From the Department of Surgery and ¶ Howard
Hughes Medical Institute/Department of Pharmacology
Center for Developmental Biology, University of Washington, School of
Medicine, Seattle, Washington 98195
Received for publication, August 20, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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Tuberous sclerosis complex (TSC) is characterized
by the formation of hamartomas in multiple organs resulting from
mutations in the TSC1 or TSC2 gene. Their protein products, hamartin
and tuberin, respectively, form a functional complex that affects cell
growth, differentiation, and proliferation. Several lines of evidence,
including renal tumors derived from TSC2+/ The phenotype of patients with
TSC1 encompasses the
development of multiple focal lesions in the brain, heart, kidney,
lung, and skin (1). While the underlying pathogenic mechanisms are unclear, the histology of these discrete, tumor-like lesions suggests defects in cell proliferation, differentiation, and cell size control.
Studies in mammalian cells have shown that overexpression of TSC1 and
TSC2 negatively regulates cell proliferation and induces G1/S arrest (2-4). In the case of tuberin, there appears
to be an inverse correlation between tuberin level and p27(Kip1)
expression and stability (5). Correspondingly, evidence supports a link between tuberin and cyclin D1 expression. Cortical tubers
microdissected from TSC patients showed elevated cyclin D1 mRNA
expression in the giant cells (6). Antisense inhibition of TSC2 in Rat1
fibroblasts resulted in up-regulation of cyclin D1 protein (3). Renal
cortical tumors from the Eker rat model for TSC express elevated cyclin D1 compared with unaffected kidney tissue (7). As an in vivo target of the Materials--
Eker rats harboring a germ-line TSC2 mutation
were described previously (12). Human embryonic kidney cells (HEK293T)
were obtained from the American Type Culture Collection (Manassas, VA).
All cell culture reagents and transfection reagents (LipofectAMINE and
Plus reagents) were purchased from Invitrogen. Monoclonal antibodies directed toward Eker Rat Tumor Tissue Analysis--
Eker rats were sacrificed at
12 months of age to collect kidney samples. Kidney lesions were
dissected from unaffected tissue. Both were homogenized in cold RIPA
buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 10 mM Tris-HCl, pH 7.2, 0.025 M Pulse-Chase Experiments--
Vectors encoding TSC1, TSC2, or
TSC2 ( Luciferase Assay--
Cells (HEK293T) were transfected with the
Tcf/LEF reporter construct (TOPFLASH) (18) using LipofectAMINE Plus
reagents (Invitrogen). Vectors encoding TSC1, TSC2, or TSC2 ( Immunoprecipitation--
For immunoprecipitation of endogenous
proteins, HEK293T cells were transfected Wnt-1 construct or empty
vector. Similarly, for ectopically expressed proteins, HEK293T cells
were transfected with TSC1 and TSC2 vectors or pcDNA3 (vector
control) in the presence of vectors for GSK3 Expression of Hamartin and Tuberin Regulate
Next, we examined the effect of hamartin and tuberin expression on
Hamartin and Tuberin Inhibit Wnt-1-stimulated Hamartin and Tuberin Function within the Wnt/
Next, TOPFLASH activity was measured in the presence of ectopically
expressed Dsh, an effector that is stimulated by the Wnt-Frizzled receptor complex upstream of Hamartin and Tuberin Interact with Components of the
If hamartin and tuberin interact with the GSK3
Finally, to examine whether tuberin interacts with the endogenous
GSK3 The TSC1 and TSC2 tumor suppressor genes have recently been
implicated to play a role in negatively regulating mTOR in the PI3K
signaling cascade (23, 24). As a result, tumors secondary to the
inactivation of these genes have elevated levels of p70 S6 kinase
activity that is reversible by rapamycin, a specific mTOR inhibitor
(7). While this pathway may explain some of the complex phenotype
exhibited by TSC pathology (i.e. cell size abnormalities),
alteration in other cellular functions may involve additional
mechanisms. Up-regulation of cyclin D1 mRNA and protein has been
noted in CNS and renal lesions (6, 7), but a recent study using
rapamycin in the Eker rat model of TSC failed to show a significant
change in cyclin D1 or p27 levels despite anti-tumor response (7).
Here, we describe an alternative pathway that may be relevant to the
abnormalities observed in cell proliferation and differentiation. Our
data show that in vivo levels of In our model, TSC1 and TSC2 complex with GSK3 animals, suggest that the
loss or inhibition of tuberin is associated with up-regulation of
cyclin D1. As cyclin D1 can be regulated through the canonical
Wnt/
-catenin signaling pathway, we hypothesize that the cell
proliferative effects of hamartin and tuberin are partly mediated
through
-catenin. In this study, total
-catenin protein levels
were found to be elevated in the TSC2-related renal tumors. Ectopic
expression of hamartin and wild-type tuberin, but not mutant tuberin,
reduced
-catenin steady-state levels and its half-life. The
TSC1-TSC2 complex also inhibited Wnt-1 stimulated Tcf/LEF luciferase
reporter activity. This inhibition was eliminated by constitutively
active
-catenin but not by Disheveled, suggesting that hamartin and
tuberin function at the level of the
-catenin degradation complex.
Indeed, hamartin and tuberin co-immunoprecipitated with glycogen
synthase kinase 3
and Axin, components of this complex in a
Wnt-1-dependent manner. Our data suggest that hamartin and
tuberin negatively regulate
-catenin stability and activity by
participating in the
-catenin degradation complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin pathway, cyclin D1 mRNA is responsive to
the activity of the Tcf/LEF family of transcription factors (8, 9).
This raises the possibility that TSC1 and TSC2 negatively regulate
-catenin signaling and, thereby, modulate the expression of cyclin D1.
-Catenin is a highly conserved 95-kDa protein that participates in
cell-cell adhesion through its association with members of the
membrane-bound cadherin family, and in cell proliferation and
differentiation as a key component of the Wnt/Wingless pathway (reviewed in Ref. 10). In its signaling role,
-catenin shuttles between the cytoplasm and the nucleus where it binds the Tcf/LEF family
of transcription factors to activate downstream target genes (reviewed
in Ref. 11). In the absence of the secreted factor, Wnt,
-catenin is
phosphorylated by GSK3
and is targeted for ubiquitination and
degradation. Upon Wnt stimulation, Disheveled (Dsh) is activated and
blocks the ability of GSK3
to phosphorylate
-catenin. Other
components of this degradation complex include Axin, serving as a
scaffolding protein, and APC, a tumor suppressor protein. Disruption at
multiple levels of this pathway has been shown to be oncogenic in
humans and rodents. In this study,
-catenin protein levels were
found to be elevated in renal tumors from Eker rats. Overexpression of
tuberin and hamartin in cells down-regulated
-catenin levels, its
half-life and its activity. Furthermore, we showed that TSC1 and TSC2
proteins co-immunoprecipitated with GSK3
and Axin, supporting a role
of hamartin and tuberin in modulating the
-catenin pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin and GSK3
were purchased from
BD Transduction Laboratories (Los Angeles, CA). Monoclonal antibodies
for c-Myc (9E10), actin, and
-tubulin were from Sigma. IgG
purified polyclonal antibodies directed toward tuberin (L3-2) and
hamartin (4050) were prepared as described earlier (13, 14). Polyclonal
antibody for tuberin (C20) was obtained from Santa Cruz (Santa Cruz,
CA). Expression vectors included rat TSC2 (14), human TSC1 (15), mWnt-1
(Gift from Marian Waterman, University of California, Irvine, CA),
constitutively active-
-catenin (CA-
-catenin) (16),
Xenopus Dsh (17), c-Myc-Xenopus Axin,
N-Tcf-4
(18), c-
-galactosidase (19), Xenopus GSK-3
(XG73) (20), and the reporter constructs TOPFLASH and FOPFLASH (18). Luciferin stock solution was purchased from Pharmingen. Galacton and
Emerald stock solutions were obtained from Tropix (Bedford, MA).
Immobilon-P (polyvinylidene difluoride) membranes used for Western blots were from Millipore (Bedford, MA). Radiolabeled [35S]methionine (EASYTAGTM express protein
labeling mix) was purchased from PerkinElmer (Boston, MA). All other
reagents were purchased from Sigma.
-glycophosphate, pH 7.2, 2 mM EDTA, 50 mM sodium fluoride, 0.05 mM
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 10 µg/ml
aprotinin, 10 µg/ml pepstatin, 1 mM orthovanadatate, 10 µg/ml leupeptin, 1 mM microcystin LR), and the protein
concentration was determined using the BCA protein assay (Pierce).
SDS-PAGE and Western blots were performed using equal amounts of total protein.
-Catenin Steady-state Levels--
HEK293T cells were
transfected with increasing concentrations of TSC1 and TSC2 (wild-type
or
Y1571H mutant) vectors or with control vector (pcDNA3) (500, 1000, and 1500 ng) either with or without Wnt-1 vector using
LipofectAMINE Plus reagents according to the manufacturer's
instructions. Empty plasmids were added accordingly to normalize total
DNA transfected. The cells were harvested in lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% Nonidet
P-40, 0.5 µg/ml leupeptin, 1.0 µg/ml pepstatin, 0.2 mM
PMSF) and lysed by freeze thawing at 48 h after transfection. Samples were analyzed by Western blotting.
Y1571H) mutant were co-transfected into HEK293T cells with or
without Wnt-1. After 48 h, pulse-chase using
[35S]methionine incorporation was performed on
transfected cells as outlined by Williams et al. (21).
Protein content in cell lysates was analyzed using the BCA protein
assay. Equal amounts of cell lysate were adjusted to equal volumes,
pretreated with ConA-Sepharose (1:1 in immunoprecipitation buffer, see
below) (Amersham Biosciences, Uppsala, Sweden) and then
subjected to immunoprecipitation for
-catenin. Samples were resolved
by SDS-PAGE and detected by autoradiography. Radiolabel band
intensities were determined using a PhosphorImager Storm 840 (Amersham Biosciences).
Y1571H)
mutant were co-transfected with or without Wnt-1, Dsh, or a
constitutively active
-catenin mutant (CA-
-cat) construct. A
vector encoding
-galactosidase was co-transfected as a transfection
control, while a dominant negative Tcf-4 (
N-Tcf-4) was used as a
negative control for activation. Reporter vector containing mutated
Tcf/LEF binding sites (FOPFLASH) (18) served as a control for
background activity. Luciferase activity was analyzed using an EG&G
Berthold Autolumet LB953 luminometer (PerkinElmer Instruments, Bad
Wildbad, Germany), and normalized to corresponding
-galactosidase values.
or c-Myc-tagged Axin.
Cells were harvested the following day in lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% Nonidet
P-40, 0.5 µg/ml leupeptin, 1.0 µg/ml pepstatin, 0.2 mM
PMSF). Equal total amounts of lysates were immunoprecipitated using
anti-tuberin polyclonal antibody, anti-GSK monoclonal antibody, or
anti-c-Myc antibodies as described previously (14).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Catenin in Eker Rat Kidney Tumors--
The Eker
rat contains a germ-line mutation in the TSC2 gene (12) and
spontaneously develops renal cortical epithelial tumors that have been
shown to possess biallellic inactivation of TSC2 due to loss of
heterozygosity, nonsense mutation, or null mutation (22). In a previous
study, cyclin D1 levels were shown to be elevated in these kidney
tumors compared with unaffected kidney tissue (7). Since cyclin D1 gene
is a known target of the
-catenin signaling pathway and the
accumulation of
-catenin has been shown to activate the
transcription of the cyclin D1 gene (8, 9).
-Catenin levels were
examined in Eker rat kidney tumors. Tumors from three separate Eker
rats were dissected from unaffected tissue, homogenized, and analyzed
by Western blotting for
-catenin expression. As shown in Fig.
1A,
-catenin levels are
higher in tumor samples compared with corresponding unaffected kidney
tissue, reflecting the trend observed for cyclin D1. Thus
-catenin
appears to accumulate upon TSC2 inactivation implying that tuberin may
affect
-catenin levels.
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Fig. 1.
Regulation of
-catenin levels by tuberin. A,
homogenates from three different Eker rat renal tumors and
corresponding unaffected kidney tissue were separated by SDS-PAGE and
blotted for
-catenin and actin (loading control). B,
steady-state levels of
-catenin with increasing TSC1 and TSC2
transfection. HEK293T cells were co-transfected with Wnt-1 and
increasing amounts of vector(s) (500, 1000, and 1500 ng). Empty vector
was added to normalize total DNA transfected. Cells were harvested at
48 h after transfection, and protein levels were analyzed by
Western blotting.
-Catenin band intensities were measured using the
NIH Image processing program and normalized against the density of
corresponding
-tubulin bands. The accumulation of
-catenin
relative to the non-stimulated sample point was plotted. C,
determination of
-catenin half-life was performed as described under
"Experimental Procedures." Radiolabel intensities of
[35S]methionine-labeled
-catenin was measured,
normalized to the zero time point, and plotted against time (0, 30, 60, and 120 min). Simultaneously exposed gels were measured and compared.
The half-life of
-catenin was determined from the slope of each
graph. Specific polyclonal antibodies were used to detect hamartin
(4050) and tuberin (L3-2). Commercially available antibodies were used to detect
-catenin, actin, and
-tubulin.
-Catenin Levels--
To
investigate a possible link between tuberin and the
-catenin
signaling pathway, we analyzed the effects of tuberin expression on
-catenin steady-state levels. Endogenous
-catenin expression was
assessed in HEK293T cells 48 h after transfection with increasing concentrations of TSC1 and TSC2 or control vectors in the presence or
absence of Wnt-1 stimulation. In the absence of Wnt-1 stimulation,
-catenin levels were unchanged with or without overexpression of
hamartin and tuberin (data not shown). Upon Wnt-1 stimulation,
-catenin accumulated over a 48-h period in vector control samples (Fig. 1B, compare lanes 1-4). However,
-catenin levels were significantly reduced with overexpression of
wild-type tuberin and hamartin (Fig. 1B, compare lanes
1-7). In contrast, overexpression of a disease-causing TSC2
mutant (
Y1571H) (14) resulted in a modest
-catenin accumulation
(Fig. 1B, compare lanes 8-10). These results show an inverse relationship between
-catenin and hamartin/tuberin expression. With increasing expression of hamartin and wild-type tuberin,
-catenin levels diminished progressively (Fig.
1B, compare lanes 5-7). In contrast,
steady-state
-catenin increased modestly with the expression of
control vector (Fig. 1B, compare lanes 2-4) or
with mutant tuberin (
Y1571H) (Fig. 1B, compare
lanes 8-10).
-catenin half-life. HEK293T cells were transfected as described
above and then subjected to pulse-chase following
[35S]methionine incorporation. Lysates collected at
specific time points were treated with ConA-Sepharose to remove
cadherin-bound
-catenin leaving only the free pool of
-catenin
for immunoprecipitation. Immunoprecipitates were resolved on SDS-PAGE,
and the radiolabel intensity of [35S]methionine
incorporated in to
-catenin was measured from the gel to determine
the half-life. Without Wnt-1 stimulation,
-catenin half-life was
unchanged either with or without expression of hamartin and tuberin
(data not shown). With Wnt-1 stimulation, the half-life of
-catenin
in vector control samples was 1.7 h (Fig. 1C). This was
the same for
-catenin in samples overexpressing hamartin and mutant
tuberin (
Y1571H) (Fig. 1C). However, the half-life of
-catenin was reduced to about 1 h upon expression of hamartin and wild-type tuberin (Fig. 1C). This 41% decrease in
-catenin half-life is consistent with the steady-state data showing
an effect of the TSC proteins on
-catenin level. In both situations, modulation of
-catenin by wild-type tuberin occurred under condition of Wnt stimulation. This function is disrupted in the presence of a
disease-causing TSC2 mutation.
-Catenin
Transcriptional Activity--
To determine whether modulation of
-catenin levels by hamartin and tuberin affects its transcriptional
activity, the ability of Wnt-1 to activate a Tcf/LEF-luciferase
reporter construct (TOPFLASH) was examined in transient transfection
assays using HEK293T cells. These cells were co-transfected with
hamartin and/or tuberin constructs along with a vector for
-galactosidase to account for transfection efficiency. Parallel
assays were performed using FOPFLASH, a mutant reporter, to monitor
background activity. Upon stimulation with Wnt-1, cells with vector
control revealed a 12-fold increase in luciferase activity relative to
the non-stimulated cells (Fig. 2A, compare lanes 1 and 2). Co-expression of both wild-type tuberin and hamartin
in Wnt-1 stimulated cells significantly reduced reporter activity (Fig.
2A, lane 3), while expression of hamartin or
tuberin alone had only minor effects (Fig. 2A, lanes
5 and 6). Importantly, co-expression of the tuberin
Y1571H mutant with hamartin did not suppress Wnt-1-stimulated
TOPFLASH activity (Fig. 2A, lane 4) consistent
with the effects on
-catenin protein levels described above (Fig. 1,
B and C). Under the same conditions, hamartin and tuberin had no effects on FOPFLASH activity (Fig. 2A,
lanes 7 and 8). Our data suggest that hamartin
and tuberin, functioning as a complex, are capable of inhibiting Wnt-1
stimulated
-catenin-dependent transcriptional
activity.
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Fig. 2.
Regulation of
-catenin activity by hamartin and tuberin.
Effects of hamartin and tuberin expression on
-catenin activity
stimulated by Wnt-1 (A), CA-
-catenin (B), or
Dsh (C).
-Catenin activity was stimulated in HEK293T
cells and monitored by TOPFLASH. FOPFLASH control was also assayed
along with
-galactosidase as transfection control. Luciferase
activity from each sample was normalized and expressed as a value
relative to
-galactosidase activity. Data from each graph are
averages from at least three separate assays and are expressed as a
value relative to
-galactosidase activity in each sample. Ectopic
expression of hamartin and tuberin was detected by Western blotting
using polyclonal antibodies (4050 and L3-2, respectively). The
relative low levels of hamartin transgene expression are due to reduced
protein stability in the absence of tuberin co-expression (27) (*,
unpaired t test, p < 0.05).
-Catenin
Signaling Pathway--
To determine at what level in the
Wnt/
-catenin signaling pathway hamartin and tuberin act, we examined
the effects of TSC1 and TSC2 on TOPFLASH activity when stimulated by
different components of the Wnt pathway. The CA-
-catenin mutant with
its serine/threonine residues (Ser-33, Ser-37, Thr-41, Ser-45) replaced
with alanine residues, thus preventing its phosphorylation and
degradation (16), acts as a downstream stimulus and activates the
TOPFLASH reporter by over 6-fold in HEK293T cells (Fig. 2B,
compare lanes 1 and 2). Co-expression of hamartin
and tuberin was ineffective in reducing CA-
-catenin stimulation of
the Tcf/LEF reporter (Fig. 2B, compare lanes 2 and 3). Western blot of samples confirmed equal expression
of CA-
-catenin (data not shown). As a control, a dominant negative
mutant of Tcf-4 (
N-Tcf-4) that lacks the N-terminal
-catenin
binding domain (18) was able to inhibit CA-
-catenin activity
completely (Fig. 2B, lane 6). These results suggest that hamartin and tuberin act upstream of
-catenin.
-catenin (see Ref. 10). Transient overexpression of Dsh stimulates TOPFLASH activity by ~3-fold in
control vector transfected cells (Fig. 2C, compare
lanes 1 and 2). This activity was inhibited to
near baseline levels upon overexpression of hamartin and tuberin (Fig.
2C, lane 3). Hamartin alone did not reduce the
activity, while tuberin alone slightly decreased activity (Fig.
2C, lanes 4 and 5). Again,
N-Tcf-4
reduced activity toward unstimulated levels (Fig. 2C,
lane 6). Together, these results are consistent with tuberin
and hamartin exerting an effect on the Wnt signaling pathway at a level
between Dsh and
-catenin (i.e. the
-catenin
degradation complex).
-Catenin Degradation Complex--
The
-catenin degradation
complex is comprised of several proteins, including APC, Axin, and
GSK3
, and is responsible for the regulation of cytoplasmic
-catenin (see Ref. 10). To determine whether hamartin and tuberin
physically interact with components of the
-catenin degradation
complex, co-immunoprecipitation assays were performed in HEK293T cells
ectopically expressing hamartin and tuberin along with GSK3
.
Anti-tuberin antibodies brought down GSK3
only in samples where both
were overexpressed (Fig. 3A,
panel i, lane 4). This band was not observed in
samples where the GSK3
construct was co-expressed with vector
control (Fig. 3A, panel i, lane 2) or
in samples without the GSK3
construct (Fig. 3A,
panel i, lanes 1 and 3). As expected,
hamartin co-immunoprecipitated with tuberin in sample where both were
overexpressed (Fig. 3A, panel ii, lanes
3 and 4). Conversely, immunoprecipitation of GSK3
brought down both tuberin and hamartin only in samples where all three
were overexpressed (Fig. 3A, panels iv and
v, lane 4). The expression of ectopic proteins
was verified in cell lysates (Fig. 3A, panels
vii, viii, and ix). Compared with the level
of overexpression, the amount of interacting protein was relatively
small, suggesting that only a fraction of tuberin/hamartin and GSK3
can associate with one another.
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Fig. 3.
Co-immunoprecipitation of tuberin and
hamartin with components of the -catenin
degradation complex. A, HEK293T cells were transfected
with vectors for pcDNA3, TSC1, TSC2, or GSK3
. Cell lysates were
immunoprecipitated (IP) either with monoclonal antibody for
GSK3
or with tuberin polyclonal antibody (C20). Specific proteins
were detected by immunoblotting (IB) using antibodies toward
tuberin, hamartin, or GSK3
. B, similarly, HEK293T cells
were transfected with vectors for pcDNA3, GSK3
, c-Myc-tagged
Axin, or hamartin and tuberin. Immunoprecipitations were performed
using anti-c-Myc antibody (9E10) for c-Myc-tagged Axin or polyclonal
antibody for tuberin (L3-2). Co-immunoprecipitated protein was
detected on Western blots using specific antibodies. C,
endogenous tuberin was immunoprecipitated from HEK293T cell lysates and
analyzed by immunoblotting for GSK3
. Tuberin was immunoprecipitated
using tuberin polyclonal antibody (C20), while monoclonal antibody was
used to detect GSK3
. Blots in each panel are from the same
exposure.
that function in the
-catenin degradation complex, one would predict that other
components of the complex such as Axin would co-immunoprecipitate with
hamartin and tuberin. To test this hypothesis, hamartin and tuberin
were ectopically expressed in the presence of c-Myc-tagged Axin and
then subjected to immunoprecipitation analyses. Using anti-tuberin and
anti-c-Myc antibodies, tuberin and tagged Axin co-precipitated in
samples where they were both overexpressed (Fig. 3B,
panels i and v, lanes 5 and
6). Hamartin also co-immunoprecipitated with Axin (Fig.
3B, panel vi, lanes 5 and
6). Tuberin, hamartin, and c-Myc-tagged Axin were not
detected in vector control samples (Fig. 3B, panels
i, iii, v, and vi, lanes
1-3) or in samples without the tagged construct (Fig.
3B, panels i, iii, v, and
vi, lane 4). We conclude that the
tuberin-hamartin complex can associate with GSK3
and Axin
possibly as part of the
-catenin degradation complex.
complex, co-immunoprecipitation assays were performed in
HEK293T cells with and without Wnt stimulation. A band corresponding to
GSK3
was found to co-immunoprecipitate with tuberin but not in
preimmune serum control samples (Fig. 3C, compare
lanes 1 and 3). Furthermore, the amount of
co-immunoprecipitated GSK3
was reduced upon Wnt-1 stimulation (Fig.
3C, compare lanes 1 and 2), suggesting
that this interaction can be modulated by Wnt-1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin are elevated
upon disruption of TSC2 in renal tumors from Eker rats. Also, hamartin
and tuberin reduce Wnt stimulation of
-catenin half-life and its
downstream Tcf/LEF transcriptional activation. The results further
suggest that tuberin and hamartin associate with the GSK3
degradation complex in a Wnt-1-dependent manner. These
findings are in contrast to those of Kugoh et al. (25) who
reported a lack of change in Tcf-dependent luciferase
activities in tuberin-null cells compared with controls. However, these
experiments were conducted in the absence of Wnt stimulation.
to promote
-catenin
degradation. Upon Wnt stimulation, tuberin dissociates from this
complex resulting in stabilization of cytoplasmic
-catenin. As a
known target of
-catenin signaling, the cyclin D1 gene contains Tcf
responsive elements in its promoter and its expression is, in part,
dependent on
-catenin activity (8, 9). We postulate that in the
absence of functional tuberin or hamartin, stimulation of the
-catenin pathway will be unopposed resulting in the sustained transcriptional activation of cyclin D1. However, at this point, we
cannot exclude the influence of the PI3K/Akt/mTOR pathway on cyclin D1
regulation. There is growing evidence that there exists substantial
cross-talk between the Wnt and PI3K signaling pathways. For example,
Wnt stimulation increases Akt activity resulting in GSK3
phosphorylation and
-catenin stabilization (26). This function of
Akt is distinct from its effects on the PI3K pathway and is dependent
on the recruitment of Dsh to the GSK3
-
-catenin-Axin complex (26).
It is conceivable that upon stimulation, activated Akt phosphorylates
tuberin and disables it from the complex. Accordingly, tuberin acting
downstream of Akt may play a role in coordinating signals tranduced
through the Wnt and PI3K pathways, thus providing a mechanism for the
pleiotropic effects of the TSC1 and TSC2 genes in tuberous sclerosis.
The molecular components and their regulation by which TSC1 and TSC2
interact with the GSK3
complex remain to be identified.
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ACKNOWLEDGEMENTS |
---|
We thank David Kimelman for providing the
GSK3 construct and members of the Yeung laboratory for critical
reading and assistance in the preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA77882 and by the Tuberous Sclerosis Alliance.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.
§ Recipient of a Tuberous Sclerosis Alliance Fellowship.
Supported by postdoctoral fellowships from the Japan Science
and Technology Corporation and Uehara Memorial Foundation.
** Investigator of the Howard Hughes Medical Institute.
To whom correspondence should be addressed: Dept. of Surgery,
University of Washington, 1959 NE Pacific St., Box 356410, Seattle, WA
98195. Tel.: 206-616-6405; Fax: 206-616-6406; E-mail:
ryeung@u.washington.edu.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.C200473200
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ABBREVIATIONS |
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The abbreviations used are:
TSC, tuberous
sclerosis complex;
PI3K, phosphoinositide 3-kinase;
Tcf/LEF, T cell
factor/lymphoid enhancer factor;
APC, adenomatous polyposis coli;
GSK3, glycogen synthase kinase 3
;
Dsh, Disheveled;
PMSF, phenylmethylsulfonyl fluoride;
ConA, concanavalin A.
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REFERENCES |
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