From the Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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Regulation of translation of mRNAs coding for
specific proteins plays an important role in controlling cell growth,
differentiation, and transformation. Two proteins have been implicated
in the regulation of specific mRNA translation: eukaryotic
initiation factor eIF4E and ribosomal protein S6. Increased
phosphorylation of eIF4E as well as its overexpression are associated
with stimulation of translation of mRNAs with highly structured
5'-untranslated regions. Similarly, phosphorylation of S6 results in
preferential translation of mRNAs containing an oligopyrimidine
tract at the 5'-end of the message. In the present study, leucine
stimulated phosphorylation of the eIF4E-binding protein, 4E-BP1, in L6
myoblasts, resulting in dissociation of eIF4E from the inactive
eIF4E·4E-BP1 complex. The increased availability of eIF4E was
associated with a 1.6-fold elevation in ornithine decarboxylase
relative to global protein synthesis. Leucine also stimulated
phosphorylation of the ribosomal protein S6 kinase,
p70S6k, resulting in increased phosphorylation of S6.
Hyperphosphorylation of S6 was associated with a 4-fold increase in
synthesis of elongation factor eEF1A. Rapamycin, an inhibitor of the
protein kinase mTOR, prevented all of the leucine-induced effects.
Thus, leucine acting through an mTOR-dependent pathway
stimulates the translation of specific mRNAs both by increasing the
availability of eIF4E and by stimulating phosphorylation of S6.
Certain amino acids, notably the essential amino acids, not only
serve as precursors for protein synthesis, but also have important
regulatory roles in the initiation phase of mRNA translation (1-3). Regulation of translation initiation is known to occur through
modulation of two of the numerous steps in the pathway. The first
regulated step is the binding of methionyl-tRNAi
(Met-tRNAi) to the 40 S ribosomal subunit to form the 43 S
preinitiation complex (reviewed in Refs. 4 and 5). This step is
mediated by eukaryotic initiation factor, eIF2, and involves formation
of an eIF2·GTP·Met-tRNAi ternary complex followed by
binding of the ternary complex to the 40 S ribosomal subunit. The
overall process of Met-tRNAi binding is regulated through
changes in the activity of the guanine nucleotide exchange factor for
eIF2, termed eIF2B, and appears to involve changes in phosphorylation
of either the The second regulated step in translation initiation is the binding of
mRNA to the 43 S preinitiation complex (reviewed in Refs. 4 and 5).
This step is mediated by a group of proteins collectively referred to
as eIF4. During this step, eIF4E binds to the m7GTP cap
structure present at the 5'-end of essentially all eukaryotic mRNAs
and, through association with eIF4G, also binds to the 40 S ribosomal
subunit. The mRNA binding step is regulated through changes in
phosphorylation of eIF4E, with phosphorylation increasing the affinity
of eIF4E for the cap structure (6) as well as by changes in the
availability of eIF4E to form the active eIF4E·eIF4G complex. Changes
in eIF4E availability occur through modulation of the association of
eIF4E with the translational repressor, 4E-BP1. eIF4E associated with
4E-BP1 cannot bind to eIF4G and therefore does not bind to the 43 S
preinitiation complex. The binding of eIF4E to 4E-BP1 is regulated
through phosphorylation of 4E-BP1. A variety of hormones stimulate
4E-BP1 phosphorylation and result in dissociation of the eIF4E·4E-BP1
complex (reviewed in Ref. 7).
In a previous study, we showed that leucine availability in L6
myoblasts caused alterations in both the Met-tRNAi and
mRNA binding steps (8). In particular, leucine caused a stimulation of eIF2B activity, an impairment of eIF4E binding to 4E-BP1 and an
enhancement of eIF4E binding to eIF4G. However, the changes in eIF4E
availability could be dissociated from both the changes in eIF2B
activity and the changes in global protein synthesis caused by leucine.
Thus, global protein synthesis was regulated by alterations in eIF2B
activity and were independent of changes in eIF4E availability.
The question remained as to the potential functional role of changes in
eIF4E availability caused by leucine. Studies by others have suggested
that changes involving eIF4E may be important in regulating the
translation of mRNAs encoding specific proteins or families of
proteins (reviewed in Ref. 4). eIF4E has been implicated in the
regulation of translation of mRNAs containing highly structured
5'-untranslated regions, such as that coding for ornithine
decarboxylase (ODC).1 In
addition, translation of specific mRNAs is regulated through changes in phosphorylation of ribosomal protein S6 (reviewed in Ref.
9). S6 phosphorylation plays an important role in regulating the
synthesis of proteins, such as elongation factors eEF1A and eEF2, which
are encoded by mRNAs containing oligopyrimidine tracts at the
5'-end of the message (TOPS mRNAs). In our previous study (8), we
found that leucine not only modulated eIF4E availability, but also
altered the phosphorylation state of the 70-kDa S6 protein kinase
(p70S6k). Thus, leucine may regulate the translation of
specific mRNAs through changes in eIF4E availability and S6
phosphorylation. The goal of the present study was to define the
mechanism(s) by which leucine modulates the synthesis of the
translationally regulated proteins ODC and eEF1A.
Materials--
ECL detection reagents and horseradish
peroxidase-conjugated sheep anti-mouse Ig and donkey anti-rabbit Ig
were purchased from Amersham Pharmacia Biotech. Polyvinylidene
difluoride membrane was obtained from Bio-Rad.
[35S]Easytag Express Protein Labeling Mix was from NEN
Life Science Products. DMEM lacking leucine was purchased from Life
Technologies, Inc. The antibody against p70S6k was
purchased from Santa Cruz Biotechnology, Inc. The antibody against
eEF1A was purchased from Upstate Biotechnology, Inc. The antibody
against Akt, and the antibody that specifically recognizes Akt
phosphorylated on Ser473 were purchased from New England
BioLabs. The anti-phosphopeptide antibody specific for phosphorylated
S6 was a kind gift from Dr. Morris J. Birnbaum (Department of Medicine,
University of Pennsylvania).
L6 Myoblast Culture--
L6 myoblasts were grown in culture in
100-mm dishes in DMEM supplemented with 10% fetal bovine serum
(Hyclone Labs, Inc), 100 units/ml benzylpenicillin, and 100 µg/ml
streptomycin sulfate. Cells were grown to approximately 70% confluence
and were washed twice with phosphate-buffered saline. Serum-free DMEM
lacking leucine was then added to the dishes, and the cells were
returned to the incubator for 60 min. At that time the cells were
randomly divided into two groups; leucine was added to the dishes in
one group to a final concentration equivalent to that present in
complete DMEM. When present, rapamycin at a final concentration of 90 nM was added 45 min after the change to serum-free DMEM.
The cells were returned to the incubator, and 30 min later, all dishes
received 10 µl of [35S]Easytag Express Protein Labeling
Mix (22 mCi/ml). Thirty min after the addition of radiolabel, cells
were harvested by scraping in buffer A (20 mM HEPES, pH
7.4, 100 mM KCl, 2.5% Triton X-100, 0.25% deoxycholate,
50 mM NaF, 0.2 mM EDTA, 2 mM EGTA,
1 mM dithiothreitol, 50 mM
Measurement of ODC Activity--
L6 myoblasts were treated as
described above except they were harvested in ODC assay buffer (50 mM Tris·HCl, pH 7.5, 2.5 mM dithiothreitol,
0.1 mM EDTA). ODC activity was determined by measuring the
release of 14CO2 from
L-[1-14C]ornithine as described previously
(10).
Measurement of eEF1A Synthesis--
L6 myoblasts were grown in
culture as described above except 60-mm dishes were used and 50 µl of
[35S]Easytag Express Protein Labeling Mix was added
1 h before harvest. eEF1A was immunoprecipitated by incubating 250 µl of cell homogenate with 7 µl of anti-eEF1A antibody and 118 µl
of phosphate-buffered saline overnight at 4 °C. The
antibody·antigen complex was collected by incubation for 1 h
with goat anti-rabbit Biomag IgG beads (PerSeptive Diagnostics). Before
use, the beads were washed in 1% nonfat dry milk in buffer B (20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% Protein Immunoblot Analysis--
Blots were developed using an
Amersham ECL Western blotting Kit and analyzed as described previously
(11).
Quantitation of 4E-BP1·eIF4E Complex--
The association of
eIF4E with 4E-BP1 was quantitated by protein immunoblot analysis of
eIF4E immunoprecipitates exactly as described previously (8).
Examination of 4E-BP1 Phosphorylation in Extracts of L6
Myoblasts--
Aliquots of cell homogenate were immunoprecipitated
using a monoclonal antibody raised in mice against rat 4E-BP1 as
described previously (8). The immunoprecipitates were solubilized with SDS sample buffer and then subjected to protein immunoblot analysis using a rabbit anti-rat 4E-BP1 antibody.
Examination of Akt and p70S6k Phosphorylation in
Extracts of L6 Myoblasts--
Aliquots of cell homogenate were
combined with an equal volume of SDS sample buffer. For analysis of Akt
phosphorylation, duplicate sets of samples were resolved on 12.5%
polyacrylamide gels, and the proteins in the gels were transferred to
two separate polyvinylidene difluoride membranes. One membrane was
probed with anti-Akt antibody, and the second was probed with an
antibody specific for Akt phosphorylated at Ser473. For
analysis of p70S6k phosphorylation, samples were subjected
to electrophoresis on a 7.5% polyacrylamide gel (12). The samples were
then analyzed by protein immunoblot analysis using rabbit anti-rat
p70S6k polyclonal antibodies as described above.
The effect of leucine availability on global protein synthesis in
L6 myoblasts was examined by incubating cells in serum-free medium
lacking leucine for 1 h followed by the readdition of the deprived
amino acid. Protein synthesis was then measured as the incorporation of
[35S]methionine and [35S]cysteine into
total cellular protein. Consistent with the results of our earlier
study (8), leucine deprivation caused a reduction in protein synthesis
to 52.8% that of the value observed in cells maintained in complete
medium (Table I). Readdition of leucine rapidly returned protein synthesis to near the control value.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of eIF2 and/or the
-subunit of eIF2B.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 0.2 mM benzamidine, 0.8 µM leupeptin, and 0.6 µM pepstatin).
-mercaptoethanol, 0.5% Triton X-100).
The beads were captured using a magnetic stand and were washed twice
with buffer B and once with buffer C (50 mM Tris·HCl, pH
7.4, 500 mM NaCl, 5 mM EDTA, 0.04%
-mercaptoethanol, 1% Triton X-100, 0.5% sodium deoxycholate, and
0.1% sodium dodecylsulfate). Protein bound to the beads was eluted by
resuspension in SDS sample buffer and boiling for 5 min. The beads were
collected by centrifugation, and the supernatants were subjected to
electrophoresis on a 12.5% polyacrylamide gel. The gel was stained
with Coomassie Blue dye followed by incubation in En3Hance
autoradiography enhancer (NEN Life Science Products) for 1 h at
room temperature. The gel was then rinsed with cold water, dried, and
exposed to film. Films were analyzed by scanning densitometry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Effects of leucine readdition on protein synthesis in L6 myoblasts
In addition to its effect on global protein synthesis, leucine
readdition to leucine-deprived myoblasts caused a relatively greater
stimulation in the activity of ODC (Fig.
1A). It has been shown
previously that intracellular fluctuations in ODC activity are mediated
by changes in the amount of enzyme rather than by post-translational
modifications (13). In the present study, leucine readdition caused a
2.8-fold increase in ODC activity, from 0.20 ± 0.02 to 0.55 ± 0.08 nmol of 14CO2 released from
[1-14C]ornithine/mg of protein/30 min, in
leucine-deprived and leucine-repleted cells, respectively. In contrast,
leucine stimulated global protein synthesis 1.7-fold (Table I). Thus,
as assessed by enzyme activity, the increase in ODC synthesis was
1.6-fold greater in magnitude than the stimulatory effect of leucine on
global protein synthesis. To confirm that the increase in ODC activity
was a result of stimulated synthesis of the protein rather than reduced
degradation, the effect of leucine on the half-life of ODC activity was
determined (14). The half-life of ODC decreased from 44.2 min in
leucine-deprived myoblasts to 31.3 min after the readdition of leucine,
suggesting that ODC activity changes caused by leucine actually
underestimate the increase in ODC protein. Overall, the results support
the conclusion that leucine caused a proportionally greater stimulation in the translation of ODC mRNA compared with global protein
synthesis.
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Previous studies have implicated eIF4E in the stimulation of ODC mRNA translation by the hormone insulin (15). An important mechanism for regulating eIF4E availability involves the reversible sequestration of eIF4E into an inactive complex with 4E-BP1 (16, 17). In the present study, leucine readdition to leucine-deprived myoblasts resulted in a 70% reduction in the amount of 4E-BP1 associated with eIF4E (Fig. 1B).
Association of 4E-BP1 with eIF4E is regulated through changes in
phosphorylation of 4E-BP1 with hyperphosphorylation being associated
with a decrease in the binding of 4E-BP1 to eIF4E (reviewed in Ref.
18). As shown in the inset to Fig. 1C, 4E-BP1 was resolved into three bands during SDS-polyacrylamide gel electrophoresis. Previous studies have shown that the three bands represent
differentially phosphorylated forms of 4E-BP1 and that the fastest
migrating, or -form, represents the least phosphorylated species of
the protein, whereas the slowest migrating, or
-form, represents the
most highly phosphorylated species. Because the
-form is the only
one that does not bind to eIF4E, the data are presented as the
proportion of 4E-BP1 in the
-form. As shown in Fig. 1C, leucine readdition caused a 2.6-fold increase in 4E-BP1
phosphorylation, consistent with the observed decrease in association
of 4E-BP1 with eIF4E.
Leucine readdition to leucine-deprived myoblasts also stimulated the
synthesis of eEF1A (Fig. 2A).
However, whereas leucine stimulated global protein synthesis 1.7-fold,
readdition of the amino acid to leucine-deprived cells stimulated eEF1A
synthesis approximately 4-fold. Thus, the increase in eEF1A synthesis
relative to global protein synthesis was 2.4-fold. Synthesis of eEF1A
is thought to be regulated through changes in phosphorylation of ribosomal protein S6 (19), which is phosphorylated in vivo
by p70S6k (20). Activation of p70S6k by
hormones occurs through phosphorylation of the protein at multiple
serine and threonine residues (21). Phosphorylation results in a
decrease in mobility of p70S6k during SDS-polyacrylamide
gel electrophoresis, with several electrophoretic forms of the protein
apparent after stimulation of cells by insulin. As described above for
4E-BP1, phosphorylation causes a decrease in the rate of migration
during SDS-polyacrylamide gel electrophoresis, with the most highly
phosphorylated forms exhibiting the greatest activity (21). As shown in
Fig. 2B, leucine readdition promoted a shift in
p70S6k distribution, consistent with the protein becoming
more phosphorylated in the presence of the amino acid. To confirm that
the changes in p70S6k migration were accompanied by
alterations in kinase activity, the phosphorylation state of S6 was
examined using an antibody that specifically recognizes the
phosphorylated form of the protein. As shown in Fig. 2C,
leucine readdition significantly increased the amount of S6 in the
phosphorylated form.
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Recent studies have shown that a protein kinase referred to as mTOR
phosphorylates 4E-BP1 both in vitro and in vivo
(22, 23). Furthermore, mTOR phosphorylates p70S6k on
Thr389, a residue whose phosphorylation is associated with
activation of the protein (24). Thus, mTOR plays a key role in
regulating the phosphorylation of both 4E-BP1 and p70S6k.
To investigate the role of mTOR in the leucine-mediated stimulation of
ODC activity and eEF1A synthesis, the studies shown in Figs. 1 and 2
were repeated in the presence of a specific inhibitor of mTOR,
rapamycin. As shown in Fig.
3A, rapamycin did not prevent the stimulation of global protein synthesis associated with leucine readdition to leucine-deprived cells. In contrast, rapamycin prevented the leucine-induced stimulation of both ODC activity (Fig.
3B) and eEF1A synthesis (Fig. 3C). Furthermore,
as shown in Fig. 3D, rapamycin prevented the leucine-induced
reduction in 4E-BP1 associated with eIF4E. Finally, rapamycin not only
prevented phosphorylation of p70S6k but resulted in a
complete shift in p70S6k to the fastest migrating form
(Fig. 3E), suggesting that p70S6k was completely
dephosphorylated. The failure of leucine to stimulate p70S6k phosphorylation in the presence of rapamycin was
reflected in the lack of change in S6 phosphorylation in response to
leucine readdition in cells treated with the inhibitor (Fig.
3F). Overall, the results support the conclusion that
leucine stimulates both 4E-BP1 and p70S6k phosphorylation
through an mTOR-dependent process.
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The mechanism through which leucine stimulates mTOR is unknown.
Hormones such as insulin stimulate mTOR through activation of
phosphatidylinositol 3-kinase (reviewed in Ref. 25). However, two
recent studies (26, 27) have shown that leucine does not stimulate
phosphatidylinositol 3-kinase activity in cells in culture. An
alternative mechanism for the activation of mTOR by leucine may involve
phosphorylation of the mTOR regulator, Akt (also known as protein
kinase B). Akt is an intermediate in the insulin signaling pathway and
lies downstream of phosphatidylinositol 3-kinase but upstream of mTOR
(28). Activation of Akt is caused by phosphorylation of the protein at
two distinct sites, one of which is Ser473. Therefore, in
the present study, the effect of leucine on the phosphorylation of Akt
at Ser473 was examined. As a positive control,
phosphorylation of Akt in response to insulin treatment was
investigated. As shown in Fig. 4, neither
stimulation by insulin nor leucine had any effect on the cellular
content of Akt. However, insulin, but not leucine, stimulated Akt
phosphorylation. The results suggest that Akt is not a component of the
signaling pathway through which leucine acts to stimulate mTOR or
4E-BP1 phosphorylation.
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DISCUSSION |
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In a previous study (8), we reported that leucine availability affects both eIF2B activity and formation of the active eIF4E·eIF4G complex. Changes in global protein synthesis were shown to be directly related to changes in eIF2B activity but not to modulation of the eIF4 complex. Therefore, we proposed that leucine-induced changes in eIF4E availability might be important in regulating the translation of mRNAs coding for specific proteins. In its role as the m7GTP cap-binding protein, eIF4E is key in determining which mRNAs will be translated. Thus, those mRNAs that bind well to eIF4E presumably are translated most efficiently, and those that bind poorly are not. This fact is emphasized by studies using cells that overexpress eIF4E. In such cells, the mRNAs encoding proteins that have roles in cellular growth and development, such as ODC (15, 29), cyclin D1 (30), myc (31), and P23 (32), are preferentially translated. In contrast, decreasing the amount of eIF4E using an antisense RNA approach specifically reduces P23 expression (32). A common link among these proteins is that the mRNAs coding for them typically contain 5'-untranslated regions that are predicted to contain a high degree of secondary structure. The importance of a highly structured untranslated region in regulating the translation of these mRNAs has been demonstrated by transfecting cells with a series of vectors expressing the chloramphenicol acetyltransferase gene containing sequences in the 5'-untranslated region predicted to form different amounts of secondary structure (33). In wild type NIH-3T3 cells, increasing the secondary structure of the 5'-untranslated region caused a dramatic decrease in chloramphenicol acetyltransferase synthesis. In contrast, in NIH-3T3 cells overexpressing eIF4E, only a minor decrease in chloramphenicol acetyltransferase synthesis was observed. Thus, an increase in the amount of eIF4E relieved the translational repression caused by a structured 5'-untranslated region.
In contrast to the artificial manipulation of eIF4E availability using overexpression and antisense approaches, the present study used a more physiological approach to vary eIF4E availability; i.e. through changes in association of eIF4E with 4E-BP1. The release of eIF4E from the eIF4E·4E-BP1 complex was shown to correlate with increased ODC activity. Because ODC activity is a direct reflection of the amount of ODC protein present in the cell (13), it can be assumed that the leucine-induced increase in ODC activity represents an increase in ODC synthesis. Overall, the results suggest that leucine specifically stimulates the synthesis of ODC through an increase in eIF4E availability.
A second group of translationally regulated mRNAs is typified by
ribosomal proteins and elongation factors eEF1A and eEF2. mRNAs
belonging to this group usually have short 5'-untranslated regions
containing little or no predicted secondary structure and have an
oligopyrimidine tract at the 5' terminus. As with proteins like ODC
(34), synthesis of the ribosomal proteins is specifically increased by
growth factors such as insulin (reviewed in Ref. 35). However,
translational regulation of ribosomal protein synthesis is still
observed in cells overexpressing eIF4E (36). In addition, in cells
containing a targeted disruption of the p70S6k gene
(p70S6k /
cells), serum does not stimulate eEF1A
mRNA translation (20). As expected, in p70S6k
/
cells, serum does not stimulate S6 phosphorylation even though 4E-BP1
is phosphorylated to the same extent as in cells expressing wild type
p70S6k. These studies suggest that regulation of ribosomal
protein synthesis occurs through an eIF4E/4E-BP1-independent mechanism
that is regulated by changes in phosphorylation of ribosomal protein
S6. In vivo, phosphorylation of S6 is mediated by
p70S6k (20), and phosphorylation of S6 by
p70S6k is associated with increased translation of TOPS
mRNAs. The mechanism involved in the stimulation of TOPS mRNA
translation in response to S6 phosphorylation is unknown. However, S6
present in ribosomes has been cross-linked to both initiation factors
and mRNA (37), suggesting that phosphorylation of S6 might alter
the interaction of the protein with either of these components and
promote translation of TOPS mRNAs.
As reported previously for insulin, in the present study the leucine-stimulated phosphorylation of S6 was associated with activation of p70S6k. Furthermore, phosphorylation of S6 correlated with increased synthesis of eEF1A. eEF1A synthesis, S6 phosphorylation, and p70S6k activation were all blocked by rapamycin, suggesting that mTOR is required for this response. Indeed, mTOR has been implicated not only in the protein synthesis response to amino acids but also in the regulation of protein degradation by amino acids (38). Hara et al. (26) demonstrated that a p70S6k variant that is rapamycin-resistant is also resistant to amino acid deprivation, suggesting that amino acids signal to p70Sk6 through mTOR. Activation of mTOR may also be involved in the amino acid-regulated phosphorylation of 4E-BP1, because in the present study, rapamycin prevented both the phosphorylation of 4E-BP1 and the stimulation of ODC activity caused by leucine in L6 myoblasts. This idea is supported by a recent study showing that 4E-BP1 is phosphorylated both in vivo and in vitro by mTOR (22).
The question remains as to the mechanism through which amino acids activate mTOR. Hormones such as insulin activate mTOR through a phosphatidylinositol 3-kinase dependent pathway involving activation of Akt (reviewed in Ref. 39). Although the mechanism through which Akt activates mTOR is unknown, a recent study showed that stimulation of Akt causes an increase in mTOR kinase activity as well as enhances 4E-BP1 phosphorylation (28). However, in that study, the authors were unable to phosphorylate mTOR with purified Akt, suggesting that activation of mTOR by Akt is indirect (i.e. there may be another kinase between Akt and mTOR in the insulin signaling pathway). In the present study, leucine stimulated phosphorylation of two proteins downstream of mTOR, 4E-BP1 and p70S6k, even though Akt was not phosphorylated. In similar studies by others, amino acids failed to stimulate either phosphatidylinositol 3-kinase (26, 27) or Akt (26). Thus, it is possible that amino acids stimulate mTOR directly or through activation of a putative mTOR kinase residing downstream of Akt. It is noteworthy that in other studies (40, 41), wortmannin, an inhibitor of phosphatidylinositol 3-kinase, reportedly prevents the stimulation of 4E-BP1 phosphorylation by amino acids. Based on this observation, it is tempting to speculate that activation of mTOR by amino acids might be cell-type-specific. However, in Chinese hamster ovary cells, amino acid-stimulated 4E-BP1 phosphorylation was found to be both inhibited by wortmannin (42) and independent of phosphatidylinositol 3-kinase and Akt activation (26). Thus, it is likely that wortmannin is inhibiting a kinase downstream of Akt or is inhibiting a step that is required but not sufficient for amino acid-mediated mTOR activation.
In summary, leucine specifically stimulates the synthesis of both ODC
and eEF1A. The stimulation is associated with increased availability of
eIF4E as well as phosphorylation of ribosomal protein S6. Finally, the
stimulation occurs through a rapamycin-sensitive pathway, suggesting
that the effect of leucine is through activation of the mTOR signaling pathway.
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ACKNOWLEDGEMENT |
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We are grateful to Joan McGwire for technical help.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK-15658 (NIDDKD).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 Cellular and
Molecular Physiology, The Pennsylvania State University College of
Medicine, P. O. Box 850, Hershey, PA 17033. Tel.: 717-531-8970; Fax:
717-531-7667; E-mail: skimball{at}psu.edu.
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ABBREVIATIONS |
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The abbreviations used are: ODC, ornithine decarboxylase; p70S6k, 70-kDa ribosomal protein S6 kinase; mTOR, mammalian target of rapamycin; Akt, protein kinase B; DMEM, Dulbecco's modified Eagle's medium; TOPS, terminal oligopyrimidine stretch.
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