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RESEARCH COMMUNICATION
1 Department of Biological Chemistry , 2 Institute of Gerontology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA , 3 Howard Hughes Medical Institute, Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06536, USA
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Abstract |
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[Keywords: TSC2; Rheb; mTOR; S6K; GAP; tuberous sclerosis complex]
Received May 6, 2003; revised version accepted June 2, 2003.
). The most
serious clinical complications are mental retardation, epilepsy, and autism
caused by tumor growth in the brain (Gomez
1991). Other symptoms include renal dysfunction, dermatological
abnormalities, and heart problems. Heterozygous deletions of either
TSC1 or TSC2 produce similar phenotypes and increase tumor
incidence particularly in the kidney, whereas homozygous deletion of either
gene is embryonic lethal (Au et al.
1998; Kobayashi et al.
2001). Genetic data from Drosophila show that TSC1 and
TSC2 negatively regulate cell growth and cell size
(Gao and Pan 2001;
Potter et al. 2001;
Tapon et al. 2001).
Recent studies have established that TSC1/TSC2 inhibits phosphorylation of
the ribosomal S6 kinase (S6K) and the eukaryotic initiation factor 4E-binding
protein (4EBP1), two key regulators of translation
(Goncharova et al. 2002;
Inoki et al. 2002;
Manning et al. 2002;
Tee et al. 2002).
Phosphorylation of S6K and 4EBP1 enhances translation
(Gingras et al. 1999). How
TSC1/TSC2 regulates the phosphorylation of S6K and 4EBP1 is a key question yet
to be answered. The mammalian target of rapamycin (mTOR) is directly
responsible for phosphorylation of both S6K and 4EBP1
(Brown et al. 1995;
Hara et al. 1998). Recent
studies have suggested that TSC1/TSC2 acts through mTOR to regulate the
phosphorylation of S6K and 4EBP1 (Gao et
al. 2002; Inoki et al.
2002; Tee et al.
2002). Consistent with this model is that TSC1 and TSC2 are
important for cellular nutrient response, which requires the function of mTOR
(Gao et al. 2002). However, it
is unclear how TSC1/TSC2 inhibits mTOR activity.
The C-terminal region of TSC2 displays significant homology to the Rap
GTPase-activating protein (GAP; The
European Chromosome 16 Tuberous Sclerosis Consortium 1993). In
fact, GAP activity of TSC2 toward Rap1 and Rab5 had previously been reported
(Wienecke et al. 1995;
Xiao et al. 1997). However,
the reported GAP activity was extremely low, and the functional significance
is unclear. Rheb is a small GTPase initially isolated as a Ras homolog
enriched in brain (Yamagata et al.
1994) and is widely expressed. Rheb shares higher sequence
identity with Ras than with Rho family members. The biological function of
mammalian Rheb is unclear. Conflicting studies report that Rheb both inhibits
and activates the Raf-MAP kinase pathway
(Clark et al. 1997;
Yee and Worley 1997).
Interestingly, mutation of the Rheb gene in Schizosaccharomyces
pombe produces a phenotype similar to nutrient starvation, indicating
that Rheb is possibly involved in nutrient signaling
(Mach et al. 2000).
In this report, we provide direct biochemical data demonstrating that TSC2 has GAP activity toward Rheb in vitro. Furthermore, we show that TSC2 regulates Rheb-GTP levels in vivo. We show that one of the roles of Rheb is to stimulate phosphorylation of S6K and 4EBP1, two of the best characterized cellular downstream targets of TSC1/TSC2. Both the effector domain and the GTP binding are essential for Rheb function. The ability of Rheb to stimulate S6K phosphorylation requires the function of mTOR, indicating that mTOR acts downstream of Rheb. Consistent with this is that Rheb stimulates the phosphorylation of mTOR on serine residue 2448. Furthermore, our data support that Rheb plays an important role in cellular responses to energy limitation and nutrient starvation. Together, this study provides a model that Rheb is a direct downstream target of TSC2 and acts upstream of mTOR to regulate translation and cell growth.
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Results and Discussion |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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The C-terminal region of TSC2 contains a putative GAP domain with
significant homology to RapGAP (The
European Chromosome 16 Tuberous Sclerosis Consortium 1993;
Scheffzek et al. 1998).
However, the precise physiological and biochemical functions of the putative
TSC2 GAP domain have not been demonstrated. Interestingly, a high frequency of
TSC-associated mutations occurs in the C-terminal putative GAP domain of TSC2,
indicating the GAP domain may be important for TSC2 function
(Jin et al. 1996;
Momose et al. 2002;
Kwiatkowski 2003). To study
the biochemical functions of TSC2, we expressed and purified the GAP domain of
TSC2 in Escherichia coli and tested for GAP activity toward the Ras
subfamily GTPases (Ras, Rap, TC21, and Rheb) and the Rho family GTPases (Rac
and Cdc42). Our in vitro assays failed to detect significant GAP activity,
whereas the positive control of RasGAP1 showed activity toward Ras (data not
shown). It is worth noting that the catalytic arginine residue essential for
GAP activity in the Rap GAP family is not conserved in TSC2
(Fig. 1A), suggesting that TSC2
may have no GAP activity (Scheffzek et al.
1998). It is also possible that TSC2 has GAP activity, but a
different arginine or a completely different catalytic mechanism may be
used.
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To further test for GAP activity of TSC2, we expressed TSC2 together with
TSC1 in HEK293 cells. The TSC1/TSC2 complex was purified by
immunoprecipitation, and in vitro GAP activity was determined using various
small GTPases. We made particular efforts on Rheb because deletion of Rheb in
S.pombe produces phenotypes similar to nutrient starvation
(Mach et al. 2000). We found
that the TSC1/TSC2 complex stimulated the GTP hydrolysis of Rheb
(Fig. 1B,C) but not Ras. The
GAP activity of TSC2 requires the full-length protein because neither the
N-terminal nor the C-terminal region, which contains the GAP homology domain,
displayed activity toward Rheb (Fig.
1D). To exclude the possibility that the GAP activity detected in
the TSC1/TSC2 immunoprecipitation is due to another coprecipitated GAP, we
made TSC2-R1701Q and TSC2-R1703Q mutants, which are found in TSC patients, and
detected no GAP activity (data not shown). Therefore, we conclude that TSC2 is
an Rheb GAP. The lack of GAP activity of TSC2C could be due to the requirement
of residues in the N-terminal region of TSC2 for GAP activity or incorrect
folding of TSC2C. The ability of TSC2 to stimulate GTPase activity of Rheb is
very interesting because TSC2 does not have the conserved active-site arginine
found in other related RapGAPs and Rheb is an unusual GTPase
(Fig. 1A). Almost all Ras
family GTPases contain a conserved glycine residue at the position equivalent
to codon 12 in Ras (Lowy and Willumsen
1993). Mutation of this glycine to almost any residue, including
valine found in many cancers, activates Ras family GTPases. The RasV12 mutant
is known to be resistant to down-regulation by RasGAP
(Trahey and McCormick 1987).
Interestingly, Rheb has an arginine residue at the corresponding position
codon 12 and has a much lower basal GTPase activity than Ras (data not shown).
However, the GTPase activity of Rheb can be effectively stimulated by
TSC1/TSC2. Therefore, Rheb and TSC2 are unusual GTPases and GAPs,
respectively.
To determine whether TSC1 is required for the GAP activity of TSC2, HA-TSC2 alone was expressed in HEK293 cells and immunoprecipitated. The precipitated TSC2, which may also coimmunoprecipitate a small amount of endogenous TSC1, showed significant GAP activity toward Rheb (Fig. 1E). In contrast, immunoprecipitated TSC1 showed a very low level of GAP activity toward Rheb possibly due to coprecipitation of endogenous TSC2 protein. These observations indicate that TSC1 stabilizes TSC2 in vivo, but is not directly required for GAP activity. TSC1/TSC2 was also immunoprecipitated under various conditions, including treatments with rapamycin, LY294002, and D-PBS, which are all known to inhibit phosphorylation of S6K and 4EBP1. We observed that these treatments did not directly affect the GAP activity of immunoprecipitated TSC1/TSC2 (Fig. 1E).
The effect of TSC2 on Rheb GTP levels was also determined in vivo. The
nucleotide-binding status of Rheb was determined by in vivo labeling with
32P-phosphate. Interestingly, Rheb contains a high basal GTP level
(Fig. 1F). The high GTP level
of wild-type Rheb is consistent with it having an arginine at a position
equivalent to codon 12 of Ras (Fig.
1A). Expression of TSC1/TSC2 decreased the GTP/GDP ratio of
cotransfected Rheb 
10-fold (Fig.
1F). As a control, expression of the N-terminal domain of TSC2 had
no significant effect on the GTP level of Rheb. These data provide in vivo
evidence that TSC2 is an Rheb GAP.
Rheb stimulates the phosphorylation of S6K and 4EBP1
If Rheb is a key downstream target of TSC2, Rheb should stimulate the phosphorylation of S6K and 4EBP1. Expression of the wild-type Rheb induced a dramatic increase in S6K phosphorylation and mobility shift of 4EBP1 (Fig. 2A). Rheb also increased the phosphorylation of endogenous S6K in transfected HEK293 cells. In contrast, Rheb did not stimulate ERK phosphorylation (data not shown). The high activity of wild-type Rheb is consistent with it being predominantly GTP bound (Fig. 1F). Rheb also stimulates S6K phosphorylation in a dose-dependent manner (Fig. 2B).
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Five residues between 38 and 42 in the effector domain of Rheb were mutated
to alanines to produce Rheb5A. We found that mutation of the effector domain
completely abolished the ability of Rheb to stimulate S6K phosphorylation
(Fig. 2C). We also tested
constitutively active Rheb (RhebL64) and observed that RhebL64 is
approximately two to three times more active than the wild type
(Fig. 2C). In an attempt to
create a dominant-negative mutant, we made an RhebN20. Surprisingly, RhebN20
does not show any dominant-negative effect nor does it stimulate S6K
(Fig. 2C). A similar
dominant-negative mutant of RasN17 is effective in blocking Ras function
through sequestration of the Ras guanine nucleotide exchange factor (GEF;
Lowy and Willumsen 1993). This
observation suggests that Rheb may not need a GEF because of its high basal
GTP level and that GTP binding is required to stimulate S6K
phosphorylation.
To determine the specificity of Rheb, several other GTPases were examined, including Cdc42, Rap1, RhoA, and Rab5. Constitutively active mutants of these GTPases fail to stimulate S6K phosphorylation (Fig. 2D). These results demonstrate that Rheb is specific to stimulate S6K phosphorylation. We tested the effect of TSC1 or TSC2 alone on S6K phosphorylation. Expression of TSC2, but not TSC1, significantly decreased the basal phosphorylation of cotransfected S6K (Fig. 3E), consistent with the fact that TSC2 alone has GAP activity. The ability of TSC2 but not TSC1 to inhibit S6K indicates that TSC2 is the active component of the TSC1/TSC2 complex, whereas TSC1 may have regulatory functions, such as localization and stabilization. Furthermore, these observations suggest that inhibition of endogenous Rheb by TSC2 decreases S6K phosphorylation, thus supporting the importance of Rheb in S6K regulation.
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TSC1/TSC2 inhibits Rheb function
The functional relationship between TSC1/TSC2 and Rheb was examined in vivo. Cotransfection of TSC1/TSC2 suppressed the ability of Rheb to stimulate S6K (Fig. 3A). Similar results were observed with 4EBP1 (Fig. 3B). These data further support the idea that TSC2 acts as a GAP toward Rheb.
mTOR acts downstream of Rheb
Previous studies have suggested that mTOR acts downstream of TSC2
(Gao et al. 2002;
Inoki et al. 2002;
Tee et al. 2002). To determine
the relationship between Rheb and mTOR, the effect of rapamycin was
investigated. Treatment of cells with rapamycin completely blocked the
stimulatory effect of Rheb on S6K phosphorylation
(Fig. 4A), indicating that Rheb
functions upstream of mTOR. To further test the functional relationship
between Rheb and mTOR, a kinase-inactive mutant of mTOR was coexpressed with
Rheb. We found that the dominant-negative mTOR-KD significantly blocked the
phosphorylation of S6K by Rheb (Fig.
4B). We had previously observed that TSC1/TSC2 inhibits the
phosphorylation of S2448 of mTOR (Inoki et
al. 2002; Kenerson et al.
2002). Coexpression of Rheb caused a significant increase of mTOR
S2448 phosphorylation, whereas phosphorylation of Akt was not affected
(Fig. 4C), indicating that Rheb
activates mTOR.
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Rheb is involved in multiple signaling pathways that regulate
S6K
Phosphorylation of S6K is regulated by numerous cellular signals including
nutrients (Proud 2002). The
functional significance of Rheb in nutrient signaling was analyzed. In
Rheb-transfected HEK293 cells, nutrient starvation (treatment with D-PBS that
contains glucose, but not amino acids) induced a minor dephosphorylation of
S6K (Fig. 4A). In contrast,
nutrient starvation inhibited S6K phosphorylation in RasV12-transfected cells
(Fig. 4A). These results
demonstrate that Rheb plays an important role in the nutrient signaling
pathway, but Ras is not directly involved in nutrient signaling. We also
examined the effect of osmotic stress by sorbitol, energy depletion by
2-deoxyglucose (2-DG), and inhibition of phospholipase D by 1-butanol. These
treatments are known to induce S6K dephosphorylation
(Fig. 4D; Dennis et al. 2001;
Fang et al. 2001;
Desai et al. 2002). Rheb
significantly blocked the effect of 2-DG and 1-butanol, but had little effect
on S6K dephosphorylation induced by sorbitol
(Fig. 4D). These data suggest
that Rheb acts in the cellular energy-sensing pathway and the phospholipase D
pathway, but is not directly involved in the osmotic stress pathway.
Consistently, TSC1/TSC2 also plays an important role in regulation of S6K in
response to cellular energy depletion (data not shown).
The importance of TSC1/TSC2 in cell growth regulation has been well
established (Potter and Xu
2001). The major cellular function of TSC1/TSC2 is to inhibit the
phosphorylation of S6K and 4EBP1, likely through mTOR. This study provides a
significant advance in the understanding of the molecular mechanism of how
TSC1/TSC2 regulates the phosphorylation of S6K and 4EBP-1. Based on both in
vitro and in vivo biochemical experiments, we conclude that TSC2 is an
Rheb-specific GAP and Rheb is a key downstream target of TSC1/TSC2
(Fig. 5). Rheb is closely
related to Ras and is an unusual GTPase in that it is predominantly GTP bound
in cells. The biological function of Rheb has not been clearly defined. Our
studies support that Rheb promotes cell growth by stimulating phosphorylation
of S6K and 4EBP1, but has no role in ERK activation.
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Inhibition of Rheb by TSC1/TSC2 is likely to be critical for the function
of these two tumor suppressor proteins because Rheb potently regulates the
phosphorylation of S6K and 4EBP1. Interestingly, mutation of the Rheb ortholog
in S.pombe displays phenotypes characteristic of nutrient starvation
(Mach et al. 2000). Our data
are consistent with the genetic observations made in S.pombe and
support the notion that Rheb plays an essential role in nutrient signaling.
S.pombe also contains TSC1- and TSC2-related molecules
(Matsumoto et al. 2002). We
have also shown that TSC2 is an unusual GAP because it lacks the conserved
arginine found in other Rap family GAPs. Yet it has very high and selective
GAP activity toward Rheb. Future studies to elucidate the catalytic mechanism
of TSC2 on Rheb-GTP hydrolysis will provide new insights into how this unusual
GTPase and GAP work.
Our studies present a model in which Rheb is involved in S6K regulation in response to nutrients and energy levels (Fig. 5). In contrast, osmotic stress regulates S6K independently of Rheb. A major function of Rheb is to stimulate mTOR; however, we did not detect a significant association between Rheb and mTOR, suggesting that Rheb may activate mTOR indirectly. Future efforts will focus on how Rheb stimulates mTOR. Our model predicts that TSC2 acts through Rheb to regulate translation (Fig. 5). It suggests that Rheb positively regulates cell growth and plays an important role in the development of TSC. Therefore, inhibition of Rheb could be a potential target for the treatment of TSC and cancer.
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Materials and methods |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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Anti-phospho S6K (T389), anti-mTOR, and anti-phospho mTOR (S2448)
antibodies were from Cell Signaling Inc., and anti-Myc antibodies were from
Santa Cruz Biotechnology. Anti-HA and anti-Flag were purchased from Covance
and Sigma, respectively. TSC1 and TSC2 constructs were described previously
(Inoki et al. 2002). The
HA-tagged S6K1 (
II) construct was generously provided by J. Blenis
(Harvard University, Cambridge, MA) and Flag-tagged mTOR and kinase inactive
mTOR (mTORKD) were from S. Schreiber (Harvard University, Cambridge, MA). Rab5
is a gift of G. Li (University of Oklahoma, Health Sciences Center, Oklahoma
City, OK). All other DNA constructs including Ras, Rheb, Rap1, Rac, Cdc42,
RhoA, 4E-BP1, Akt, and ERK2 were laboratory stocks. All mutant constructs of
TSC2 and Rheb were created by PCR mutagenesis and verified by DNA
sequencing.
Cell culture, transfection, and immunoprecipitation
HEK293 cells were seeded and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Transfection was performed using Lipofectamine Reagent (Invitrogen) following the manufacturer's instructions. Cells were lysed in lysis buffer (10 mM Tris-HCl at pH 7.5, 100 mM NaCl, 1% NP-40, 1% Triton X-100, 50 mM NaF, 2 mM EDTA, 1 mM PMSF, 10 µg/mL leupeptin, 10 µg/mL aprotinin) and immunoprecipitated with the indicated antibodies and protein G-Sepharose beads. Immunocomplexes were subjected to SDS-PAGE.
In vitro GTPase assay
GST-Rheb, GST-Ras, GST-Rac, and GST-Rap were expressed in the bacterial
strain BL21 and purified using glutathione-Sepharose 4B beads (Sigma) as
described. Small GTPases were loaded with 32P-
-GTP before
GAP assays. Here, 10 µg of small GTPase protein bound on glutathione beads
was incubated with 50 µCi of 32P--GTP in loading buffer
(20 mM Tris at pH 8, 5 mM EDTA, 1 mM DTT, 0.1 mg/mL BSA) in a volume of 20
µL. The loading reaction was stopped by addition of MgCl2 to 10
mM. Then the small GTPase protein was eluted.
HA-TSC2 was transfected to HEK293 cells and immunoprecipitated by anti-HA
antibody. The immune complexes were washed three times with wash buffer (20 mM
Tris at pH 7.4, 800 mM NaCl, 2 mM EDTA, 1% NP-40) and two times with GAP assay
wash buffer (20 mM Tris at pH 8, 100 mM NaCl, 5 mM MgCl2, 1 mM
DTT). The final GAP assays included 0.5 µg of 32P--GTP
loaded small GTPase, and immunoprecipitated TSC2 from 1 x 107
transfected to HEK293 cells. Reactions were carried out in 40 µL of GAP
assay buffer (20 mM Tris at pH 8, 10 mM MgCl2, and 4 mM DTT), at
room temperature for 20 min. The reaction was stopped by 300 µL of charcoal
buffer (5% charcoal, 20 mM phosphoric acid, 0.6 M HCl), then vortexed for 10
min and spun at 13,000 rpm for 10 min. Then 100 µL of supernatant was
taken, and the 32P release was determined by scintillation
counting.
In vivo labeling of Rheb
The cells were washed once with phosphate-free DMEM and incubated with 0.5 mCi/mL 32P-orthophosphate (ICN) for 4 h. The cells were lysed with labeling lysis buffer (1% Triton X-100, 50 mM HEPES at pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mg/mL BSA, 1 mM DTT, 1 mM PMSF, 10 µg/mL leupeptin, 10 µg/mL aprotinin). Myc-tagged Rheb was immunoprecipitated. The Rheb-bound nucleotides were eluted with elution buffer (2 mM EDTA, 0.2% SDS, 1 mM GDP, 1 mM GTP) at 68°C for 20 min. Then the eluted nucleotides were subjected to thin layer chromatography using PEI cellulose plates (Baker-flex) in 0.75 M KH2PO4 (pH 3.4).
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Acknowledgments |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Article published online ahead of print. Article and publication date are at http://www.genesdev.org/cgi/doi/10.1101/gad.1110003.
4 These authors contributed equally to this work. 
5 E-MAIL
kunliang{at}umich.edu;
FAX (734) 763-4581.
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) and presence (
) of TSC1/TSC2. (D) Neither
the N-terminal nor the C-terminal region of TSC2 has GAP activity toward Rheb.
Two truncated TSC2 constructs (TSC2N, amino acids 1-1007; TSC2C, amino acids
1008-1765) were coexpressed with TSC1 and immunoprecipitated and assayed for
GAP activity in vitro. The amount of HA-TSC2 used in this assay is equivalent
to 1 µL in B. The expression levels of these proteins were
determined by Western blot. (E) TSC2, but not TSC1, has GAP activity.
Transfected HEK293 cells were untreated or treated with D-PBS, rapamycin (20
nM), or LY294002 (50 µM) for 30 min as indicated. The relative amount of
TSC2 used in the GAP assay was determined by Western blot and is shown
below each bar. (F) TSC1/TSC2 decreases the Rheb-GTP levels
in vivo. Myc-Rheb was transfected in HEK293 cells and labeled with
32P-phosphate. Myc-Rheb was immunoprecipitated, and the bound
nucleotides were eluted and resolved on a cellulose plate. Cotransfection with
TSC1 and TSC2 are indicated. Myc-RacL61 was included as a control. The ratio
of GTP/GDP was calculated by the formula GTP counts/3 divided by GDP counts/2,
and indicated on top of each lane.
