Rapamycin-insensitive Regulation of 4E-BP1 in Regenerating Rat Liver*

Ya-Ping Jiang, Lisa M. Ballou, and Richard Z. LinDagger §

From the Departments of Pharmacology and Dagger  Medicine, University of Texas Health Science Center and the § Research Service, Audie L. Murphy Memorial Veterans Hospital, San Antonio, Texas 78229

Received for publication, August 24, 2000, and in revised form, January 24, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In cultured cells, growth factor-induced phosphorylation of two translation modulators, p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), is blocked by nanomolar concentrations of the immunosuppressant rapamycin. Rapamycin also attenuates liver regeneration after partial hepatectomy, but it is not known if this growth-suppressive effect is due to dephosphorylation of p70 S6 kinase and/or 4E-BP1. We found that partial hepatectomy induced a transient increase in liver p70 S6 kinase activity and 4E-BP1 phosphorylation as compared with sham-operated rats. The amount of p70 S6 kinase protein in regenerating liver did not increase, but active kinase from partially hepatectomized animals was highly phosphorylated. Phosphorylated 4E-BP1 from regenerating liver was unable to form an inhibitory complex with initiation factor 4E. Rapamycin blocked the activation of p70 S6 kinase in response to partial hepatectomy in a dose-dependent manner, but 4E-BP1 phosphorylation was not inhibited. By contrast, functional phosphorylation of 4E-BP1 induced by injection of cycloheximide or growth factors was partially reversed by the drug. The mammalian target of rapamycin (mTOR) has been proposed to directly phosphorylate 4E-BP1. Western blot analysis using phospho-specific antibodies showed that phosphorylation of Thr-36/45 and Ser-64 increased in response to partial hepatectomy in a rapamycin-resistant manner. Thus, rapamycin inhibits p70 S6 kinase activation and liver regeneration, but not functional phosphorylation of 4E-BP1, in response to partial hepatectomy. These results indicate that the effect of rapamycin on 4E-BP1 function in vivo can be significantly different from its effect in cultured cells.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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 (alpha , beta , and gamma ). The alpha  band is the least phosphorylated form, and the beta  and gamma  bands are more highly phosphorylated species. 4E-BP1 in the liver of CONT rats appeared on Western blots mainly as the beta  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 gamma  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 beta  band was more intense than the gamma  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.

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.

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 gamma  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.

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 gamma  isoform in comparison to the alpha  and beta  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 alpha  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 gamma  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."

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).

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.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha 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 alpha 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.


    ACKNOWLEDGEMENTS

We thank E. M. McReynolds for excellent technical support, Dr. Lei Chen of Cell Signaling Technology, Inc. for supplying the Thr-36 and Thr-45 phospho-specific 4E-BP1 antibodies, and Shih-Chia Tso and Linda Yu of Oklahoma State University for purifying the GST-4E-BP1 fusion protein.


    FOOTNOTES

* This work was supported in part by a grant from the American Federation for Aging Research, a Paul Beeson Physician Faculty Scholar Award (to R. Z. L.), and a grant from the Children's Cancer Research Center, San Antonio (to L. M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, Mail Code 7764, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-2978; Fax: 210-567-4303; E-mail: linr@uthscsa.edu.

Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M007758200


    ABBREVIATIONS

The abbreviations used are: eIF, eukaryotic initiation factor; p70 S6 kinase, Mr = 70,000 ribosomal protein S6 kinase; 4E-BP1, eIF4E-binding protein 1; mTOR, mammalian target of rapamycin; PH, rats subjected to partial hepatectomy; SHAM, rats subjected to SHAM operation; RAPA, rats treated with rapamycin and partial hepatectomy; Me2SO, rats treated with dimethyl sulfoxide and partial hepatectomy; CONT, unhandled control rats; HA, hemagglutinin; GST, glutathione S-transferase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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