Cardiovascular and ** Pulmonary and Critical Care
Divisions, Department of Medicine, Brigham and Women's Hospital and
the ¶ Cardiovascular Research Center and Division of Cardiology,
Massachusetts General Hospital, Harvard Medical School, Boston,
Massachusetts 02115 and the
Laboratory of Cellular and Molecular
Biology, Division of Basic Sciences, NCI, National Institutes of
Health, Bethesda, Maryland 20892
Received for publication, December 14, 2000, and in revised form, February 28, 2001
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ABSTRACT |
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Inactivation of glycogen synthase kinase 3 Wnt signaling regulates several developmental processes in both
insects and vertebrates. Aberrant Wnt signaling occurs both in
oncogenic processes (1-3) and in some cardiovascular diseases (4-6).
The interaction between secreted Wnt glycoproteins and their cognate
Frizzled receptors leads to the activation of
Dvl1 protein, which transmits
the Wnt signal to downstream effectors. By inhibiting phosphorylation
of The serine/threonine kinase Akt (protein Kinase B/related to A and
protein kinase C) is a major effector of the PI3K pathway and is
activated by many polypeptide growth factors (14-16). The recruitment
of Akt from the cytoplasm to the plasma membrane by the lipid products
of PI3K leads to Akt phosphorylation by
3-phosphoinositide-dependent protein kinases. This
phosphorylation of Akt at Thr308 and Ser473
results in its activation. Akt acts in part through its phosphorylation of GSK3 Cell Culture--
PC12, Int5 and Tni3 cells were cultured as
described (17). 293T cells were maintained in Dulbecco's modified
Eagle's medium (DMEM, Life Technologies, Inc.) supplemented with 10%
fetal bovine serum (FBS, Hyclone Laboratories), 20 mM HEPES
(pH 7.35), 1 mM sodium pyruvate, and 1 mM
non-essential amino acids (all from Life Technologies, Inc.). NIH-3T3
cells were cultured in DMEM supplemented with 10% FBS.
Plasmid Constructs and Adenoviruses--
Mouse Dvl-1, Akt-1,
Axin, and rat GSK3 Wnt-3a-conditioned Medium--
Wnt-3a- and control-conditioned
medium were prepared essentially as described (21). We verified that
Wnt-3a medium specifically up-regulated the cellular Reporter Assays--
293T cells were transfected using
LipofectAMINETM (Life Technologies, Inc.) in 6-well plates with 30 ng/well of TOPFlash or FOPFlash luciferase reporter (Upstate
Biotechnology) together with effector plasmids or empty pcDNA3
vector (amount added to keep the total DNA content at 1 µg/well) as
described in the figure legends. Cell extracts were prepared 24 h
after transfection by a detergent lysis method (Promega). Transfection
efficiencies were normalized by pCMV Western Blotting--
Cell lysates were prepared in a buffer
containing 20 mM Tris, pH 8.0, 140 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 10%
glycerol, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Immunoprecipitation and Akt Kinase Activity Assays--
Cell
lysates were precleared with protein A or G-Sepharose beads (Amersham
Pharmacia Biotech or Calbiochem, respectively) for 1 h at 4 °C,
and then incubated with the indicated antibody and protein A or
G-Sepharose beads for 3.5 h at 4 °C. The immunocomplexes were
pelleted and washed three times with cold lysis buffer. The samples
were subjected to immunoblotting with the indicated antibodies. Akt
activity was measured using an Akt kinase assay kit (New England BioLabs) with recombinant GSK3 as a substrate for
immunoprecipitated Akt.
Analysis of Free Neuronal Differentiation--
Cells were incubated in
differentiation medium (DMEM supplemented with 1% horse serum and 100 ng/ml NGF (Austral Biologicals)) or growth medium (DMEM supplemented
with 10% horse serum, 5% FBS). Medium was refreshed every 48 h.
Neurite out-growth was defined as a phase dark process with a clearly
defined growth cone that was at least 1.5 cell diameters in length.
Experiments were carried out in triplicate and repeated at least three
times, and in each experiment cells were counted by at least two
individuals who were blinded to the groups.
Statistics--
Where indicated, comparisons between groups were
made by factorial analysis of variance followed by Scheffe's test
(Fig. 1B) or by unpaired Student's t test (Fig.
5C) when appropriate. Significance was accepted at
p < 0.05.
Wnt Signaling Induces Phosphorylation and Activation of
Akt--
We first explored the possibility that Wnt signaling
stimulates Akt activity. In a Wnt-1-overexpressing PC12 cell line
(Int5) (17), but not in either the parental PC12 or in an
antisense Wnt-1-expressing PC12 cell line (Tni3), Akt was
phosphorylated at both Thr308 and Ser473 (Fig.
1A). The phosphorylation
of both residues is necessary and sufficient for full Akt activation
(16). Accordingly, Akt activity, measured by its ability to
phosphorylate recombinant GSK3 Dominant-negative Akt Inhibits Wnt Signaling Pathway--
To
determine whether activated Akt is required for the canonical Wnt
signaling, we used a dominant-negative form of Akt (AAA-Akt) (26). Int5
cells infected with an adenovirus expressing AAA-Akt (Ad.AAA-Akt)
showed reductions in phosphorylated GSK3 Akt Up-regulates Free
Recalling that a dominant-negative form of Akt inhibited Wnt signaling
induced by Dvl, we postulated that Akt might act in conjunction with
Dvl to promote Wnt signaling. Overexpression of Akt in 293T cells
produced a dose-dependent increase in Akt activity (Fig.
3B). As observed with PC12 cell derivatives (Fig. 3A), Akt activity was not sufficient to induce Wnt signaling
in the reporter assay. However, Akt enhanced TOPFlash activity that was
stimulated by Dvl-1 (Fig. 3B) but not by S37A Akt Interacts with the Axin Complex in the Presence of Dvl--
To
delineate the mechanism of Akt and Dvl signal transduction, we
investigated their interaction with the Axin complex. Others have shown
that activated Dvl binds Axin and recruits GSK3
Dvl protein family members contain three highly conserved domains: an
N-terminal DIX (Dishevelled-Axin) domain, a
central PDZ
(PSD-95-Discs-large-ZO-1) domain,
and a C-terminal DEP
(Dishevelled-EGL-10-Pleckstrin) domain (13, 27). Although the roles of each domain have not been
elucidated completely, either the DIX or PDZ domain is sufficient for
Axin binding (11). To confirm that Dvl binding to Axin is necessary for
Akt involvement in the Axin complex, Dvl mutants were generated.
Consistent with previous reports (11, 28), C-Dvl-(369-695), which
lacks both the DIX and PDZ domains, did not bind to Axin, whereas
N-Dvl-(1-497), which lacks the C-terminal region adjacent to the DEP
domain, bound to Axin (Fig. 4C). Full-length Dvl and
N-Dvl-(1-497), but not C-Dvl-(369-695), induced Akt interaction with
the Axin complex (Fig. 4C). Although the magnitude of Akt association with the Axin complex induced by N-Dvl (1) was less than that seen with full-length Dvl-1, this difference was consistent with the relative amounts of the recombinantly expressed Dvl proteins (Fig. 4C). C-Dvl-(369-695) and N-Dvl-(1-497) were
expressed at similar levels (Fig. 4C), indicating that the
absence of Akt in the Axin complex is not due to reduced
C-Dvl-(369-695) expression levels. These results suggest that the
N-terminal region of Dvl, which can bind to Axin, is required to induce
Akt association with the Axin complex.
Akt did not interact directly with Axin or Dvl (Fig. 4B and
data not shown). However, Akt binding to the Axin complex may be
mediated by GSK3 Dominant-negative Akt Restores Neuronal Differentiation of
Int5 Cells--
Wnt signaling frequently is responsible for changes in
cell morphology and differentiation (1-3). For instance, the
Wnt-overexpressing Int5 cells have a flat shape and do not
differentiate with NGF treatment, whereas the parental PC12 cells are
round and differentiate readily in response to NGF (17). sFRP-1-treated
Int5 cells differentiated in response to NGF (Fig.
5A); this implies that the
phenotype of Int5 cells depends on Wnt signaling. To test the impact of Akt on this Wnt-dependent cellular phenotype, we infected
Int5 cells with either a dominant-negative Akt (Ad.AAA-Akt) or a
control (Ad. Here we show that Wnt signaling stimulates Akt and that
activated Akt, in association with Dvl, enhances the
phosphorylation of GSK3 Past reports have suggested that Akt is not involved in Wnt signaling
(30, 32). Similar to these accounts, we observed that Akt by itself was
not sufficient to mimic a Wnt signal. Also consistent with this
literature, constitutively active membrane-bound forms of Akt (14) did
not enhance TOPFlash activity, even in the presence of Dvl
overexpression (data not shown). Presumably, membrane-anchored Akt is
unable to interact with the Axin complex in the cytosol and
consequently cannot regulate GSK3 It is still unclear how Akt is activated by a prolonged Wnt stimulus.
Recently, integrin-linked kinase was reported to interact with
activated Dvl (33), to phosphorylate Akt (34), and to induce Wnt target
genes (34, 35). Future studies will determine whether Dvl activates Akt
through the integrin-linked kinase pathway.
(GSK3
) and the resulting stabilization of free
-catenin are
critical steps in the activation of Wnt target genes. While Akt
regulates GSK3
/
in the phosphatidylinositide 3-OH kinase
signaling pathway, its role in Wnt signaling is unknown. Here we report
that expression of Wnt or Dishevelled (Dvl) increased Akt activity.
Activated Akt bound to the Axin-GSK3
complex in the presence of Dvl,
phosphorylated GSK3
and increased free
-catenin levels.
Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt
decreased free
-catenin and derepressed nerve growth factor-induced
differentiation. Therefore, Akt acts in association with Dvl as an
important regulator of the Wnt signaling pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin by GSK3
, activated Dvl prevents the ubiquitination
and subsequent proteosomal degradation of
-catenin. The resulting
accumulation of free
-catenin enhances its interaction with
transcription factors of the lymphoid enhancer factor-T cell factor
(LEF/TCF) family and induces the transcription of target genes such as
cyclin D1 and c-myc (1-3). Two mechanisms have been
advanced to account for the inhibition of GSK3
by Dvl: 1) the
catalytic activity of GSK3
is blocked by serine phosphorylation (1,
7-10); 2) the interaction of GSK3
and
-catenin is disrupted by a
conformation change of the Axin-
-catenin-GSK3
-adenomatosis polyposis coli protein complex (Axin complex) (2, 11-13). However, in
the first instance, the process whereby Dvl stimulates serine phosphorylation has not been elucidated; nor is it clear whether these
two mechanisms function in a concerted manner to promote Wnt signaling.
/
, which in turn regulates cell metabolism. Recognizing the pivotal role of GSK3
in Wnt signaling, we tested the hypothesis that Akt functions in this pathway to control GSK3
activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were prepared by reverse transcriptase-polymerase
chain reaction (PCR) using the high fidelity thermostable DNA
polymerase PfuTurbo (Stratagene). Dvl-1 was subcloned in
frame into pcDNA3.1(
)/Myc-His (Invitrogen) to introduce Myc-His
epitope tags at the C terminus. GSK3
was subcloned into
pcDNA3.1(
) (Invitrogen). Axin and Akt-1 were subcloned either in
frame or with a stop codon into pFLAG-CMVTM-5 (Sigma) to yield protein
with or without FLAG tag at the C terminus, respectively. Deletion
mutants of Dvl-1, N-Dvl-(1-497), and C-Dvl-(369-695) were
generated by PCR and subcloned in frame into pcDNA3.1(
)/Myc-His. The C-terminal (
-catenin binding) region of E-cadherin (18) was
cloned by reverse transcriptase-PCR in frame into pGEX-5X-1 (Amersham
Pharmacia Biotech) for GST-E-cadherin pull-down assay. Four recombinant
first-generation type 5 adenoviruses were used in these studies.
Ad.EGFP has been described previously (19). Ad.AAA-Akt and
Ad.
-gal.EGFP, which also express EGFP, were constructed using the
method described previously (20). The wild type Akt-expressing adenovirus was constructed using Akt-HA (kindly provided by T. Franke)
in pAdRSV-4 (kindly provided by D. Dichek) and obtaining homologous
recombinants through co-transfection with pJM17 (Microbix Biosystems)
in 293 cells.
-catenin levels
in L cells.
-gal reporter activity
(CLONTECH). Each construct was transfected at least
three times in triplicate. Reporter activities (mean ± S.D.) are
presented as the -fold increase of TOPFlash activity from the cells
transfected with empty vector.
-glycerophosphate, 1 mM sodium
vanadate, dithiothreitol, and protease inhibitors (CompleteTM-without
EDTA, Roche). Cell extracts were clarified by centrifugation and
subjected to SDS-polyacrylamide gel electrophoresis followed by
immunoblotting with indicated antibodies.
-Catenin Levels--
Free
-catenin levels
were detected by GST-E-cadherin pull-down assay as described (18).
GST-E-cadherin fusion protein was expressed in bacteria and purified by
binding to glutathione-Sepharose beads (Amersham Pharmacia Biotech). 1 mg of cell lysate was incubated with GST-E-cadherin-beads for 1 h
at 4 °C. The beads were collected by centrifugation and washed, and
proteins were dissolved in Laemmli buffer and subjected to
SDS-polyacrylamide gel electrophoresis followed by immunoblotting with
anti-
-catenin antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, was much higher in Int5 cells than
in control cells (Fig. 1A). Furthermore, phosphorylation of
GSK3
at Ser9, which results in its inactivation (10),
was higher in Int5 than in control cells (Fig. 1, A and
B). Phosphorylation of Akt and GSK3
was inhibited by the
Wnt antagonist-secreted Frizzled-related protein-1 (sFRP-1) (Fig.
1C) accompanied by a reduction in free
-catenin as we
showed previously (22). These results indicated that phosphorylation of
Akt and GSK3
, and free
-catenin levels in Int5 cells, were
dependent on secreted Wnt-1 protein. To extend these observations to
other cells, we also analyzed Akt phosphorylation in NIH-3T3 cells
transiently transfected with Wnt-1, Dvl-1, and a
-catenin mutant
(S37A
-catenin). Previous studies had shown that overexpression of
these gene products cause an increase in free
-catenin, indicative
of Wnt signaling (2, 8, 23-25). Transient expression of Wnt-1 or
Dvl-1, but not S37A
-catenin, increased Akt phosphorylation (Fig.
1D). These results indicated that Akt activation is a
consequence of Wnt signaling that occurs downstream of Dvl.
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Fig. 1.
Wnt signaling induces phosphorylation and
activation of Akt. A, Akt is phosphorylated and
activated in Int5 cells. 100 µg of cell lysate from PC12, Tni3, and
Int5 cells were probed with anti-phospho-Akt (P-Thr308-Akt),
anti-phospho-Akt (P-Ser473-Akt), anti-Akt,
anti-phospho-GSK3 (Ser9; P-GSK3
) (New
England BioLabs), and anti-GSK3
antibodies (Transduction
Laboratories). B, GSK3
phosphorylation levels are
significantly higher in Int5 cells than in PC12 or Tni3 cells.
The phospho-GSK3
(Ser9) signal from Western blots
was normalized to the total GSK3
signal using NIH Image software;
the results are expressed in arbitrary units. Data are presented as the
mean ± S.D. from 3 experiments. *, significant difference
compared with PC12 or Tni3 cells (p < 0.05).
C, sFRP-1 suppresses both Wnt signaling and Akt
phosphorylation. Recombinant sFRP-1 protein was prepared as described
(22). Int5 cells were incubated with the indicated concentrations of
sFRP-1 in DMEM with 1% horse serum for 12 h. Cell lysates were
analyzed by immunoblotting with anti-phospho-Akt (Ser473;
P-Akt), anti-Akt, anti-phospho-GSK3
(Ser9;
P-GSK3
), and anti-
-catenin antibodies (Transduction
Laboratories). D, Wnt-1 or Dvl-1 expression causes Akt
phosphorylation. NIH-3T3 cells were transfected with the expression
plasmids pcDNA3, HA-tagged S37A
-catenin (S37A-HA),
Myc-tagged Dvl-1 (Dvl-Myc), or HA-tagged Wnt-1
(Wnt-HA, Upstate Biotechnology) using LipofectAMINETM (Life
Technologies, Inc.). Immunoblots were probed with anti-phospho-Akt
(Ser473; P-Akt) and anti-Akt antibodies.
Anti-Myc (clone 9E10) and anti-HA (clone 12CA5) antibodies were
used to confirm expression.
and free
-catenin
protein levels (Fig. 2A).
These changes did not occur in cells infected with control virus
(Ad.
-gal.EGFP). In transient transfection assays using 293T cells,
Wnt-1, Dvl-1, and S37A
-catenin each enhanced TCF-binding site
reporter gene activity (TOPFlash) (Fig. 2B). AAA-Akt
suppressed TOPFlash activity induced by Wnt-1 or Dvl-1 but did not
affect the S37A
-catenin induced activity (Fig. 2B). The
activity of the mutated TCF-binding site reporter (FOPFlash) was not
affected in these experiments (data not shown and Fig.
3B). These results implied
that Akt is involved in Wnt signaling, functioning at a point in the
pathway between Dvl and
-catenin.
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Fig. 2.
Dominant-negative Akt inhibits Wnt
signaling. A, dominant-negative Akt reduces both
phosphorylated GSK3 and free
-catenin levels in Int5 cells. Int5
cells were infected with the control adenovirus Ad.
-gal.EGFP or
Ad.AAA-Akt at a multiplicity of infection (MOI) of 50 and harvested
after 48 h. Proteins were analyzed as described for Fig. 1.
B, dominant-negative Akt suppresses the expression of Wnt
target gene. 293T cells were transfected with TOPFlash luciferase
reporter together with AAA-Akt, Wnt-1 (330 ng), Dvl-1 (170 ng), S37A
-catenin (S37A) (8 ng), or empty pcDNA3 vector.
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Fig. 3.
Akt enhances Wnt signaling in the presence of
Dvl. A, Akt expression increases free -catenin
levels in Int5 cells but not in Tni3 cells. Tni3 and Int5 cells were
infected with Ad.EGFP or Ad.Akt, and cells were harvested after 3 days. The cell lysates were probed with anti-Akt antibody.
B, Akt increases the expression of a Wnt target gene in the
presence of Dvl. 293T cells were transfected with the TOPFlash or the
FOPFlash reporter together with plasmids of Akt and Dvl-1 (170 ng/well)
or pcDNA3 as indicated. TOPFlash (closed bars) or
FOPFlash (open bars) reporter activities are presented as
the -fold increase of TOPFlash activity from the cells transfected with
empty vector.
-Catenin Levels and Wnt Target Gene
Expression in the Presence of Dvl--
To test whether Akt activity is
sufficient to stimulate an increase in free
-catenin, we infected
Int5 and Tni3 cells with either an adenovirus-expressing wild type Akt
(Ad.Akt) or a control adenovirus (Ad.EGFP). Although the amount and
activity of Akt protein were similarly increased in both lines infected
with Ad.Akt, a rise in free
-catenin levels was seen only in the
Int5 cells (Fig. 3A). These results demonstrated that Akt
activity was not sufficient to up-regulate free
-catenin but that it
could augment the activity of a Wnt stimulus.
-catenin
(data not shown). Neither Akt nor Dvl-1 affected control FOPFlash
activity (Fig. 3B). These results suggested that Dvl is
required for Akt to up-regulate Wnt-dependent gene expression.
-binding protein to
the Axin complex. This binding induces a conformational change in the
complex, resulting in the inhibition of GSK3
phosphorylation of
-catenin (2, 12, 13). We first investigated the association between
endogenous Akt and Axin. Similar to Wnt-1, Wnt-3a-conditioned medium
induces the canonical Wnt signaling pathway (2, 9, 25). Axin was
co-immunoprecipitated with Akt in 293T cells treated for 24 h with
Wnt-3a-conditioned medium but not control medium (Fig.
4A). Thus, the association of
Akt with Axin requires a Wnt stimulus. Consistent with results using
Wnt-1-overexpressing PC12 cells (Fig. 1A), these
Wnt-3a-treated 293T cells had increased phosphorylation levels of Akt
and GSK3
(Fig. 4A, Cell lysate). We also
examined total GSK3
in cell lysates and GSK3
in the Axin complex,
the latter defined as the GSK3
protein co-immunoprecipitated with
Axin. In transfected 293T cells, Dvl expression stimulated the
phosphorylation of both total GSK3
(Fig. 4B,
Cell lysate) and GSK3
in the Axin complex (Fig.
4B, IP). Akt expression alone did not affect the
phosphorylation of GSK3
in the Axin complex (Fig. 4B,
IP), although it did stimulate the phosphorylation of total
GSK3
(Fig. 4B, Cell lysate). However, in the
presence of Dvl, Akt increased the amount of phosphorylated GSK3
in
the Axin complex (Fig. 4B, IP). These results
suggested that Dvl was necessary for Akt-dependent
phosphorylation of GSK3
in the Axin complex. Furthermore,
phosphorylated Akt was included in the Axin complex only in the
presence of Dvl (Fig. 4B).
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Fig. 4.
Akt interacts with the Axin complex in the
presence of Dvl. A, endogenous Akt and Axin associate
in Wnt-3a-treated cells. 293T cells were cultured in Wnt-3a-conditioned
medium or control medium for 24 h, which was refreshed every
6 h. Cell lysates (5 mg) were immunoprecipitated with an anti-Akt
antibody (1:100). The immunoprecipitates and cell lysates were probed
with anti-Axin, anti-phospho-Akt (Ser473;
P-Akt), anti-Akt, anti-phospho-GSK3 (Ser9;
P-GSK3
), and anti-GSK3
antibodies. IB,
immunoblot; IP, immunoprecipitation. B,
Akt associates with the Axin complex only in the presence of Dvl-1.
293T cells were transfected with Axin-FLAG, Dvl-Myc, Akt, or pcDNA3
as indicated and harvested after 24 h. For immunoprecipitation,
the cell lysates (1 mg protein) were incubated with 5 µg of
anti-FLAGTM antibody (Sigma). The immunoprecipitates and cell lysates
were probed with anti-phospho-Akt (Ser473;
P-Akt), anti-Akt, anti-phospho-GSK3
(Ser9;
P-GSK3
), anti-GSK3
, anti-FLAG, or anti-Myc antibodies.
C, a Dvl mutant, which does not bind to Axin, cannot induce
Akt association with the Axin complex. 293T cells were transfected with
Axin-FLAG, Akt, and Dvl-Myc, C-Dvl-(369-695)-Myc, or
N-Dvl-(1-497)-Myc as indicated. Immunoprecipitation and immunoblotting
were performed as described above. D, Akt levels are
correlated with GSK3
levels in the Axin complex. 293T cells were
transfected with Dvl-Myc and Akt and with GSK3
and/or Axin-FLAG as
indicated. Immunoprecipitation and immunoblotting were performed as
described above.
, judging from the correlation of Akt and GSK3
levels in the complex (Fig. 4D). As shown in Fig.
4D, the amount of GSK3
and Akt protein in the Axin
complex was higher in GSK3
-overexpressing cells than in control
cells. Taken together, our results indicate that Dvl binding to the
Axin complex enables Akt to associate with and to phosphorylate GSK3
in the complex.
-gal.EGFP) adenovirus. In contrast to the cells treated
with control virus, Int5 cells infected with Ad.AAA-Akt had a more differentiated appearance with extensive neurite outgrowth in response
to NGF (Fig. 5B and C). Neuronal differentiation
of Int5 cells was confirmed by changes in peripherin levels. Consistent with a previous report (29), in response to NGF, Wnt-1-overexpressing PC12 cells (Int5) did not up-regulate peripherin protein expression, whereas parental PC12 cells did (Fig. 5D). However,
Ad.AAA-Akt-infected Int5 cells exhibited increased levels of peripherin
when cultured in differentiation medium (Fig. 5D). Increased
levels of peripherin are the result of Int5 cell differentiation but
not of a direct effect of AAA-Akt, because peripherin levels of PC12
and Int5 cells in growth medium were not enhanced by Ad.AAA-Akt
infection (Fig. 5D, growth). These results demonstrated that
inhibition of Akt activity reversed the Wnt-dependent
phenotype of Int5 cells, reinforcing the evidence that Akt has an
important role in the Wnt signaling pathway.
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Fig. 5.
Dominant-negative Akt restores
neuronal differentiation of Int5 cells. A, Int5 cells
were incubated in differentiation medium including vehicle (control) or
sFRP-1 (15 µg/ml) for 4 days. Cells were observed using phase
contrast microscopy (original magnification, 200×). B, Int5
cells were infected with control Ad. -gal.EGFP (left
panels) or Ad.AAA-Akt (right panels) at MOI 50 for 2 , and then were maintained for 3 days in the differentiation medium.
Cells were observed using phase contrast (upper panels) and
fluorescence microscopy (lower panels) (original
magnification, 400×). Cells infected by the adenovirus were identified
by GFP expression. C, The number of neurite-bearing cells
compared with total cells is indicated as % differentiation.
Data are presented as the mean ± S.D. *, p < 0.001 versus Ad.
-gal.EGFP. D, PC12 and Int5
cells were infected with control Ad.
-gal.EGFP or Ad.AAA-Akt, and
cells were maintained in growth medium or differentiation medium for 3 days. Western blots were probed with a peripherin antibody. To confirm
equal protein loading, the same blot was also probed with an
-tubulin antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in the Axin complex. The concerted
action of Dvl and Akt is a distinctive feature of Wnt signaling, which
targets phosphorylation of the GSK3
pool that is directly
responsible for phosphorylation of
-catenin and its subsequent rapid
degradation. This finding explains why other factors, such as
insulin and epidermal growth factor, that stimulate Akt through the
PI3K pathway but do not activate Dvl fail to cause an increase in
cytosolic
-catenin (30, 31). We believe that Dvl interaction with
Axin induces a conformational change, allowing Axin-bound GSK3
to be
phosphorylated by Akt. This phosphorylated GSK3
cannot modify
-catenin efficiently, which consequently accumulates in the cytosol
and translocates to the nucleus where it combines with LEF/TCF family
members to up-regulate Wnt-dependent gene expression.
activity in this critical pool. In
contrast to these constitutively active forms, wild type Akt is
activated at the plasma membrane and then translocates to the cellular
interior (15). Previous studies did not detect Akt activation and
resulting GSK3
phosphorylation at Ser9 when cells were
treated with Wnt/Wg-conditioned medium for short time periods, whereas
free
-catenin was accumulated immediately by this treatment (8, 31).
In contrast, we documented Akt activation and involvement in Wnt
signaling using models characterized by either prolonged or
constitutive Wnt stimulation. Our data may suggest that the
contribution of Akt is to sustain or enhance, but not initiate, the
signaling process.
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ACKNOWLEDGEMENTS |
---|
We thank M. E. Greenberg and A. E. West for PC12 cells, G. M. Shackleford for Int5 and Tni3 cells, H. Band for 293T cells, J. R. Woodgett for the AAA-Akt expression
plasmid, S. W. Byers for the S37A -catenin expression plasmid,
K. Willert and R. Nusse for the anti-Axin antibody, and S. Takada for
the pPGKWnt-3aneo expression plasmid.
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FOOTNOTES |
---|
* This study was supported in part by National Institutes of Health Grants HL57977 (to S.-F. Y.) and HL60788 and GM53249 (to M. A. P.).
This manuscript is dedicated to the memories of Arthur Mu-En Lee and Edgar Haber.
To whom correspondence should be addressed: Pulmonary Div.,
Dept. of Medicine, Brigham and Women's Hospital, 75 Francis St., Thorn
Bldg., Rm. TH1133, Boston, MA 02115. Tel.: 617-732-6809; Fax:
617-582-6148; E-mail: mperrella@rics.bwh.harvard.edu.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.C000880200
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ABBREVIATIONS |
---|
The abbreviations used are:
Dvl, Dishevelled;
GSK3, glycogen synthase kinase 3
;
LEF, lymphoid enhancer factor;
TCF, T cell factor;
PI3K, phosphatidylinositide 3-OH kinase;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum;
PCR, polymerase chain reaction;
GST, glutathione S-transferase;
EGFP, enhanced green fluorescent protein;
NGF, nerve growth factor;
sFRP-1, Frizzled-related protein-1.
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REFERENCES |
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