From the Department of Cell Biology, Georgetown
University School of Medicine and Lombardi Cancer Research Center,
Washington, D. C. 20007 and § Onyx Pharmaceuticals,
Richmond, California 94806
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ABSTRACT |
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The tumor suppressor function of the adenomatous
polyposis coli protein (APC) depends, in part, on its ability to bind
and regulate the multifunctional protein, Mutations in the tumor suppressor adenomatous polyposis coli
(APC)1 gene are responsible
for tumors that arise in both familial adenomatous polyposis and
sporadic colon cancers (1-7). APC mutations are almost
always truncating, giving rise to proteins lacking C termini (6, 8, 9).
Efforts to understand how these mutations contribute to cancer have
focused on the ability of APC to bind and subsequently down-regulate
the cytoplasmic levels of The mechanism of APC-mediated APC-mediated tumorigenesis might depend, in part, on its ability to
regulate Reagents, Antibodies, and Cells--
ALLN, ALLM, lactacystin- Transfections and LEF-Luciferase Reporter Assays--
Cells were
seeded in 12-well plates at 1 × 105 cells/well. The
following day cells were transiently transfected with 1 µg of APC
constructs and 0.4 µg of the LEF reporter, pTOPFLASH (optimal motif),
or pFOPFLASH (mutant motif) (31), and 0.008 µg of
pCMV-Renilla luciferase (Promega) per well, using
LipofectAMINE-Plus reagent according to the manufacturer's
instructions (Life Technologies, Inc.) for 5 h. In experiments
designed to monitor the effect of APC on
Cells were treated with indicated levels of the inhibitors for 12-24
h. Luciferase activity was monitored using the dual luciferase assay
system (Promega). The experimental LEF-luciferase reporter activity was
controlled for transfection efficiency and potential toxicity of
treatments using the constitutively expressed pCMV-Renilla luciferase. The specificity of APC-mediated effects on LEF reporters was confirmed using pFOPFLASH, which harbors mutated LEF binding sites
(31), and an unrelated AP-1 reporter (32).
Immunological Procedures--
Double immunofluorescent staining
for APC and APC-mediated Down-regulation of APC Down-regulates WT The Bisindoylmaleimide-type PKC Inhibitor GF-109203X Decreases the
Ability of APC to Down-regulate LEF Signaling in a
Dose-dependent Manner--
PKC activity is required for
Wnt-1 growth factor signaling to inhibit GSK-3
The bisindoylmaleimide-type PKC inhibitor GF-109203X prevents
Lithium (Li+) Does Not Inhibit the Ability of
APC to Down-regulate
We tested the hypothesis that Li+ can inhibit the ability
of APC to down-regulate
Our observations suggest that one function of APC is to down-regulate
-catenin.
-Catenin
binds the high mobility group box transcription factors, lymphocyte enhancer-binding factor (LEF) and T-cell factor, to directly regulate gene transcription. Using LEF reporter assays we find that
APC-mediated down-regulation of
-catenin-LEF signaling is
reversed by proteasomal inhibitors in a dose-dependent
manner. APC down-regulates signaling induced by wild type
-catenin
but not by the non-ubiquitinatable S37A mutant,
-catenin.
Bisindoylmaleimide-type protein kinase C inhibitors, which prevent
-catenin ubiquitination, decrease the ability of APC to
down-regulate
-catenin-LEF signaling. All these effects on LEF
signaling are paralleled by changes in
-catenin protein levels.
Lithium, an inhibitor of glycogen synthase kinase-3
, does not alter
the ability of APC to down-regulate
-catenin protein and
-catenin-LEF signaling in the colon cancer cells that were tested.
These results point to a role for
-catenin ubiquitination, proteasomal degradation, and potentially a serine kinase other than
glycogen synthase kinase-3
in the tumor-suppressive actions of
APC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-catenin (10-13).
-Catenin is a multifunctional protein that participates in
cadherin-mediated cell-cell adhesion and in transduction of the Wnt
growth factor signal that regulates development (14, 15). Activation of
the Wnt growth factor signaling cascade results in the inhibition of
the serine/threonine kinase, GSK-3
, and in response,
-catenin
accumulates in the cytoplasm (16-18). At elevated cytoplasmic levels,
-catenin translocates to the nucleus, interacts with the high
mobility group box transcriptional activator lymphocyte
enhancer-binding factor (LEF)/T-cell factor, and directly regulates
gene expression (19-22). Mutations that stabilize
-catenin protein
are likely to be oncogenic, although this has not been proven directly
(23).
-catenin regulation is unknown.
Recently,
-catenin was shown to be regulated at the level of protein
stability via proteasomal degradation (24, 25). Proteins targeted for
degradation by the ubiquitin-proteasome system are first tagged with
multiple copies of the small protein ubiquitin by highly regulated
ubiquitination machinery (27). Polyubiquitinated proteins are
recognized and rapidly degraded by the proteasome, a large multisubunit
proteolytic complex. Proteasomal degradation plays a critical role in
the rapid elimination of many important regulatory proteins,
e.g. cyclins and transcriptional activators like
NF
B-I
B (28). Proteins regulated via proteasomal degradation can
be specifically studied using the well characterized proteasome-specific peptidyl-aldehyde inhibitors (29, 30).
-catenin signaling (26). In this report, we show that the
ubiquitin-proteasome pathway and the activity of a serine kinase other
than GSK-3
modulate APC-mediated regulation of
-catenin-LEF signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
lactone, and MG-132 were purchased from Calbiochem. GF-109203X was
purchased from Roche Molecular Biochemicals. Ro31-8220 was a gift from
Dr. Robert Glazer. The monoclonal anti-
-catenin antibody (Clone 14)
and the anti-FLAGTM antibody were purchased from
Transduction Laboratories, Lexington, KY and Eastman Kodak Co.,
respectively. Affinity-purified rabbit polyclonal anti-APC2 and
anti-APC3 antibodies (12) were generously provided by Dr. Paul Polakis
(Onyx Pharmaceuticals). Affinity-purified fluorescein
isothiocyanate-conjugated goat anti-rabbit and Texas Red-conjugated
goat anti-mouse antibodies were purchased from Kirkegaard and Perry
Laboratories. The SW480 and CACO-2 colon cancer cell lines were
acquired from the ATCC and maintained in Dulbecco's modified Eagle's
medium with 5% fetal bovine serum and 1% penicillin/streptomycin.
-catenin protein, 0.3 µg
of FLAG-tagged WT or S37A
-catenin (25) was cotransfected with 0.6 µg of empty vector or APC constructs. This approach facilitated
analysis of only the transfected cells, using anti-FLAG antibodies.
-catenin was performed according to Munemitsu et
al. (11, 40). In experiments where FLAG-tagged
-catenin was
cotransfected with APC, anti-FLAGTM antibodies (Kodak) were
used to detect the exogenous
-catenin.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-Catenin-LEF Signaling Is
Reversed by Proteasomal Inhibitors--
In the SW480 colon cancer cell
line, which produces only a mutant APC protein containing amino acids
1-1337 of the complete 2843-amino acid sequence, overexpression of WT
APC or deletion construct APC 25 (amino acids 1342-2075), but not APC
3 (amino acids 2130-2843) (Fig.
1A), can effect a
posttranslational down-regulation of
-catenin (11, 26). We tested
the hypothesis that APC effects the down-regulation of
-catenin-LEF
signaling by targeting
-catenin for proteasomal degradation. SW480
cells were transiently transfected with various APC deletion constructs
(Fig. 1A) and treated with proteasomal inhibitors, and
-catenin-LEF signaling was assayed using LEF reporters (31). Fig.
1B shows that the APC-mediated down-regulation of
-catenin-LEF signaling is reversed by a panel of proteasomal
inhibitors including ALLN, lactacystin-
lactone, and MG-132, but not
Me2SO (vehicle) or ALLM (calpain inhibitor II), that
effectively inhibits calpain proteases but has a 100-fold lower potency
as a proteasomal inhibitor. The specificity of APC-mediated effects on
LEF reporters was confirmed using pFOPFLASH, which harbors mutated LEF
binding sites, and an unrelated AP-1 reporter, neither of which was
influenced by APC (31, 32). The proteasomal inhibitor ALLN reverses the
APC- mediated down-regulation of
-catenin-LEF signaling in a
dose-dependent manner (Fig. 1C). The effects of APC 25 can be completely reversed by the proteasomal inhibitor ALLN,
and the effects of WT APC can be restored to 50-60% of control values. However, the full-length WT APC construct, and not the APC 25 deletion construct, was used for all immunostaining experiments because
it was more physiologically relevant (incorporating all the functional
domains). SW480 cells were transfected with empty vector or WT APC and
were treated with Me2SO (vehicle) or the proteasomal
inhibitors ALLN or lactacystin-
lactone. Double immunofluorescent staining for APC (Fig. 2, A,
C, and E) and
-catenin (Fig. 2, B,
D, and F) shows that the APC induced reduction in
-catenin protein (Fig. 2, A and B) is reversed
by proteasomal inhibitors ALLN (Fig. 2, C and D)
and lactacystin-
lactone (Fig. 2,E and F).
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Fig. 1.
A, the structure of WT APC and APC
deletion constructs (26); B, APC-mediated down-regulation of
-catenin- LEF signaling is reversed by proteasomal inhibitors. SW480
cells were transiently transfected with various APC constructs, using
LipofectAMINE-Plus reagent (Life Technologies, Inc.). 12 h
posttransfection, the cells were treated with proteasomal inhibitors
ALLN, lactacystin-
lactone, and MG-132 or with Me2SO
(DMSO, vehicle) and ALLM (calpain inhibitor II) for 12 h.
-Catenin-LEF signaling was assayed using the LEF reporters
pTOPFLASH (and pFOPFLASH; data not shown) (31). Raw data were
normalized for transfection efficiency and potential toxicity of
treatments, using pCMV-Renilla luciferase and the dual
luciferase assay system (Promega). The experiment was repeated at least
three times, with each treatment repeated in triplicate. Error
bars represent S.D. C, APC-mediated down-regulation of
-catenin-LEF signaling is reversed by the proteasomal inhibitor,
ALLN, in a dose-dependent manner. The transfections were
performed as described in B and were followed by treatment
with the various doses (µM) of the proteasomal inhibitor,
ALLN. a.a., amino acid(s); DLG, Discs Large
protein.
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Fig. 2.
APC-mediated down-regulation of
-catenin protein is reversed by proteasomal
inhibitors. SW480 cells were transfected with WT APC and treated
with Me2SO (DMSO, A and
B), 10 µM ALLN (C and
D), or 5 µM lactacystin-
lactone
(E and F). Double immunofluorescent staining for
APC (A, C, and E) and
-catenin
(B, D, and F) was performed according
to Munemitsu et al. (11, 40).
-Catenin but Not the Non-ubiquitinatable
S37A Mutant Form of
-Catenin-induced LEF Signaling--
Mutation of
a single serine residue (S37A) within the ubiquitination-targeting
sequence prevents
-catenin ubiquitination (25). Serine mutations in
the ubiquitin-targeting sequence of
-catenin occur in a number of
different cancers (33-38). At least one of these, S37A, is a
stabilizing mutation that renders
-catenin resistant to
ubiquitination (25). If indeed APC regulates
-catenin-LEF signaling
by targeting
-catenin for proteasomal degradation, then it should
not be able to down-regulate the non-ubiquitinatable S37A mutant
-catenin protein or the LEF signaling induced by this stable form of
-catenin. To test this hypothesis, vector, FLAG-tagged WT, or S37A
mutant
-catenin constructs were cotransfected with vector or WT APC
and the LEF reporters into SW480 cells.
-Catenin-LEF signaling was
monitored by assaying LEF reporter activity. Overexpression of both WT
and S37A mutant forms of
-catenin increased the basal LEF reporter
activity by about 30%, even against the background of high levels of
endogenous
-catenin and
-catenin-LEF signaling in the SW480
cells. S37A
-catenin is more stable than WT
-catenin (in cells
that actively degrade
-catenin, e.g. SKBR3 cells), but
both forms increased LEF signaling by comparable levels in SW480 cells
(which lack the ability to degrade
-catenin). Fig.
3 shows that APC down-regulates LEF
signaling induced by WT
-catenin but not by the S37A mutant
-catenin. The ability of APC to down-regulate the cotransfected
FLAG-tagged WT
-catenin and the S37A
-catenin protein levels was
examined by double immunofluorescent staining using anti-APC antibodies
and anti-FLAG antibodies (Kodak) (40). By double immunofluorescent
staining for both the FLAG epitope and APC, we were able to monitor
effects of APC specifically on the coexpressed forms of
-catenin.
Fig. 4A (anti-APC) and Fig.
4B (anti-FLAG) show that WT APC effectively down-regulates WT
-catenin. Fig. 4C (anti-FLAG) shows that in concurrent
transfections with empty vector and FLAG-tagged WT
-catenin, the
FLAG-tagged WT
-catenin is expressed and the anti-FLAG antibody
efficiently detects it. Fig. 4, D and E shows
that APC does not down-regulate the S37A mutant
-catenin protein.
These findings complement the observations of Munemitsu et
al. (41) and Li et al. (42) that APC associates with
but does not down-regulate
-catenin with an N-terminal deletion.
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Fig. 3.
APC down-regulates LEF signaling induced by
WT -catenin but not by the non-ubiquitinatable
S37A mutant
-catenin. SW480 cells were
transfected with empty vector or FLAG-tagged WT
-catenin or
FLAG-tagged S37A
-catenin and empty vector or WT APC constructs, LEF
reporters, and pCMV-Renilla luciferase. 24 h
posttransfection, LEF reporter activity was monitored using the dual
luciferase assay system (Promega).
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Fig. 4.
APC down-regulates WT
-catenin but not the non-ubiquitinatable S37A
mutant
-catenin protein. SW480 cells were
transfected with FLAG-tagged WT
-catenin (A,
B, and C) or FLAG-tagged S37A
-catenin
(D and E) and WT APC constructs (A,
B, D, and E) or empty vector
(C). Double immunofluorescent staining for APC (A
and D) and
-catenin (B, C, and
E) were performed according to Munemitsu et al.
(11, 40), except that the tranfected FLAG-tagged
-catenin was
detected using anti-FLAG antibodies (Kodak).
activity (18).
TPA-induced down-regulation of diacylglycerol
(DAG)-dependent PKCs prevents Wnt from inhibiting GSK-3
(18). However, our earlier studies demonstrate that neither the PKC
inhibitor calphostin C nor TPA-induced down-regulation of PKCs
stabilizes
-catenin (25). In contrast, the bisindoylmaleimide-type PKC inhibitor GF-109203X causes a dramatic accumulation of
-catenin in the cytoplasm (25). The bisindoylmaleimides inhibit both DAG-dependent and -independent PKC isoforms by competing
with ATP for binding to the kinase, whereas calphostin C and long term TPA treatment inhibit only DAG-dependent PKC activities.
The inhibitor profile implicates DAG-independent, atypical PKC activity
in regulating
-catenin stability. These kinase(s) may offer a level
of regulation distinct from the DAG-dependent PKC isoforms
that regulate Wnt-dependent and GSK-3
-mediated
-catenin signaling (25).
-catenin ubiquitination but does not inhibit GSK-3
(25). We
tested the hypothesis that GF-109203X will inhibit the ability of APC
to regulate
-catenin-LEF signaling. Fig.
5 shows that the PKC inhibitor GF-109203X
decreases the ability of APC to down-regulate LEF signaling in a
dose-dependent manner in SW480 cells. The changes in
-catenin-LEF signaling are paralleled by changes in
-catenin protein (Fig. 6). Similar results were
obtained with another bisindoylmaleimide-type PKC inhibitor Ro31-8220
(data not shown).
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Fig. 5.
The bisindoylmaleimide-type PKC inhibitor,
GF-109203X, which prevents -catenin
ubiquitination, inhibits APC-mediated down-regulation of
-catenin-LEF signaling in a
dose-dependent manner. SW480 cells were transfected
with empty vector or WT APC, LEF reporters, and pCMV-Renilla
luciferase. 12 h posttransfection, cells were treated with various
concentrations of GF-109203X. 12 h later, LEF reporter activity
was monitored using the dual luciferase assay system (Promega).
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Fig. 6.
The bisindoylmaleimide-type PKC inhibitor,
GF-109203X, but not lithium, reverses the APC-mediated down-regulation
of -catenin protein. SW480 cells were
transfected with WT APC and were treated with 5 µM
GF-109203X (A and B) for 12 h as described
in Fig. 5. 20 mM NaCl (C and D) or
LiCl (E and IF) were added immediately following
transfections and were present throughout the 24-h assay period to
assure GSK-3
repression. Double immunofluorescent staining for APC
(A, C, and E) and
-catenin
(B, D, and F) was performed according
to Munemitsu et al. (11, 40).
-Catenin-LEF Signaling--
Physiologically
effective concentrations of Li+ specifically and reversibly
inhibit GSK-3
activity in vitro and in vivo
and can mimic the effects of Wnt signaling on
-catenin in mammalian cells (43-46). Treatment of breast cancer cell lines with lithium results in the accumulation of the cytoplasmic signaling pool of
-catenin (25). Axin, the recently described product of the mouse
Fused locus, forms a complex with GSK-3
,
-catenin, and APC (47). Axin promotes GSK-3
-dependent phosphorylation
of
-catenin and may therefore help target
-catenin for
degradation (48). However, overexpression of Axin inhibits
-catenin-LEF signaling in SW480 colon cancer cells in the absence of
functional, WT APC. It is not known if APC promotes
GSK-3
-dependent phosphorylation of
-catenin.
Rubinfeld et al. (49) have shown that the APC protein is
phosphorylated by GSK-3
in vitro and suggest that this
phosphorylation event is linked to
-catenin turnover. It has also
been suggested that APC and Axin may regulate the degradation of
-catenin by different mechanisms (50).
-catenin-LEF signaling. The colon cancer cell line SW480 was transfected with empty vector or WT APC and treated
with 10, 20, or 40 mM LiCl or NaCl for 24 h. The
treatments were initiated immediately following the 5-h transfection
period, and the cells were exposed to LiCl or NaCl throughout the 24-h assay period to assure GSK-3
repression. Fig. 6 shows that lithium does not alter the ability of WT APC to down-regulate
-catenin protein. Fig. 7 shows that lithium does
not reverse the ability of WT APC to down-regulate LEF reporter
activity in SW480 cells. Even at 40 mM lithium, a level
well above that required to completely inhibit GSK-3
, exogenous WT
APC continues to significantly down-regulate LEF reporter activity.
These experiments were repeated in several different formats
incorporating variations in the amount of WT APC transfected, duration
of treatment with lithium, and timing of treatment initiation following
transfections. Regardless of these variations, lithium does not inhibit
the ability of exogenous APC to down-regulate
-catenin-LEF signaling
in the colon cancer cells tested. Lithium treatment also leads to
activation of AP-1-luciferase reporter activity in Xenopus
embryos, consistent with previous observations that GSK-3
inhibits
c-jun activity (46, 51). Concurrent AP-1 transactivation
assays also confirmed that GSK-3
was inhibited in SW480 cells
following treatment with lithium (data not shown). These results
indicate that GSK-3
activity (the molecular target of lithium
action, in the Wnt signaling cascade) is not required for the ability
of exogenously expressed APC to down-regulate
-catenin. Recent data
indicated that the role of GSK-3
may be to potentiate assembly of
the APC·Axin·
-catenin complex (48). In our experiments, the high
level of APC expressed in the transiently transfected cells may well
drive complex assembly in the absence of GSK-3
activity. Indeed, in
SKBR3 cells, lithium treatment causes the accumulation of cytoplasmic
-catenin and increases
-catenin-LEF
signaling2 (25).
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Fig. 7.
Lithium, an inhibitor of
GSK-3 , does not significantly alter the
ability of exogenous WT APC to down-regulate LEF reporter
activity. SW480 cells were transfected with empty vector or WT
APC, LEF reporters, and pCMV-Renilla luciferase. Various
concentrations of NaCl or LiCl were added immediately after
transfection to assure GSK-3
repression. 24 h later, LEF
reporter activity was monitored using the dual luciferase assay system
(Promega).
-catenin-LEF signaling via the ubiquitin-proteasome pathway.
In vitro reconstitution experiments designed to explore
-catenin ubiquitination suggested the requirement of key components other than GSK-3
and APC.2 During the course of this
study there has been an explosion of data describing novel proteins,
including Axin, Conductin, and Slimb·
-TrCP as regulators of
-catenin stability (47, 52-57). In Drosophila, loss of
function of Slimb results in accumulation of high levels of Armadillo
and the ectopic expression of Wg-responsive genes (56). Recently, the
receptor component of the I
B·ubiquitin ligase complex has been
identified as a member of the Slimb·
-TrCP family (39). Considering
the increasing number of similarities between the regulation of I
B
and
-catenin (25), it is tempting to speculate that like I
B,
-catenin ubiquitination occurs in a multiprotein complex that
includes kinases, ubiquitin-conjugating enzymes, and co-factors.
Context-dependent potentiation of this complex by GSK-3
and other serine kinase(s) may be regulated by
DAG-dependent and -independent PKC activity, respectively. The challenge for future studies will be to determine the exact role of
APC in this process.
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ACKNOWLEDGEMENTS |
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We thank Patrice Morin, Hans Clevers, and
Keith Orford for the WT APC expression plasmid, LEF reporters, and S37A
-catenin construct, respectively.
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FOOTNOTES |
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* This work was supported by Grants DAMD1794J-4171 (to V. E.) and DAMD17-98-1-8089 (to S. B.) from the Department of Defense.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: E415 The Research Building, GUMC, 3970 Reservoir Rd., NW, Washington, D. C. 20007. Tel.: 202-687-1813; Fax: 202-687-7505; E-mail: byerss{at}gunet.georgetown.edu.
2 V. Easwaran and S. Byers, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
APC, adenomatous
polyposis coli;
GSK-3, glycogen synthase kinase-3
;
LEF, lymphocyte enhancer-binding factor;
ALLN, N-acetyl-Leu-Leu-norleucinal;
ALLM, N-acetyl-Leu-Leu-methional;
WT, wild type;
PKC, protein
kinase C;
TPA, 12-O-tetradecanoylphorbol 13-acetate;
DAG, diacylglycerol;
NK
B, nuclear factor
B;
I
B, inhibitor of
NF
B.
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