fibroblasts, showing that p70 S6 kinase is
most active during the G1 and S phases of the cell cycle
(47). The kinetics of p70 S6 kinase activation obtained here are
substantially different from those reported by Mizuta et al.
(37), who measured total S6 kinase activity in liver extracts. In that
work, S6 kinase activity increased 1.5-fold 5-7 h after partial
hepatectomy and returned to the level in SHAM-operated animals by
24 h. Based on chromatographic and catalytic properties, the
authors suggested that the activity measured in their assays was due to
proteolytically modified protein kinase C (37). By contrast, Nemenoff
et al. (38) measure a 6-fold increase in total liver S6
kinase activity 2 h after partial hepatectomy. The activity
remained elevated as compared with SHAM-operated controls through
36 h and then dropped sharply by 48 h, similar to the pattern
we obtained (Fig. 2). The chromatographic behavior of the S6 kinase
from regenerating liver in Nemenoff et al. (38) suggests
that a major component was due to p70 S6 kinase.
Experiments using a variety of cell culture systems have shown that
rapamycin at low nanomolar concentrations inhibits the growth
factor-induced activation of p70 S6 kinase and disruption of the
4E-BP1·eIF4E complex (17-20). To our knowledge, the effect of
rapamycin on these two proteins has never been examined in proliferating tissues in vivo. We show here that activation
of p70 S6 kinase in regenerating liver was strongly inhibited by a
single injection of 0.1 mg/kg rapamycin, and a reduction to basal
levels was obtained with 0.4 mg/kg of the drug (Fig. 4A). Daily injections of 0.4 mg/kg rapamycin maintained these low levels of
p70 S6 kinase activity for up to 4 days post-partial hepatectomy (Fig.
4C) and blocked regrowth of the liver remnant (Fig. 1). p70
S6 kinase from rats treated with the immunosuppressant was hypophosphorylated, as indicated by the faster-migrating species seen
on Western blots (Fig. 4B). mTOR has been reported to
phosphorylate p70 S6 kinase at Thr-389 in vitro, a site
critical for kinase activation (27, 28). The in vitro kinase
activity of mTOR is inhibited in the presence of rapamycin-FKBP12 (26,
48, 49), leading to the speculation that the cellular effects of rapamycin are exerted through the inhibition of mTOR kinase activity. Our results are consistent with the hypothesis that rapamycin inhibits
mTOR-mediated phosphorylation of p70 S6 kinase in vivo, resulting in inactivation of the enzyme. However, in some reports mTOR
has been shown to exhibit a significant amount of kinase activity in
the presence of the rapamycin-FKBP12 complex in vitro (24,
27, 50). In addition, a truncation mutant of p70 S6 kinase is
phosphorylated on Thr-389 in rapamycin-treated cells, suggesting that
the drug does not inhibit the activity of the kinase that modifies this
putative mTOR site (51). These results suggest that mTOR might regulate
p70 S6 kinase activity indirectly by inhibiting a protein phosphatase
that inactivates the enzyme.
This conclusion is further supported by genetic studies in yeast
suggesting that Tap42 and type 2A-related protein phosphatases are
components of a TOR signaling pathway that controls translation initiation. The TOR proteins in yeast appear to phosphorylate Tap42 and
promote its association with the phosphatases in a rapamycin-sensitive manner (52, 53). A mammalian protein related to Tap42,
4, has also
been shown to associate with phosphatase 2A and related enzymes, but
there is disagreement about whether the binding is disrupted in the
presence of rapamycin (54-56). We showed earlier that the major
phosphatase activity in cell extracts that inactivates p70 S6 kinase is
protein phosphatase 2A (57). The same phosphatase was recently
demonstrated to exhibit increased catalytic activity when isolated from
rapamycin-treated cells (58). Together, these results suggest a model
in which mTOR-mediated phosphorylation of
4 results in binding to
and inhibition of phosphatase 2A, thus preventing the dephosphorylation
of p70 S6 kinase. Rapamycin would relieve the inhibitory effect of mTOR
on the phosphatase, leading to dephosphorylation and inactivation of
p70 S6 kinase. It should be noted that control of protein
phosphorylation directly by mTOR kinase activity or indirectly by mTOR
inhibition of a phosphatase is not necessarily mutually exclusive.
Additional experiments will be required to determine whether rapamycin
utilizes either of these two mechanisms to inhibit p70 S6 kinase
activity in the liver of partially hepatectomized rats.
In contrast to its potent effect on p70 S6 kinase, rapamycin did not
inhibit the functional phosphorylation of 4E-BP1 in regenerating liver
(Fig. 5). This result was unexpected, as growth factor-induced disruption of the 4E-BP1·eIF4E complex has been shown to be blocked by rapamycin in a variety of cell types in vitro. Expression
of a rapamycin-resistant mutant of mTOR confers rapamycin resistance to
4E-BP1 phosphorylation (26), indicating that mTOR regulates 4E-BP1 in
cultured cells. mTOR is thought to control 4E-BP1 by directly
phosphorylating the protein, but there is disagreement about which
sites are involved. Brunn et al. (49) reported that immunoprecipitated mTOR can phosphorylate recombinant 4E-BP1 at Thr-36,
Thr-45, Ser-64, Thr-69, and Ser-82, but it appears that Thr-36 and
Thr-45 are preferentially modified by mTOR in vitro (27,
40-42). In intact cells, phosphorylation of Ser-64 and Thr-69 is more
sensitive to rapamycin than that of Thr-36 and Thr-45 (21, 43). We
found that partial hepatectomy induced the phosphorylation of Thr-36/45
and Ser-64 and that Thr-69 was already phosphorylated in CONT animals
(Fig. 8). However, in contrast to the situation in cultured cells, none
of these sites exhibited rapamycin sensitivity (Fig. 8). One possible
explanation for these results is that mTOR kinase activity in
regenerating liver is not inhibited by rapamycin, so mTOR is able to
fully phosphorylate 4E-BP1 in the presence of the drug. However, in
light of the potent effect of rapamycin on p70 S6 kinase, it is equally
likely that a kinase other than mTOR phosphorylates 4E-BP1 in
regenerating liver. Ongoing experiments are being conducted to examine
these two possibilities.
In contrast to the results in regenerating liver, phosphorylation of
4E-BP1 in the liver of rats injected with cycloheximide or growth
factors did display rapamycin sensitivity (Fig. 6). Rapamycin has also
been shown to inhibit the phosphorylation of 4E-BP1 induced by acute
exposure to high concentrations of amino acids in a perfused rat liver
model system (59). These findings indicate that an
mTOR-dependent pathway that can control 4E-BP1 phosphorylation exists in the liver. One explanation for the lack of
rapamycin sensitivity in regenerating liver is that partial hepatectomy
might activate mTOR much more potently than these acute agonist
treatments, so an inhibitory effect of rapamycin on mTOR kinase
activity might only be detected using these agonists. A second reason
why this rapamycin-sensitive pathway is not manifest after partial
hepatectomy may be related to the proliferative state of the tissue.
The remaining liver cells in partially hepatectomized animals are
rapidly proliferating, whereas cells in an intact liver exposed for a
short time to growth factors are in a quiescent state. We speculate
that mTOR may be the dominant factor controlling the functional
phosphorylation of 4E-BP1 in the quiescent liver, but a distinct
rapamycin-insensitive 4E-BP1 kinase that takes over the function of
mTOR is induced in proliferating liver. Because the five
phosphorylation sites in 4E-BP1 are found in Ser/Thr-Pro motifs, we
expect that this kinase is a proline-directed enzyme, such as those in
the mitogen-activated protein kinase or cdc2 families. Indeed, the Erk2
isoform of mitogen-activated protein kinase has been shown to
phosphorylate all five Ser/Thr-Pro sites in vitro (21), and
an Erk2-dependent pathway has been proposed to regulate
4E-BP1 phosphorylation in vascular smooth muscle cells (60). However,
using Western blots probed with phospho-specific antibodies, we found
no evidence that the Erk1, Erk2, Jnk, or p38 mitogen-activated protein
kinases are activated by partial hepatectomy at any of the times
investigated (data not shown). In this study, we showed that cdc2 can
phosphorylate 4E-BP1 at Thr-69 in vitro (Fig. 7), and others
have shown that cyclin-dependent kinases are activated in
regenerating liver (61). Further studies are needed to identify
relevant 4E-BP1 kinases in this in vivo model system.
Results from our study indicate that rapamycin shows significant
anti-proliferative effects in vivo that might be mediated by
selective inhibition of p70 S6 kinase. However, other
rapamycin-sensitive enzymes such as S6 kinase 2 (11) might be
responsible for the rapamycin effect in vivo. The specific
contribution of p70 S6 kinase to liver regeneration could possibly be
tested in rapamycin-treated transgenic mice that express a
rapamycin-resistant mutant of p70 S6 kinase (51) in the liver.
Inhibition of global protein synthesis by injection of puromycin blocks
DNA synthesis in regenerating rat liver (3). However, p70 S6 kinase
does not control global protein synthesis but rather is thought to
regulate translation of a subset of growth-regulated mRNAs
(5'-terminal oligopyrimidine mRNAs) including those that encode
ribosomal proteins (13). Indeed, studies in perfused liver have shown
that treatment with rapamycin exerts a relatively small effect on
general protein synthesis (59). The anti-proliferative effect of the
drug observed in this liver regeneration model is presumably due to a
selective inhibitory action on the synthesis of ribosomal proteins
and/or other proteins encoded by 5'-terminal oligopyrimidine mRNAs.
The importance of ribosomal protein production during liver
regeneration was highlighted in a recent report in which synthesis of
40 S ribosomal subunits was blocked by conditionally knocking out the ribosomal protein S6 gene in mice (62). Liver cells in the knockout mice were able to grow larger in response to refeeding but were unable
to proliferate after partial hepatectomy. This selective effect on
proliferation suggests that rapamycin and related compounds might be
useful therapeutic agents in the treatment of cancer. Indeed, CCI-779,
a rapamycin analog, shows antitumor activity in mice and is currently
undergoing phase I clinical trials in humans (63). A better
understanding of how rapamycin inhibits cell proliferation in
vivo will aid in the design of more specific and potent drugs to
treat patients with malignancies and after organ transplantation.