The Ubiquitin-Proteasome Pathway and Serine Kinase Activity Modulate Adenomatous Polyposis Coli Protein-mediated Regulation of beta -Catenin-Lymphocyte Enhancer-binding Factor Signaling*

Vijayasurian EaswaranDagger , Virginia SongDagger , Paul Polakis§, and Stephen ByersDagger

From the Dagger  Department of Cell Biology, Georgetown University School of Medicine and Lombardi Cancer Research Center, Washington, D. C. 20007 and § Onyx Pharmaceuticals, Richmond, California 94806

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The tumor suppressor function of the adenomatous polyposis coli protein (APC) depends, in part, on its ability to bind and regulate the multifunctional protein, beta -catenin. beta -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 beta -catenin-LEF signaling is reversed by proteasomal inhibitors in a dose-dependent manner. APC down-regulates signaling induced by wild type beta -catenin but not by the non-ubiquitinatable S37A mutant, beta -catenin. Bisindoylmaleimide-type protein kinase C inhibitors, which prevent beta -catenin ubiquitination, decrease the ability of APC to down-regulate beta -catenin-LEF signaling. All these effects on LEF signaling are paralleled by changes in beta -catenin protein levels. Lithium, an inhibitor of glycogen synthase kinase-3beta , does not alter the ability of APC to down-regulate beta -catenin protein and beta -catenin-LEF signaling in the colon cancer cells that were tested. These results point to a role for beta -catenin ubiquitination, proteasomal degradation, and potentially a serine kinase other than glycogen synthase kinase-3beta in the tumor-suppressive actions of APC.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 beta -catenin (10-13).

beta -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-3beta , and in response, beta -catenin accumulates in the cytoplasm (16-18). At elevated cytoplasmic levels, beta -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 beta -catenin protein are likely to be oncogenic, although this has not been proven directly (23).

The mechanism of APC-mediated beta -catenin regulation is unknown. Recently, beta -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 NFkappa B-Ikappa B (28). Proteins regulated via proteasomal degradation can be specifically studied using the well characterized proteasome-specific peptidyl-aldehyde inhibitors (29, 30).

APC-mediated tumorigenesis might depend, in part, on its ability to regulate beta -catenin signaling (26). In this report, we show that the ubiquitin-proteasome pathway and the activity of a serine kinase other than GSK-3beta modulate APC-mediated regulation of beta -catenin-LEF signaling.

    EXPERIMENTAL PROCEDURES
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Reagents, Antibodies, and Cells-- ALLN, ALLM, lactacystin-beta 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-beta -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.

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 beta -catenin protein, 0.3 µg of FLAG-tagged WT or S37A beta -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.

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 beta -catenin was performed according to Munemitsu et al. (11, 40). In experiments where FLAG-tagged beta -catenin was cotransfected with APC, anti-FLAGTM antibodies (Kodak) were used to detect the exogenous beta -catenin.

    RESULTS AND DISCUSSION
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APC-mediated Down-regulation of beta -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 beta -catenin (11, 26). We tested the hypothesis that APC effects the down-regulation of beta -catenin-LEF signaling by targeting beta -catenin for proteasomal degradation. SW480 cells were transiently transfected with various APC deletion constructs (Fig. 1A) and treated with proteasomal inhibitors, and beta -catenin-LEF signaling was assayed using LEF reporters (31). Fig. 1B shows that the APC-mediated down-regulation of beta -catenin-LEF signaling is reversed by a panel of proteasomal inhibitors including ALLN, lactacystin-beta 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 beta -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-beta lactone. Double immunofluorescent staining for APC (Fig. 2, A, C, and E) and beta -catenin (Fig. 2, B, D, and F) shows that the APC induced reduction in beta -catenin protein (Fig. 2, A and B) is reversed by proteasomal inhibitors ALLN (Fig. 2, C and D) and lactacystin-beta 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 beta -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-beta lactone, and MG-132 or with Me2SO (DMSO, vehicle) and ALLM (calpain inhibitor II) for 12 h. beta -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 beta -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 beta -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-beta lactone (E and F). Double immunofluorescent staining for APC (A, C, and E) and beta -catenin (B, D, and F) was performed according to Munemitsu et al. (11, 40).

APC Down-regulates WT beta -Catenin but Not the Non-ubiquitinatable S37A Mutant Form of beta -Catenin-induced LEF Signaling-- Mutation of a single serine residue (S37A) within the ubiquitination-targeting sequence prevents beta -catenin ubiquitination (25). Serine mutations in the ubiquitin-targeting sequence of beta -catenin occur in a number of different cancers (33-38). At least one of these, S37A, is a stabilizing mutation that renders beta -catenin resistant to ubiquitination (25). If indeed APC regulates beta -catenin-LEF signaling by targeting beta -catenin for proteasomal degradation, then it should not be able to down-regulate the non-ubiquitinatable S37A mutant beta -catenin protein or the LEF signaling induced by this stable form of beta -catenin. To test this hypothesis, vector, FLAG-tagged WT, or S37A mutant beta -catenin constructs were cotransfected with vector or WT APC and the LEF reporters into SW480 cells. beta -Catenin-LEF signaling was monitored by assaying LEF reporter activity. Overexpression of both WT and S37A mutant forms of beta -catenin increased the basal LEF reporter activity by about 30%, even against the background of high levels of endogenous beta -catenin and beta -catenin-LEF signaling in the SW480 cells. S37A beta -catenin is more stable than WT beta -catenin (in cells that actively degrade beta -catenin, e.g. SKBR3 cells), but both forms increased LEF signaling by comparable levels in SW480 cells (which lack the ability to degrade beta -catenin). Fig. 3 shows that APC down-regulates LEF signaling induced by WT beta -catenin but not by the S37A mutant beta -catenin. The ability of APC to down-regulate the cotransfected FLAG-tagged WT beta -catenin and the S37A beta -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 beta -catenin. Fig. 4A (anti-APC) and Fig. 4B (anti-FLAG) show that WT APC effectively down-regulates WT beta -catenin. Fig. 4C (anti-FLAG) shows that in concurrent transfections with empty vector and FLAG-tagged WT beta -catenin, the FLAG-tagged WT beta -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 beta -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 beta -catenin with an N-terminal deletion.


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Fig. 3.   APC down-regulates LEF signaling induced by WT beta -catenin but not by the non-ubiquitinatable S37A mutant beta -catenin. SW480 cells were transfected with empty vector or FLAG-tagged WT beta -catenin or FLAG-tagged S37A beta -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 beta -catenin but not the non-ubiquitinatable S37A mutant beta -catenin protein. SW480 cells were transfected with FLAG-tagged WT beta -catenin (A, B, and C) or FLAG-tagged S37A beta -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 beta -catenin (B, C, and E) were performed according to Munemitsu et al. (11, 40), except that the tranfected FLAG-tagged beta -catenin was detected using anti-FLAG antibodies (Kodak).

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-3beta activity (18). TPA-induced down-regulation of diacylglycerol (DAG)-dependent PKCs prevents Wnt from inhibiting GSK-3beta (18). However, our earlier studies demonstrate that neither the PKC inhibitor calphostin C nor TPA-induced down-regulation of PKCs stabilizes beta -catenin (25). In contrast, the bisindoylmaleimide-type PKC inhibitor GF-109203X causes a dramatic accumulation of beta -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 beta -catenin stability. These kinase(s) may offer a level of regulation distinct from the DAG-dependent PKC isoforms that regulate Wnt-dependent and GSK-3beta -mediated beta -catenin signaling (25).

The bisindoylmaleimide-type PKC inhibitor GF-109203X prevents beta -catenin ubiquitination but does not inhibit GSK-3beta (25). We tested the hypothesis that GF-109203X will inhibit the ability of APC to regulate beta -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 beta -catenin-LEF signaling are paralleled by changes in beta -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 beta -catenin ubiquitination, inhibits APC-mediated down-regulation of beta -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 beta -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-3beta repression. Double immunofluorescent staining for APC (A, C, and E) and beta -catenin (B, D, and F) was performed according to Munemitsu et al. (11, 40).

Lithium (Li+) Does Not Inhibit the Ability of APC to Down-regulate beta -Catenin-LEF Signaling-- Physiologically effective concentrations of Li+ specifically and reversibly inhibit GSK-3beta activity in vitro and in vivo and can mimic the effects of Wnt signaling on beta -catenin in mammalian cells (43-46). Treatment of breast cancer cell lines with lithium results in the accumulation of the cytoplasmic signaling pool of beta -catenin (25). Axin, the recently described product of the mouse Fused locus, forms a complex with GSK-3beta , beta -catenin, and APC (47). Axin promotes GSK-3beta -dependent phosphorylation of beta -catenin and may therefore help target beta -catenin for degradation (48). However, overexpression of Axin inhibits beta -catenin-LEF signaling in SW480 colon cancer cells in the absence of functional, WT APC. It is not known if APC promotes GSK-3beta -dependent phosphorylation of beta -catenin. Rubinfeld et al. (49) have shown that the APC protein is phosphorylated by GSK-3beta in vitro and suggest that this phosphorylation event is linked to beta -catenin turnover. It has also been suggested that APC and Axin may regulate the degradation of beta -catenin by different mechanisms (50).

We tested the hypothesis that Li+ can inhibit the ability of APC to down-regulate beta -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-3beta repression. Fig. 6 shows that lithium does not alter the ability of WT APC to down-regulate beta -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-3beta , 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 beta -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-3beta inhibits c-jun activity (46, 51). Concurrent AP-1 transactivation assays also confirmed that GSK-3beta was inhibited in SW480 cells following treatment with lithium (data not shown). These results indicate that GSK-3beta 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 beta -catenin. Recent data indicated that the role of GSK-3beta may be to potentiate assembly of the APC·Axin·beta -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-3beta activity. Indeed, in SKBR3 cells, lithium treatment causes the accumulation of cytoplasmic beta -catenin and increases beta -catenin-LEF signaling2 (25).


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Fig. 7.   Lithium, an inhibitor of GSK-3beta , 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-3beta repression. 24 h later, LEF reporter activity was monitored using the dual luciferase assay system (Promega).

Our observations suggest that one function of APC is to down-regulate beta -catenin-LEF signaling via the ubiquitin-proteasome pathway. In vitro reconstitution experiments designed to explore beta -catenin ubiquitination suggested the requirement of key components other than GSK-3beta and APC.2 During the course of this study there has been an explosion of data describing novel proteins, including Axin, Conductin, and Slimb·beta -TrCP as regulators of beta -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 Ikappa B·ubiquitin ligase complex has been identified as a member of the Slimb·beta -TrCP family (39). Considering the increasing number of similarities between the regulation of Ikappa B and beta -catenin (25), it is tempting to speculate that like Ikappa B, beta -catenin ubiquitination occurs in a multiprotein complex that includes kinases, ubiquitin-conjugating enzymes, and co-factors. Context-dependent potentiation of this complex by GSK-3beta 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.

    ACKNOWLEDGEMENTS

We thank Patrice Morin, Hans Clevers, and Keith Orford for the WT APC expression plasmid, LEF reporters, and S37A beta -catenin construct, respectively.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: APC, adenomatous polyposis coli; GSK-3beta , glycogen synthase kinase-3beta ; 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; NKkappa B, nuclear factor kappa B; Ikappa B, inhibitor of NFkappa B.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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