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Previous Article | Next Article 
Molecular and Cellular Biology, December 2003, p. 9117-9126, Vol. 23, No. 24
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.24.9117-9126.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
Paul Lasko,2 and Nahum Sonenberg1*
Department of Biochemistry and McGill Cancer Center, McGill University, Montréal, Québec H3G 1Y6,1 Department of Biology, McGill University, Montréal, Québec H3A 1B1, Canada2
Received 18 June 2003/ Returned for modification 5 August 2003/ Accepted 16 September 2003
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ABSTRACT |
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AND METHODSResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials
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(where X is any residue and
is a hydrophobic residue)
(29). Upon binding to
eIF4E, the 4E-BPs adopt an energetically favorable 
-helical
structure that mimics the eIF4E-binding site of eIF4G
(30). The block to
translation caused by the 4E-BPs is reversible by their phosphorylation
at certain key residues
(4,
15,
17). Mammalian 4E-BP1
(hereafter referred to as 4E-BP1) may be phosphorylated on at least
seven sites: Thr37, Thr46, Ser65, Thr70, Ser83, Ser101, and Ser112
(10,
20,
55). Thr37 and Thr46 are
coordinately phosphorylated, priming the hierarchical phosphorylation
of Thr70, followed by that of Ser65, which ultimately results in 4E-BP1
release from eIF4E (15,
17). Although Ser83 is
phosphorylated, it is not required for 4E-BP1 release from eIF4E
(6). A recent report
suggests that a Ser or Glu residue at position 101 is necessary for
phosphorylation of Ser65 and that Ser112 phosphorylation affects 4E-BP1
binding to eIF4E (55). In
d4E-BP, Thr37, Thr46, Ser65, and Thr70 are identical to 4E-BP1, but
Ser83 is a Thr residue, Ser101 is Gln, and Ser112 is absent
(33). In mammals, the phosphoinositide 3-OH kinase (PI3K) and mammalian target of rapamycin (mTOR) signaling pathways impinge on two known downstream effectors, namely, 4E-BP1 and ribosomal protein S6 kinase 1 (S6K1) (for a review, see reference 18). In the fruit fly, D. melanogaster, genetic studies have underscored the importance of the insulin-PI3K and TOR signaling pathways in controlling the sizes of cells and organs, as well as that of the entire animal (for a review, see reference 45). These evolutionarily conserved pathways are also critical for the control of cell growth in mammals (1, 11, 24, 49, 54). The signaling pathways have been assumed to function similarly in mammalian and invertebrate systems (23), but recent studies of Drosophila S6K (dS6K) argue that this may not be true. Indeed, dS6K-regulated cell growth, which was believed to be triggered following activation of PI3K, has been shown to be independent of dPI3K and its downstream effector, dPKB (41, 42).
The insulin-induced phosphorylation of the single Drosophila 4E-BP (d4E-BP), which is reduced by inhibition of PI3K and TOR signaling with the inhibitors LY294002 and rapamycin, respectively, was previously described(33). However, the recent finding that S6K signaling in Drosophila is independent of PI3K and Akt led us to undertake a detailed study of the regulation of d4E-BP phosphorylation by the insulin, PI3K, and TOR signaling pathways using double-stranded RNA interference (dsRNAi) (7).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials
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Plasmids, transfections, and induction of protein expression. Drosophila 4E-BP phosphorylation mutants were generated by PCR mutagenesis using Pwo polymerase (Boehringer). Wild-type and mutant d4E-BP coding regions were inserted into pcDNA-3HA, and the coding regions, including the three-hemagglutinin (3HA) tags, were once more amplified and inserted into the pUAST vector (2). For transfections, S2 cells (3 x 106) were propagated in a 100-mm-diameter plate and left to attach overnight. Next, the cells were cotransfected with equal quantities of pUAST-3HA-d4E-BP and pMK-GAL4 vectors (700 ng or 1 µg) using Effectene (Qiagen) and left to grow for 48 h. The cells were then placed in Schneider's medium lacking serum for 24 h as described above. For induction of protein expression, cells were treated with 0.7 mM copper sulfate (CuSO4) for 30 min. For insulin treatment, the cells were treated for 5 min as described above after the 25-min copper sulfate treatment, for a total of 30 min.
Phosphatase treatment. Cells were lysed in phosphatase reaction buffer, and the resulting extract (50 µg) was incubated with or without the addition of 10 U of calf intestine alkaline phosphatase (CIP; New England Biolabs) at the required reaction temperature (4 or 30°C). The composition of CIP reaction buffer was 50 mM Tris · HCl, pH 7.5, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride.
IEF-SDS-PAGE.
Cell lysate prepared in cap binding
buffer (
20 µl) was mixed with 27 mg of urea and 7
µl of isoelectric focusing (IEF) sample buffer (17%
[vol/vol] Pharmalytes at pH 3 to 10, 4%
[vol/vol] Pharmalytes at pH 8 to 10.5, 14%
[vol/vol] ß-mercaptoethanol, 35% CHAPS
{3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate},
1.8% [wt/vol] sodium dodecyl sulfate [SDS]),
and incubated at room temperature until the urea was dissolved.
Two-dimensional IEF-SDS-polyacrylamide gel electrophoresis
(PAGE) was performed as previously described
(17), with the exception
that the first-dimension gel contained 1% (vol/vol) Pharmalytes
at pH 8 to 10.5 instead of 1% (vol/vol) Pharmalytes at pH 2.5 to
5.
Antibodies and immunoblotting analysis. Antibody 1868 to d4E-BP (33), anti-deIF4A (25), and antibody 1739 to deIF4E (50) were described previously. Anti-phospho-S6K(Thr389) (which recognizes Thr389 of dS6K), anti-Akt, and anti-phospho-4E-BP1(Thr37/46), -4E-BP1(S65), and -4E-BP1(T70) antibodies were obtained from Cell Signaling Technology. Anti-dS6K and anti-dTSC1 were gifts from G. Thomas, Basel, Switzerland, and D. Pan, Dallas, Tex., respectively. Anti-HA.11 was obtained from BAbCO. Polypeptides were resolved on SDS-polyacrylamide gels (15% for d4E-BP, deIF4E, dS6K, and dAkt immunoblotting and 6% for dTsc1 immunoblotting) and transferred to 0.22-µm-pore-size nitrocellulose membranes. The membranes were blocked with 2% (wt/vol) nonfat dry milk in PBS containing 0.5% (vol/vol) Tween-20 (all subsequent incubations were performed in the same solution) for 2 h and incubated for 2 h to overnight with the individual primary antibody 1868 (1:2,000) or 1739 (1:3,000) or antibody directed to deIF4A (1:3,000), phospho-S6K (Thr389; 1:1,000), dS6K (1:200), Akt (1:1,000), dTSC1 (1:5,000), or HA.11 (1:2,500). Incubation with secondary antibody was performed with peroxidase-coupled donkey anti-rabbit immunoglobulin or with peroxidase-coupled sheep anti-mouse immunoglobulin, followed by enhanced chemiluminescence (Amersham).
dsRNA interference. Fragments from Dp110 (positions 375 to 1075), dAkt1/dPKB (positions 784 to 1510), dPTEN (positions 205 to 912), dPDK1 (positions 2521 to 3008), dTSC1 (positions 435 to 1098), and dTOR (positions 5401 to 6130) were amplified by PCR using gene-specific primers flanked by the T7 RNA polymerase-binding site (5'-GAATTAATACGACTCACTATAGGGAGA-3'). The PCR products were purified using the Ultraclean GelSpin DNA purification kit (MoBIO Laboratories). The fragments (4 µl) were used in 50-µl in vitro transcription reaction mixtures, which also contained 2 mM nucleotide mixture, 50 U of RNasin RNase inhibitor (Promega), and 30 U of T7 RNA polymerase (MBI Fermentas). The synthesized RNA was phenol-chloroform extracted, ethanol precipitated, resuspended in water, and annealed by heating it at 65°C for 30 min, followed by slow cooling to room temperature. Each dsRNA was visualized as a single band on a 1% agarose gel. For RNAi, S2 cells (5 x 106) were propagated in 60-mm-diameter plates and left to attach overnight. Next, the medium was removed and replaced with 1.5 ml of serum-free medium containing the dsRNA. The same effect was produced with either 10 or 20 µg of dsRNA for dPDK1; 10, 15, or 20 µg of dsRNA produced the same effect for dAkt. For Dp110, dPTEN, and dTSC1, 20 µg of dsRNA was used. After 30 min of incubation, 2.5 ml of complete medium was added, and the cells were left to grow for 3 days at 25°C. The cells were then serum starved for 36 h as described above, with the exception that a fresh dose of dsRNA was added to the serum-free medium.
Northern analysis to measure RNAi effects. No antibodies were available to confirm the knockdown of the expression of Dp110, dPTEN, dPDK1, or dTOR. Therefore, knockdown was analyzed by Northern hybridization of a blot containing 5 µg of total RNA extracted from a plate of S2 cells treated with the dsRNA of interest concomitantly with the other experimental plates. The probes were derived as follows: Dp110 was an 824-nucleotide (nt) fragment from the BglII/PvuII digest of the pUAS-Dp110 vector (a kind gift from Sally Leevers), dPTEN was a 395-nt fragment from the HincII/HindIII digest of pUAST-dPTEN (a kind gift of D. Pan), and dPDK1 was a 656-nt fragment from the XhoI/HindIII digest of the dPDK1 cDNA excised a priori from the pUAST-dPDK1 vector by digestion with EcoRI and XbaI (44). Hybridization was performed with ExpressHyb (BD Biosciences Clontech) according to the manufacturer's instructions.
Quantitation. Band intensities were quantified using the image analysis software Image-J, a Java program inspired by NIH-Image that runs on any computer platform (http://rsb.info.nih.gov/nih-image/about.html).
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RESULTS |
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TopAbstractIntroductionMaterials
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form and a small amount of
ß form (Fig.
1A, bottom) (33). After
insulin treatment, a rapid shift toward the ß form was
observed, which was completed by 30 min (Fig.
1A, bottom). We tested the
reactivities of phosphospecific antiserum directed against
4E-BP1 phosphopeptides corresponding to human phosphorylated Thr37/46,
Ser65, and Thr70 (15,
17).
Anti-phospho-4E-BP1(Ser65) and -4E-BP1(Thr70) failed to detect d4E-BP
before and after insulin treatment (data not shown). In serum-starved
cells, however, anti-phospho-4E-BP1(Thr37/46) detected a faint signal
corresponding to the ß form, which intensified immediately
after insulin treatment (Fig.
1A, top). This effect was
blocked by treating the cells with rapamycin, indicating that this
phosphorylation is susceptible to the inhibition of dTOR (data not
shown) (33).
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and ß forms were detected in untreated extracts, as
well as in treated extracts kept on ice. Phosphatase-treated extracts
incubated at 30°C contained only the form (Fig.
1B, bottom) and lacked the
phosphoreactive ß form (Fig.
1B, top). Although Thr37,
Thr46, Ser65, and Thr70 are conserved between humans and flies, only
the amino acids flanking Thr46 are perfectly conserved between 4E-BP1
and d4E-BP (Fig. 1C). The
residues flanking the serine/threonine proline sites of Ser65 and Thr70
in d4E-BP are different from their 4E-BP1 counterparts (Fig.
1C), which could explain
the lack of reactivity of these antibodies. It is also possible,
however, that these residues are not phosphorylated in response to
insulin treatment. For 4E-BP1, the residues surrounding Thr37 and Thr46
are identical (Fig. 1C),
and therefore the antibodies directed against the individual Thr37 and
Thr46 phosphopeptides cross-react
(15). The region
surrounding Thr46 in d4E-BP is perfectly conserved, whereas the Thr37
site has a mismatch in the -1 position (Fig.
1C). Thus, the
phosphospecific phospho-4E-BP1(Thr37/46) antibodies most likely
recognize phospho-Thr46 in
d4E-BP. Analysis of d4E-BP mutant phosphorylation sites. To study the phosphorylation states of different residues of d4E-BP, HA-tagged d4E-BPs (3HA-d4E-BPs) were generated and transfected into S2 cells. First, we mutated Thr37 and Thr46 to Ala, either individually or in combination. Transfected wild-type d4E-BP migrated as three distinct bands (Fig. 2A, bottom). This protein is phosphorylated to some extent in serum-starved cells, but after insulin treatment, the amount of phosphorylated protein is increased 1.7-fold (Fig. 2A, middle). This effect is paralleled by a characteristic upward shift of d4E-BP after phosphorylation (Fig. 2A, bottom). Mutation of either Thr37 or Thr46 to Ala abolished both the insulin-stimulated upward shift and the phosphorylation of d4E-BP, as detected with the antibody to phospho-4E-BP1(Thr37/46) (Fig. 2A, middle). These results suggest that the phosphorylations of these residues in Drosophila 4E-BP are interdependent, as in human 4E-BP1 (15).
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Analysis of
d4E-BP phosphorylation by IEF-SDS-PAGE.
To better understand d4E-BP
phosphorylation, we used two-dimensional IEF combined with
immunoblotting with phosphospecific antibodies (IEF-SDS-PAGE),
a method developed to show the hierarchical phosphorylation of 4E-BP1
(17). This method
separates the d4E-BP molecules based on their charges and reflects the
number of phosphate residues added. Extracts from serum-starved S2
cells, insulin-treated cells, and rapamycin-treated cells were analyzed
using this method. The starved cells expressed little of the ß
form relative to the form; the 15-min sample had both the
and ß forms, and the 30-min sample contained the
ß form almost exclusively (Fig.
3A). Rapamycin treatment prevented insulin-induced d4E-BP phosphorylation
(data not shown) (33).
In extracts from serum-starved or rapamycin-treated cells,
d4E-BP exists as three predominant isoforms, as well as a fourth,
less-abundant isoform (Fig. 3B and
F, isoforms a to d). The form
therefore consists of either a nonphosphorylated and three
phosphorylated isoforms of d4E-BP or of four phosphorylated isoforms,
depending on the phosphorylation state of isoform a, which is
not known. After insulin treatment, a stronger signal for isoform
d was detected, and a fifth weak and more acidic isoform,
e, was also detected (Fig. 3C
and D). The longer 30-min treatment generally caused the
signal intensity for isoforms a and b to weaken
relative to the 15-min treatment. Duplicate blots were probed with
anti-phospho-4E-BP1(Thr37/46). In starved or rapamycin-treated cells,
no signal was detected. Thus, there is very little, if any, of the
ß form in these extracts. After insulin treatment, isoforms
b to d were detected, but not isoform e (the
30-min treatment is shown in Fig.
3E).
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form is composed of a mixture of isoforms a to
c, indicating that d4E-BP is phosphorylated at a number of
sites that do not prevent its interaction with deIF4E. The most likely
candidates are Thr37, Ser65, and Thr70, since they are conserved from
Drosophila to humans. After insulin stimulation, a regulatory
interplay between Thr37 and Thr46 culminates in phosphorylation of
Thr46, which causes d4E-BP to dissociate from deIF4E. Thus, these
results support the conclusion that phosphorylations at Thr37 and Thr46
represent major events in the regulation of d4E-BP after insulin
stimulation.
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First, we reduced the expression of the upstream
components of the signaling pathway in S2 cells: the Dp110 catalytic
subunit of dPI3K, as well as the phosphatase dPTEN, which antagonizes
dPI3K activity by hydrolyzing the 3' phosphate of
phosphatidylinositol-3,4,5-P3 (PIP3) (Fig.
4A). dPI3K and dPTEN act in combination as a dedicated growth rheostat that
controls the production of PIP3
(35). RNAi of Dp110
reduced the insulin-stimulated phosphorylation of d4E-BP
1.5-fold, whereas knockdown of dPTEN caused an increase in the
basal level of phosphorylated d4E-BP of 3.4-fold in
serum-starved cells (Fig.
4B, middle). This is
consistent with dPI3K and dPTEN being upstream regulators of d4E-BP
phosphorylation. The phosphorylation of dS6K at Thr398 after insulin
treatment of Dp110-RNAi cells was more robust (2.2-fold) than
in control cells (Fig. 4B,
top). However, the significance and mechanism of this increase are not
clear. Phosphorylation of dS6K at Thr398 in dPTEN-RNAi cells was
similar to that in control cells before and after insulin treatment
(Fig. 4B, top). These
results are consistent with the notion that the threshold activity of
dPI3K required for dS6K activation is relatively low, while that
required for d4E-BP phosphorylation is higher.
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Treatment of S2 cells with
dsRNA targeting dAkt abrogated dAkt expression in S2 cells (Fig.
5A, top). Knockdown of dAkt resulted in a 2.1-fold decrease in d4E-BP
phosphorylation after insulin treatment (Fig.
5A, fourth gel from top),
consistent with dAkt being an upstream regulator of d4E-BP
phosphorylation. Surprisingly, dS6K phosphorylation at Thr398 after
insulin treatment was also reduced relative to control cells
(3.3-fold less) (Fig.
5A, second gel from top),
which is inconsistent with previous reports
(41,
42). This result
demonstrates that in our system, dS6K is a downstream target of
dPI3K/dAkt signaling (see below).
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Finally, we targeted dTOR for RNAi. Treatment with dsRNA mimicked the effects of rapamycin on d4E-BP and dS6K phosphorylation. The phosphorylation of d4E-BP and dS6K after insulin treatment was abolished (Fig. 5C). Moreover, the basal level of phosphorylated d4E-BP in serum-starved cells was reduced in dTOR-RNAi cells (Fig. 5C, third and fourth gels from top). Taken together, our RNAi experiments confirm that d4E-BP is a downstream target of the PI3K-Akt-TSC-TOR signaling cascade.
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DISCUSSION |
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TopAbstractIntroductionMaterials
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Our results suggest a simpler mode of regulation of d4E-BP by phosphorylation compared with the hierarchical phosphorylation of 4E-BP1. The phosphorylation of 4E-BP1 is the best understood, but not all mammalian 4E-BPs are regulated in a similar manner. 4E-BP2 is phosphorylated on fewer residues (27) and is dephosphorylated more slowly than 4E-BP1 (B. Raught and N. Sonenberg, unpublished data). Also, 4E-BP3 is weakly stimulated by insulin treatment, causing poor release from eIF4E. This has been attributed to the lack of the four-residue RAIP motif found in the N terminus of 4E-BP1 and -2 but not in 4E-BP3 (53). The RAIP motif seems to be required for the efficient overall phosphorylation of 4E-BP1, and intriguingly, this motif is also lacking in d4E-BP. Another important motif for 4E-BP1 regulation is the TOR signaling (TOS) motif (47), which is conserved (FQLDL, at the C terminus) in d4E-BP. Hence, a consequence of the lack of the RAIP motif may be the simpler regulation of d4E-BP to improve its release from eIF4E when d4E-BP is recruited to the dTOR/dRaptor complex through the TOS motif (34, 48). Moreover, because of a divergent eIF4E-binding site, d4E-BP does not interact as strongly with deIF4E as 4E-BP1 (33). It is conceivable that because of the poorer interaction of d4E-BP with deIF4E, phosphorylation at Thr37 and Thr46 is sufficient to bring about its release from eIF4E. Our IEF-SDS-PAGE results indicate that in serum-starved S2 cells, some d4E-BP is already phosphorylated. Although these additional sites do not prevent the interaction between deIF4E and d4E-BP, it is possible that they contribute to the release from deIF4E once Thr46 becomes phosphorylated.
Drosophila 4E-BP is a downstream target of PI3K-PTEN-Akt-TSC-TOR signaling. A large body of work has established the paramount importance of the InR-IRS-PI3K-PTEN signaling module in the control of cell growth (8, 23, 45, 57). This module coordinates cellular metabolism with the nutritional state (3). The primary outcome of its activation is the modulation of the PI3K/PTEN cycle and consequent PIP3 production (35). The increase in PIP3 facilitates the recruitment of pleckstrin homology domain-containing proteins, such as dAkt and dPDK1, to the plasma membrane. dPTEN mutant flies die because of increased membrane translocation and activation of dAkt. Phosphorylation of the Tsc2 subunit of TSC by Akt results in inhibition of the complex by causing its dissociation or by blocking its interaction with other proteins (for reviews, see references 31 and 32). TSC inhibits S6K and 4E-BP1 by repressing the GTPase Rheb, preventing it from activating mTOR through an unknown mechanism (14). Thus, mTOR, is clearly a critical regulator of S6K and 4E-BP1 in mammals and Drosophila (14, 21, 36, 46, 52, 58).
Is d4E-BP regulated by a PI3K/Akt-independent pathway similar to that described for dS6K (42)? Our analysis of signaling to d4E-BP using RNAi indicates that it is not. It is more likely that d4E-BP is a direct downstream target of the dInR-dPI3K-dPTEN-dAkt-dTSC-dTOR signaling cascade. Thus, a linear pathway from InR to Akt that is important for 4E-BP regulation is conserved between Drosophila and mammals (9, 16).
PDK1 signaling to 4E-BP versus S6K in Drosophila. dPDK1 is critical for regulating growth by phosphorylating dAkt and dS6K (5, 44). RNAi of dPDK1 did not significantly affect insulin-induced phosphorylation of d4E-BP. However, consistent with the direct phosphorylation of dS6K by dPDK1 (42, 44), the phosphorylation of dS6K at Thr398 was completely blocked by RNAi of PDK1. Thus, our results favor a model in which d4E-BP regulation is effected through dAkt, even when dPDK1 levels are dramatically reduced, whereas dS6K requires both dAkt and dPDK1. The differential effects of dPDK1 RNAi on d4E-BP and dS6K phosphorylation can be explained as follows: dPDK1 levels may be reduced below a threshold that is required to phosphorylate dS6K but is still adequate to activate dAkt, allowing d4E-BP phosphorylation. Since dS6K requires direct phosphorylation by dPDK1, it may be more susceptible to variations in its levels. On the other hand, d4E-BP, which relies on a signal relayed by dAkt, may be less affected by variations in dPDK1. In mammalian PDK1-hypomorphic mutants, a kinase activity that is 10-fold lower than normal still results in normal Akt and S6K1 activation, yet these animals are greatly reduced in size (26). This observation supports the notion that reduced PDK1 activity may differentially activate downstream targets.
Regulation of dS6K signaling by dAkt but not dPI3K/dPTEN? In Drosophila, coexpression of dS6K with dPI3K does not cause additive cellular overgrowth, unlike coexpression of dAkt and dPI3K (42). RNAi of dPTEN in Kc 167 cells and overexpression of dPTEN in Drosophila larvae had little effect on dS6K activity (41). Moreover, removal of both dS6K and dPTEN in cell clones did not prevent the dPTEN-dependent overgrowth phenotype (41). Together, these results and the results of our dPI3K and dPTEN RNAi experiments would seemingly support the notion that dS6K-dependent cell growth is not influenced by dPI3K and dPTEN. However, a different effect of dPTEN RNAi on dS6K has been reported by another group. Gao et al. observed an increase in dS6K phosphorylation following RNAi of dPTEN (13). Consistent with this observation, Lizcano et al. (28) (published while this paper was under review) demonstrated that RNAi directed against dPI3K and dPTEN could modulate dS6K phosphorylation. A reasonable explanation for these discrepancies is that the knockdown of dPI3K and dPTEN achieved in our experiments was not sufficient to completely deplete these proteins and affect dS6K phosphorylation.
The role of dAkt in regulating dS6K is subject to debate. In Drosophila, Akt plays a predominant role in mediating the effects of increased PIP3 levels (51), and all Akt-mediated growth signals are thought to be transduced via Tsc1/2 (39). Tsc2 is directly phosphorylated by Akt (for a review, see reference 31), implying that S6K is downstream of Akt in the PI3K signaling pathway. Our observation that RNAi of dAkt reduces dS6K phosphorylation at Thr398 supports a direct link among dAkt, dTSC, and dS6K but contradicts the finding that TSC modulates dS6K activity in a dAkt-independent manner (41, 42). The recent data of Lizcano et al. (28) also support our conclusion of a link between dAkt and dS6K. Clones of cells doubly mutant for dPTEN and dTsc1 display an additive overgrowth phenotype, suggesting that the tumor suppressors act on two independent pathways, from dPTEN to dAkt and from dTSC to dS6K (12, 41). Our findings demonstrate clear effects of dPTEN, dAkt, and dTSC on d4E-BP, which does not preclude the possibility that two pathways regulate d4E-BP; however, a simpler interpretation is that a single pathway is important for its regulation. A possibility is that d4E-BP requires higher dAkt activity than dS6K in order to be phosphorylated. In circumstances of low PI3K activation, low levels of PIP3 are produced, resulting in weaker dAkt activity that is sufficient for dS6K activation but not for d4E-BP phosphorylation. A differential threshold of activation could be the source of the discrepancies between our results and those of Lizcano et al. (28). This model is strongly supported by recent data showing that in cells lacking both Akt1 and Akt2 isoforms, the low level of Akt activity remaining is sufficient for robust S6K1 phosphorylation, but phosphorylation of 4E-BP1 is dramatically reduced (38).
Alternatively, our results could also be explained by the existence of a negative feedback loop between dPI3K and dS6K that dampens insulin signaling by suppressing dAkt activity. This negative feedback loop has been described (19, 41, 42). Similar observations were made in mammals, as insulin-induced activation of Akt is inhibited in Tsc2-deficient mouse embryonic fibroblasts (22). Thus, depletion of dAkt may trigger this negative feedback loop, which diminishes dS6K phosphorylation and activation. Interestingly, engagement of this feedback mechanism can also provide an explanation for the reduction in total d4E-BP levels observed in dPDK1 RNAi-treated cells. Under these conditions, the reduction of dS6K signaling is accompanied by a concomitant reduction in growth signaling on the dPI3K-dAkt branch of the pathway. Thus, a reduced level of d4E-BP is required to accommodate the reduced need for deIF4E inhibition.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Cancer Institute of Canada and a Howard Hughes Medical Institute grant. M.M. was supported by a graduate fellowship from the Cancer Research Society.
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FOOTNOTES |
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Present
address: McGill University and Genome Quebec Innovation Centre,
Montreal, Quebec, Canada H3A 1A4.
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REFERENCES |
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TopAbstractIntroductionMaterials
AND METHODSResultsDiscussionReferences |
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2. Brand, A. H., and N. Perrimon. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401-415.[Abstract]
3. Britton, J. S., W. K. Lockwood, L. Li, S. M. Cohen, and B. A. Edgar. 2002. Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2:239-249.[CrossRef][Medline]
4. Brunn,
G. J., P. Fadden, T. A. J. Haystead, and
J. C. Lawrence, Jr. 1997. The mammalian
target of rapamycin phosphorylates sites having a (Ser/Thr)-Pro motif
and is activated by antibodies to a region near its COOH terminus.J. Biol. Chem.
272:32547-32550.
5. Cho,
K. S., J. H. Lee, S. Kim, D. Kim, H. Koh, J. Lee,
C. Kim, J. Kim, and J. Chung. 2001.
Drosophila phosphoinositide-dependent kinase-1 regulates
apoptosis and growth via the phosphoinositide 3-kinase-dependent
signaling pathway. Proc. Natl. Acad. Sci. USA
98:6144-6149.
6. Choi,
K. M., L. P. McMahon, and J. C. Lawrence,
Jr. 2003. Two motifs in the translational repressor
PHAS-I required for efficient phosphorylation by mammalian target of
rapamycin and for recognition by raptor. J. Biol.
Chem.
278:19667-19673.
7. Clemens,
J. C., C. A. Worby, N. Simonson-Leff, M. Muda, T.
Maehama, B. A. Hemmings, and J. E. Dixon.2000
. Use of double-stranded RNA interference in
Drosophila cell lines to dissect signal transduction pathways.Proc. Natl. Acad. Sci. USA
97:6499-6503.
8. Conlon, I., and M. Raff. 1999. Size control in animal development. Cell 96:235-244.[CrossRef][Medline]
9. Dufner,
A., M. Andjelkovic, B. M. Burgering, B. A.
Hemmings, and G. Thomas. 1999. Protein kinase B
localization and activation differentially affect S6 kinase 1 activity
and eukaryotic translation initiation factor 4E-binding protein 1
phosphorylation. Mol. Cell. Biol.
19:4525-4534.
10. Fadden,
P., T. A. Haystead, and J. C. Lawrence, Jr.1997
. Identification of phosphorylation sites in the
translational regulator, PHAS-I, that are controlled by insulin and
rapamycin in rat adipocytes. J. Biol. Chem.
272:10240-10247.
11. Fingar,
D. C., S. Salama, C. Tsou, E. Harlow, and J. Blenis.2002
. Mammalian cell size is controlled by mTOR and its
downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev.
16:1472-1487.
12. Gao,
X., and D. Pan. 2001. TSC1 and TSC2 tumor suppressors
antagonize insulin signaling in cell growth. Genes Dev.
15:1383-1392.
13. Gao, X., Y. Zhang, P. Arrazola, O. Hino, T. Kobayashi, R. S. Yeung, B. Ru, and D. Pan. 2002. Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat. Cell Biol. 4:699-704.[CrossRef][Medline]
14. Garami, A., F. J. Zwartkruis, T. Nobukuni, M. Joaquin, M. Roccio, H. Stocker, S. C. Kozma, E. Hafen, J. L. Bos, and G. Thomas. 2003. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol. Cell 11:1457-1466.[CrossRef][Medline]
15. Gingras,
A. C., S. P. Gygi, B. Raught, R. D.
Polakiewicz, R. T. Abraham, M. F. Hoekstra, R.
Aebersold, and N. Sonenberg. 1999. Regulation of
4E-BP1 phosphorylation: a novel two-step mechanism. Genes
Dev.
13:1422-1437.
16. Gingras,
A. C., S. G. Kennedy, M. A. O'Leary,
N. Sonenberg, and N. Hay. 1998. 4E-BP1, a repressor of
mRNA translation, is phosphorylated and inactivated by the Akt(PKB)
signaling pathway. Genes Dev.
12:502-513.
17. Gingras,
A. C., B. Raught, S. P. Gygi, A. Niedzwiecka, M.
Miron, S. K. Burley, R. D. Polakiewicz, A.
Wyslouch-Cieszynska, R. Aebersold, and N. Sonenberg.2001
. Hierarchical phosphorylation of the translation
inhibitor 4E-BP1. Genes Dev.
15:2852-2864.
18. Gingras,
A. C., B. Raught, and N. Sonenberg. 2001.
Regulation of translation initiation by FRAP/mTOR. Genes
Dev.
15:807-826.
19. Haruta,
T., T. Uno, J. Kawahara, A. Takano, K. Egawa, P. M. Sharma,
J. M. Olefsky, and M. Kobayashi. 2000. A
rapamycin-sensitive pathway down-regulates insulin signaling via
phosphorylation and proteasomal degradation of insulin receptor
substrate-1. Mol. Endocrinol.
14:783-794.
20. Heesom, K. J., M. B. Avison, T. A. Diggle, and R. M. Denton. 1998. Insulin-stimulated kinase from rat fat cells that phosphorylates initiation factor-4E binding protein 1 on the rapamycin-insensitive site (serine-111).Biochem. J. 336:39-48.
21. Inoki,
K., Y. Li, T. Xu, and K. L. Guan. 2003. Rheb
GTPase is a direct target of TSC2 GAP activity and regulates mTOR
signaling. Genes Dev.
17:1829-1834.
22. Jaeschke,
A., J. Hartkamp, M. Saitoh, W. Roworth, T. Nobukuni, A. Hodges, J.
Sampson, G. Thomas, and R. Lamb. 2002. Tuberous
sclerosis complex tumor suppressor-mediated S6 kinase inhibition by
phosphatidylinositide-3-OH kinase is mTOR independent.J. Cell Biol.
159:217-224.
23. Kozma, S. C., and G. Thomas. 2002. Regulation of cell size in growth, development and human disease: PI3K, PKB and S6K.Bioessays 24:65-71.[CrossRef][Medline]
24. Kwon, C. H., X. Zhu, J. Zhang, L. L. Knoop, R. Tharp, R. J. Smeyne, C. G. Eberhart, P. C. Burger, and S. J. Baker. 2001. PTEN regulates neuronal soma size: a mouse model of Lhermitte-Duclos disease. Nat. Genet. 29:404-411.[CrossRef][Medline]
25. Lachance,
P. E., M. Miron, B. Raught, N. Sonenberg, and P. Lasko.2002
. Phosphorylation of eukaryotic translation initiation
factor 4E is critical for growth. Mol. Cell. Biol.
22:1656-1663.
26. Lawlor, M. A., A. Mora, P. R. Ashby, M. R. Williams, V. Murray-Tait, L. Malone, A. R. Prescott, J. M. Lucocq, and D. R. Alessi.2002 . Essential role of PDK1 in regulating cell size and development in mice. EMBO J. 21:3728-3738.[CrossRef][Medline]
27. Lin,
T. A., and J. C. Lawrence, Jr.1996
. Control of the translational regulators PHAS-I and
PHAS-II by insulin and cAMP in 3T3-L1 adipocytes. J.
Biol. Chem.
271:30199-30204.
28. Lizcano, J. M., S. Alrubaie, A. Kieloch, M. Deak, S. J. Leevers, and D. R. Alessi. 2003. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B. Biochem. J. 374:297-306.[CrossRef][Medline]
29. Mader,
S., H. Lee, A. Pause, and N. Sonenberg. 1995. The
translation initiation factor eIF-4E binds to a common motif shared by
the translation factor eIF-4
and the translational repressors
4E-binding proteins. Mol. Cell. Biol.
15:4990-4997.[Abstract]
30. Marcotrigiano, J., A. C. Gingras, N. Sonenberg, and S. K. Burley. 1999. Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G. Mol. Cell 3:707-716.[CrossRef][Medline]
31. Marygold, S. J., and S. J. Leevers. 2002. Growth signaling: TSC takes its place. Curr. Biol. 12:R785-R787.[CrossRef][Medline]
32. McManus, E. J., and D. R. Alessi. 2002. TSC1-TSC2: a complex tale of PKB-mediated S6K regulation. Nat. Cell Biol. 4:E214-E216.[CrossRef][Medline]
33. Miron, M., J. Verdu, P. E. Lachance, M. J. Birnbaum, P. F. Lasko, and N. Sonenberg. 2001. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat. Cell Biol. 3:596-601.[CrossRef][Medline]
34. Nojima,
H., C. Tokunaga, S. Eguchi, N. Oshiro, S. Hidayat, K. Yoshino, K. Hara,
N. Tanaka, J. Avruch, and K. Yonezawa. 2003. The
mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR
substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS)
motif. J. Biol. Chem.
278:15461-15464.
35. Oldham,
S., H. Stocker, M. Laffargue, F. Wittwer, M. Wymann, and E. Hafen.2002
. The Drosophila insulin/IGF receptor
controls growth and size by modulating PtdInsP(3) levels.Development
129:4103-4109.
36. Patel,
P. H., N. Thapar, L. Guo, M. Martinez, J. Maris, C.
L. Gau, J. A. Lengyel, and F. Tamanoi. 2003.
Drosophila Rheb GTPase is required for cell cycle progression
and cell growth. J. Cell Sci.
116:3601-3610.
37. Pause, A., G. J. Belsham, A.-C. Gingras, O. Donzé, T. A. Lin, J. C. Lawrence, Jr., and N. Sonenberg. 1994. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature 371:762-767.[CrossRef][Medline]
38. Peng,
X.-D., P.-Z. Xu, M.-L. Chen, A. Hahn-Windgassen, J. Skeen, J. Jacobs,
D. Sundararajan, W. S. Chen, S. E. Crawford,
K. G. Coleman, and N. Hay. 2003. Dwarfism,
impaired skin development, skeletal muscle atrophy, delayed bone
development, and impeded adipogenesis in mice lacking Akt1 and Akt2.Genes Dev.
17:1352-1365.
39. Potter, C. J., L. G. Pedraza, and T. Xu.2002 . Akt regulates growth by directly phosphorylating Tsc2. Nat. Cell Biol. 4:658-665.[CrossRef][Medline]
40. Poulin,
F., A.-C. Gingras, H. Olsen, S. Chevalier, and N. Sonenberg.1998
. 4E-BP3, a new member of the eukaryotic initiation
factor 4E-binding protein family. J. Biol.
Chem.
273:14002-14007.
41. Radimerski,
T., J. Montagne, M. Hemmings-Mieszczak, and G. Thomas.2002
. Lethality of Drosophila lacking TSC tumor
suppressor function rescued by reducing dS6K signaling. Genes
Dev.
16:2627-2632.
42. Radimerski, T., J. Montagne, F. Rintelen, H. Stocker, J. van Der Kaay, C. P. Downes, E. Hafen, and G. Thomas. 2002. dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1. Nat. Cell Biol. 4:251-255.[CrossRef][Medline]
43. Raught, B., A. C. Gingras, and N. Sonenberg. 2000. Regulation of ribosomal recruitment in eukaryotes, p.245 -293. In N. Sonenberg, J. W. B. Hershey, and M. B. Mathews (ed.), Translational control of gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
44. Rintelen,
F., H. Stocker, G. Thomas, and E. Hafen. 2001. PDK1
regulates growth through Akt and S6K in Drosophila.Proc. Natl. Acad. Sci. USA
98:15020-15025.
45. Saucedo, L., and B. Edgar. 2002. Why size matters: altering cell size. Curr. Opin. Genet. Dev. 12:565-571.[CrossRef][Medline]
46. Saucedo, L. J., X. Gao, D. A. Chiarelli, L. Li, D. Pan, and B. A. Edgar. 2003. Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5:566-571.[CrossRef][Medline]
47. Schalm, S. S., and J. Blenis. 2002. Identification of a conserved motif required for mTOR signaling. Curr. Biol. 12:632-639.[CrossRef][Medline]
48. Schalm, S. S., D. C. Fingar, D. M. Sabatini, and J. Blenis. 2003. TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function. Curr. Biol. 13:797-806.[CrossRef][Medline]
49. Shioi, T., P. M. Kang, P. S. Douglas, J. Hampe, C. M. Yballe, J. Lawitts, L. C. Cantley, and S. Izumo. 2000. The conserved phosphoinositide 3-kinase pathway determines heart size in mice. EMBO J. 19:2537-2548.[CrossRef][Medline]
50. Sigrist, S. J., P. R. Thiel, D. F. Reiff, P. E. D. Lachance, P. Lasko, and C. F. Schuster. 2000. Postsynaptic translation affects the efficacy and morphology of neuromuscular junctions.Nature 405:1062-1065.[CrossRef][Medline]
51. Stocker,
H., M. Andjelkovic, S. Oldham, M. Laffargue, M. P. Wymann,
B. A. Hemmings, and E. Hafen. 2002. Living
with lethal PIP3 levels: viability of flies lacking PTEN restored by a
PH domain mutation in Akt/PKB. Science
295:2088-2091.
52. Stocker, H., T. Radimerski, B. Schindelholz, F. Wittwer, P. Belawat, P. Daram, S. Breuer, G. Thomas, and E. Hafen. 2003. Rheb is an essential regulator of S6K in controlling cell growth in Drosophila. Nat. Cell Biol. 5:559-565.[CrossRef][Medline]
53. Tee,
A. R., and C. G. Proud. 2002.
Caspase cleavage of initiation factor 4E-binding protein 1 yields a
dominant inhibitor of cap-dependent translation and reveals a novel
regulatory motif. Mol. Cell. Biol.
22:1674-1683.
54. Tuttle,
R. L., N. S. Gill, W. Pugh, J. P. Lee, B.
Koeberlein, E. E. Furth, K. S. Polonsky, A. Naji,
and M. J. Birnbaum. 2001. Regulation of
pancreatic beta-cell growth and survival by the serine/threonine
protein kinase Akt1/PKB. Nat. Med.
7:1133-1137.[CrossRef][Medline]
55. Wang,
X., W. Li, J. L. Parra, A. Beugnet, and C. G.
Proud. 2003. The C terminus of initiation factor
4E-binding protein 1 contains multiple regulatory features that
influence its function and phosphorylation. Mol. Cell.
Biol.
23:1546-1557.
56. Wang, X., W. Li, M. Williams, N. Terada, D. R. Alessi, and C. G. Proud. 2001. Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J. 20:4370-4379.[CrossRef][Medline]
57. Weinkove, D., and S. J. Leevers. 2000. The genetic control of organ growth: insights from Drosophila.Curr. Opin. Genet. Dev. 10:75-80.[CrossRef][Medline]
58. Zhang, Y., X. Gao, L. J. Saucedo, B. Ru, B. A. Edgar, and D. Pan. 2003. Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat. Cell Biol. 5:578-581.[CrossRef][Medline]
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