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

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

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)(2)(3)(4)(5)(6)(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 ␤-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)/Tcell 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).
The mechanism of APC-mediated ␤-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 NFB-IB (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 ␤-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
Reagents, Antibodies, and Cells-ALLN, ALLM, lactacystin-␤ 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-FLAG TM 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 ϫ 10 5 cells/well. The following day cells were transiently transfected with 1 g of APC constructs and 0.4 g of the LEF reporter, pTOPFLASH (optimal motif), or pFOPFLASH (mutant motif) (31), and 0.008 g 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 ␤-catenin protein, 0.3 g of FLAG-tagged WT or S37A ␤-catenin (25) was cotransfected with 0.6 g 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 sys-* 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Immunological Procedures-Double immunofluorescent staining for APC and ␤-catenin was performed according to Munemitsu et al. (11,40). In experiments where FLAG-tagged ␤-catenin was cotransfected with APC, anti-FLAG TM antibodies (Kodak) were used to detect the exogenous ␤-catenin.

RESULTS AND DISCUSSION
APC-mediated Down-regulation of ␤-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 Me 2 SO (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 APCmediated effects on LEF reporters was confirmed using pFOP-FLASH, 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 Me 2 SO (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).
APC Down-regulates WT ␤-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)(34)(35)(36)(37)(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 en- 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 Me 2 SO (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 (M) of the proteasomal inhibitor, ALLN. a.a., amino acid(s); DLG, Discs Large protein.
dogenous ␤-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. 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␤ 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 DAGindependent, 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).
The bisindoylmaleimide-type PKC inhibitor GF-109203X prevents ␤-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 abil-ity 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).
Lithium (Li ϩ ) Does Not Inhibit the Ability of APC to Downregulate ␤-Catenin-LEF Signaling-Physiologically effective concentrations of Li ϩ 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)(44)(45)(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).
We tested the hypothesis that Li ϩ can inhibit the ability of  (11,40), except that the tranfected FLAG-tagged ␤-catenin was detected using anti-FLAG antibodies (Kodak).
APC to down-regulate ␤-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 APC⅐Axin⅐␤-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 signaling 2 (25).
Our observations suggest that one function of APC is to down-regulate ␤-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 Slimb⅐␤-TrCP as regulators of ␤-catenin stability (47,(52)(53)(54)(55)(56)(57). In Drosophila, loss of function of Slimb results in accumulation of high levels of Armadillo and the ectopic expression of Wgresponsive genes (56). Recently, the receptor component of the IB⅐ubiquitin ligase complex has been identified as a member of the Slimb⅐␤-TrCP family (39). Considering the increasing number of similarities between the regulation of IB and ␤-catenin (25), it is tempting to speculate that like IB, ␤-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.
Acknowledgments-We thank Patrice Morin, Hans Clevers, and Keith Orford for the WT APC expression plasmid, LEF reporters, and S37A ␤-catenin construct, respectively. 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).

FIG. 6.
The bisindoylmaleimide-type PKC inhibitor, GF-109203X, but not lithium, reverses the APC-mediated downregulation of ␤-catenin protein. SW480 cells were transfected with WT APC and were treated with 5 M 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). 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).