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. Author manuscript; available in PMC: 2008 Mar 29.
Published in final edited form as: J Biol Chem. 2004 Aug 24;279(44):45669–45675. doi: 10.1074/jbc.M405025200

Activator Protein 2α Associates with Adenomatous Polyposis Coli/β-Catenin and Inhibits β-Catenin/T-cell Factor Transcriptional Activity in Colorectal Cancer Cells*

Qingjie Li ‡, Roderick H Dashwood ‡,§,
PMCID: PMC2276578  NIHMSID: NIHMS41685  PMID: 15331612

Abstract

In most human colorectal cancers, mutations in the adenomatous polyposis coli gene (APC) or CTNNB1 constitutively activate the β-catenin/T-cell factor (TCF)/lymphoid enhancer factor (LEF) signaling pathway. Here, we show that the transcription factor activator protein (AP)-2α inhibited a β-catenin/TCF-responsive reporter in human embryonic kidney 293 cells and in two human colorectal cancer lines, despite the fact that β-catenin and TCF-4 protein levels were unchanged in the nucleus. Co-immunoprecipitation studies revealed that AP-2α formed a complex with APC and β-catenin and that AP-2α disrupted β-catenin/TCF-4 interactions in the nucleus. Thus, AP-2α·APC·β-catenin complex formation appears to suppress β-catenin transactivation by shifting the pool of nuclear β-catenin toward an inactive form, having reduced binding to TCF/LEF transcription factors. Glutathione S-transferase pull-down assays showed that AP-2α physically associated with APC rather than with β-catenin, and the AP-2α binding site was identified in the N terminus of APC, involving both the heptad and armadillo repeat domains, whereas the APC binding site in AP-2α was in the basic region of the C-terminal DNA binding domain. These findings provide the first evidence for a specific interaction between the tumor suppressor protein APC and the transcription factor AP-2α, and they suggest a link between the Wnt signaling pathway and various other pathways of development and differentiation associated with AP-2α.

Mutations in the adenomatous polyposis coli (APC)1 gene or β-catenin gene (CTNNB1) stabilize β-catenin in the majority of human colorectal cancers, thereby activating a host of downstream β-catenin/TCF/LEF target genes (1–4). Consequently, there is considerable interest in mechanisms that down-regulate β-catenin and/or its target genes, since this provides an avenue for the prevention of colorectal and other cancers. We became interested in the transcription factor AP-2α as a putative negative regulator of β-catenin/TCF/LEF signaling.

The AP-2 family of transcription factors consists of five members, AP-2α, AP-2β, AP-2γ, AP-2δ, and AP-2ε, which have been implicated as critical regulators of gene expression during vertebrate development, embryogenesis, and transformation (5–8). AP-2α−/− mice die perinatally with cranio-abdominoschisis and severe dismorphogenesis of the face, skull, sensory organs, and cranial ganglia (9), whereas AP-2β knockout mice die postnatally because of polycystic kidney disease (10). In cell lines representing six different types of cancer, AP-2α inhibited cell growth by inducing cell cycle arrest and apoptosis (11). Reduction or loss of AP-2α expression has been reported in breast cancer, colon carcinoma, prostate cancer, and cutaneous malignant melanoma, suggesting a role for AP-2α as a tumor suppressor (12–15). AP-2α interacts with multiple protein partners, including retinoblastoma protein, poly(ADP-ribose) polymerase, and Myc (16–19). The finding (19) that Myc is under negative control of AP-2 is noteworthy, because Myc is a known downstream target of β-catenin/TCF/LEF signaling (4). We became interested in the cross-talk between AP-2α and β-catenin/TCF signaling and sought to test the hypothesis that AP-2α might act further upstream as a negative regulator of β-catenin/TCF/LEF signaling.

EXPERIMENTAL PROCEDURES

Plasmids

pCMV/APC was kindly provided by Dr. Bert Vogelstein. Dr. Hans Clevers and Dr. Marc van de Wetering generously supplied the wild type β-catenin cDNA construct, the positive control reporter TOPflash, and also the corresponding negative control FOPflash, containing mutated TCF/LEF binding sites. Expression vectors for S33Y and Δ45 β-catenins were made by site-directed mutagenesis, starting with the wild type β-catenin construct. pcDNA3.1/AP-2α was generated by cloning cDNA-encoding full-length AP-2α into pcDNA3.1(+) (In-vitrogen) between HindIII and EcoRV. pGEX/AP-2α was constructed by cloning AP-2α cDNA into pGEX-5x-2 (Amersham Biosciences) between EcoRI and NotI. Plasmids expressing GST-tagged AP-2α fragments were constructed by PCR amplification from pcDNA3.1/AP-2α, followed by cloning into pGEX-5x-2 between EcoRI and NotI. APC and AP-2α fragments were generated from pCMV/APC and pcDNA3.1/AP-2α, respectively, using standard PCR-based methods, and PCR products were cloned into pcDNA3.1(+). All constructs were confirmed by sequencing in both directions.

Cells and Transient Transfections

HT29 and HCT116 human colorectal cancer cells were grown in McCoy’s 5A Medium supplemented with 10% bovine fetal serum (Invitrogen), whereas human embryonic kidney 293 (HEK293) cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% horse serum, maintained at 37 °C in a humidified 5% CO2-containing atmosphere. Transfection was performed using LipofectAMINE™ 2000 (Invitrogen) or TransFast (Promega), and cells were harvested 48 h post-transfection.

Reporter Assays

β-Galactosidase and luciferase assays were performed as reported (20), including the appropriate controls for transfection efficiency.

Western Blotting and Co-immunoprecipitation Studies

Whole cell lysates were prepared using reporter lysis buffer (Promega), and the protein concentration was determined as reported (20), whereas nuclear extracts were obtained using NE-PER extraction reagents (Pierce). Proteins were separated on 4–12% bis-tris gels (Novex) or on 3–8% Tris-acetate gels (for APC); they were transferred to nitrocellulose membranes (Invitrogen); and after incubation with primary antibody followed by secondary antibody conjugated to horseradish peroxidase, detection was by Western Lighting Chemiluminescence Reagents Plus (PerkinElmer Life Sciences). Antibodies were as follows: rabbit anti-AP-2α polyclonal (C-18; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-β-catenin monoclonal (C19220; Transduction Laboratory), mouse anti-β-actin monoclonal (AC-15; Sigma), mouse anti-TCF-4 monoclonal (6H5–3; Upstate Biotechnology, Inc., Lake Placid, NY), mouse anti-APC monoclonal (F-3; Santa Cruz Biotechnology), and mouse anti-HA tag monoclonal (262K; Cell Signaling). Although β-actin served as a loading control in most experiments, we routinely confirmed the clean separation of nuclear and cytoplasmic fractions by immunoblotting with anti-α-tubulin antibody, which detected a signal only for cytoplasmic extracts (data not presented).

For co-immunoprecipitation, cells were washed with PBS and incubated in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 μg/ml leupeptin). Supernatants were precleared with protein A-Sepharose for 1.5 h at 4 °C. Immunoprecipitation was conducted overnight at 4 °C with 4 μg of the indicated antibody. Immunocomplexes were collected by adding 100 μl of 50% protein A-Sepharose slurry and incubating for 1.5 h at 4 °C. The beads were washed five times with lysis buffer, resuspended in 20 μl of SDS sample buffer, heated to 90 °C for 10 min, and subjected to Western blotting analysis with corresponding antibodies. In some experiments, TCF-4 and β-catenin were co-immunoprecipitated from nuclear lysates, followed by immunoblotting for β-catenin and TCF-4 as above.

GST Pull-down Assays

To study the interactions between AP-2α, APC, and β-catenin, GST-tagged AP-2α and AP-2α fragments were expressed in the BL21 strain of Escherichia coli and purified on glutathione-Sepharose 4B beads (Amersham Biosciences). β-Catenin and selected fragments of APC were translated in vitro with the TNT® quick coupled transcription/translation system (Promega) in the presence of [35S]methionine (Amersham Biosciences). The translated proteins and the GST affinity matrix were incubated for 2 h at room temperature in NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, and 0.5% Nonidet P-40) (21). The beads were then washed five times with NETN buffer, and the bound 35S-labeled protein was analyzed by SDS gel electrophoresis with autoradiography.

Densitometric Analysis

Image analysis and quantification were performed on an AlphaImager™ 2200 documentation and analysis system (Alpha Innotech Corp., San Leandro, CA).

RESULTS

AP-2α Inhibits β-Catenin/TCF/LEF-dependent Transcriptional Activity in Colorectal Cancer Cells

To investigate whether AP-2α modulates β-catenin/TCF/LEF-dependent transcriptional activity, experiments were performed with the reporter TOPflash (1, 20, 22–25) or with the corresponding negative control FOPflash, and pSV-β-galactosidase was included to normalize for transfection efficiency. Initially, wild type β-catenin was overexpressed by transient transfection in HEK293 cells (Fig. 1A). Exogenous β-catenin increased TOPflash but not FOPflash reporter activity, and interestingly, transient transfection of AP-2α suppressed TOPflash activity by ~65% under the experimental conditions used. Western blotting of whole cell lysates confirmed that AP-2α expression was increased in cells transfected with exogenous AP-2α, but surprisingly, total (endogenous plus transfected) β-catenin levels were similar in cells transfected with or without AP-2α (Fig. 1A, lower panel). Thus, the AP-2α-mediated attenuation of TOPflash reporter activity did not appear to involve changes in total β-catenin expression levels in HEK293 cells. Oncogenic forms of β-catenin, namely S33Y and Δ45, also responded to the inhibitory effects of AP-2α in the reporter assays (Fig. 1, B and C).

Fig. 1. TOPflash reporter activation by exogenous β-catenin is suppressed by AP-2α in HEK293 cells.

Fig. 1

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Cells were transfected with TOPflash, a β-catenin/TCF/LEF-responsive luciferase reporter plasmid containing TCF-4-binding sites, or the corresponding negative control FOPflash, as well as with AP-2α and β-catenin constructs (as indicated in the figure). pSV-β-Gal was used as internal control. Luciferase and β-galactosidase activities were determined for whole cell lysates 48 h post-transfection, as described under “Experimental Procedures.” Luciferase activity was normalized to the corresponding β-galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D., n = 3. A, induction of TOPflash activity by wild type β-catenin is suppressed by AP-2α. The cell lysates also were immunoblotted for AP-2α, β-catenin, and β-actin (lower panel). B, overexpression of AP-2α inhibits TOPflash activity induced by oncogenic S33Y β-catenin. C, AP-2α inhibits induction of TOPflash activity by Δ45 β-catenin.

We next studied the effects of AP-2α in cells transiently transfected with TOPflash but no exogenous β-catenin. In addition to studies with HEK293 cells, which have relatively low levels of cytoplasmic β-catenin, we chose two human colorectal cancer lines known to contain high endogenous β-catenin expression, namely HCT116 and HT29 cells (Fig. 2). AP-2α inhibited TOPflash reporter activity by >50% in HEK293 and HCT116 cells and by ~25% in HT29 colorectal cancer cells. The latter finding is noteworthy, because HT29 cells lack wild type APC (see below for further discussion).

Fig. 2. AP-2α inhibits TOPflash reporter activity in HEK293, HT29, and HCT116 cells.

Fig. 2

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Cells were transfected as described in the legend to Fig. 1, but in the absence of exogenous β-catenin. Lysates were prepared and analyzed for luciferase and β-galactosidase activities 48 h post-transfection, as described under “Experimental Procedures.” Luciferase activity was normalized to the corresponding β-galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D., n = 3.

AP-2α Does Not Attenuate β-Catenin or TCF-4 Levels in the Nucleus

Cells treated as described in the legend to Fig. 2 were subjected to immunoblot analyses. In all three cell lines, the nuclear expression levels of β-catenin and TCF-4 remained unchanged by AP-2α (Fig. 3). Taken together, the results in Figs. 1 and 3 clearly indicated that AP-2α decreased TOPflash reporter activity without reducing nuclear levels of β-catenin or TCF/LEF. Cytoplasmic levels of β-catenin and TCF-4 also remained unchanged in response to AP-2α (data not shown).

Fig. 3. Nuclear β-catenin and TCF-4 levels are unchanged by AP-2α.

Fig. 3

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Cells were transfected with plasmid pcDNA3.1/AP-2α or empty vector pcDNA3.1(+). After 48 h, nuclear lysates were prepared and immunoblotted for β-catenin, TCF-4, and AP-2α, using β-actin as loading control.

AP-2α Associates with APC and β-Catenin but Not with TCF-4

Immunoprecipitation experiments were performed next, in order to examine possible direct interactions between AP-2α and APC, β-catenin, or TCF-4. Immunoblotting with anti-TCF-4 antibody identified a strong band in all three cell lines when the same antibody was used for immunoprecipitation (Fig. 4A, right lane) but not when an irrelevant HA-tag antibody or no antibody was used for immunoprecipitation. Importantly, AP-2α antibody also failed to immunoprecipitate TCF-4, which implies no physical interaction between AP-2α and TCF-4.

Fig. 4. AP-2α associates with APC and β-catenin, but not with TCF-4.

Fig. 4

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Cell lysates from nontransfected cells were normalized for protein content and subjected to immunoprecipitation (IP) followed by immunoblotting (IB). A, immunoprecipitation was performed with the antibodies indicated, followed by immunoblotting with anti-TCF-4 antibody. Negative controls included no antibody (No Ab) and irrelevant antibody (HA-Tag). Note that AP-2α antibody failed to pull down TCF-4. B, immunoprecipitation was first performed with the antibodies indicated, followed by immunoblotting with anti-β-catenin. β-Catenin was associated both with APC and with AP-2α. C, immunoprecipitation with the antibodies indicated followed by immunoblotting with anti-APC identified full-length APC bound to AP-2α in HEK293 and HCT116 cells. D, in HT29 cells, immunoprecipitation with anti-AP-2α antibody pulled down 200- and 100-kDa truncated forms of APC, and anti-APC antibody pulled down AP-2α (48 kDa).

In contrast, AP-2α antibody successfully pulled down β-catenin in all three cell lines (Fig. 4B). As expected, β-catenin and APC antibodies also immunoprecipitated β-catenin (Fig. 4B). In HEK293 and HCT116 cells, antibody to AP-2α immunoprecipitated full-length APC (Fig. 4C), whereas in HT29 cells, which contain two truncated forms of APC, AP-2α antibody pulled down one protein of 200 kDa and another of 100 kDa (Fig. 4D). In the reverse experiment, immunoprecipitation with APC antibody pulled down AP-2α (Fig. 4D, lower blot). Collectively, these results suggested a complex involving AP-2α, β -catenin, and APC, but not TCF-4.

AP-2α, Alone or in Combination with APC, Reduces β-Cate-nin/TCF-4 Interaction in the Nucleus

We next examined whether AP-2α disrupts the association of β-catenin with TCF-4, as a mechanism for attenuating TOPflash reporter activity. Nuclear fractions from HEK293, HT29, and HCT116 cells were immunoprecipitated using anti-human TCF-4 antibody, followed by immunoblotting for β-catenin and reprobing for TCF-4 (Fig. 5). In cells treated with no exogenous AP-2α or APC, anti-TCF-4 antibody pulled down β-catenin, as expected. These β-catenin levels were quantified relative to TCF-4 in the same blot, and each was assigned an arbitrary expression value of 1.00 (see Relative β-cat level in Fig. 5). Transient transfection with AP-2α, APC, or the combination of AP-2α and APC attenuated the levels of TCF-associated β-catenin in each of the cell lines (Fig. 5). In HCT116 cells, for example (Fig. 5, lower blot), overexpression of AP-2α, APC, or AP-2α plus APC produced relative expression levels for TCF-associated β-catenin of 0.72, 0.65, and 0.55, respectively.

Fig. 5. AP-2α and APC, alone or in combination, inhibits the interaction of β-catenin with TCF-4 in the nucleus.

Fig. 5

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AP-2α and/or APC were overexpressed by transient transfection, as indicated, and anti-TCF-4 antibody was used to pull-down β-catenin from nuclear extracts, 48 h post-transfection. β-Catenin levels were analyzed by immunoblotting, and the same blots were reprobed for TCF-4. Nuclear lysates from control groups were used for negative controls of co-immu-noprecipitation, including no antibody (No Ab) and irrelevant antibody (HA-Tag). Relative β-catenin levels were determined by densitometric analysis and normalized to TCF-4; β-catenin expression recovered in the nontransfected controls was assigned an arbitrary value of 1.00.

AP-2α Binds to the Heptad and Armadillo Repeats of APC Rather than to β-Catenin

The APC protein contains several “repeat domains,” namely the N-terminal heptad repeats, the armadillo (“Arm”) repeats, the central 15- and 20-amino acid repeats that bind β-catenin, and also a C-terminal basic region (26). We generated expression constructs containing each of the major APC domains (designated APC1–APC5; Fig. 6A) and attempted to pull down each APC fragment, as well as β-catenin, using GST-tagged AP-2α. As shown in Fig. 6B (upper panel), AP-2α interacted strongly with the heptad repeats (APC1) and Arm repeats (APC3), and with an APC fragment containing both heptad and Arm repeats (APC2). However, no interaction was seen for AP-2α with the 15-/20-amino acid central repeat region (APC4) or basic region (APC5), or with β-catenin, despite the fact that these proteins were detected in the input controls (Fig. 6B, lower panel). The β-catenin that was immunoprecipitated using anti-AP-2α antibody (Fig. 4B) most likely was associated with APC in the same complex, rather than directly binding to AP-2α. Collectively, the results suggest that AP-2α physically associates with APC rather than β-catenin and that the AP-2α binding domain in APC is located at the N terminus, involving the heptad and armadillo repeats.

Fig. 6. AP-2α binds to the N-terminal repeats in APC.

Fig. 6

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A, scheme showing wild type (full-length) APC and truncated proteins (APC1-APC5) with one or two of the repeat domains. *, positions of nuclear export signals. B, 35S-labeled APC fragments as well as β-catenin were synthesized in vitro and incubated with Sepharose-bound GST-AP-2α. Beads were washed, and bound 35S-labeled proteins were eluted, followed by SDS-PAGE and autoradiography. The lower panel shows 50% of the total radioactivity in each reaction (50% input controls).

APC Binds to the Basic Region of AP-2α

As illustrated in Fig. 7A, the AP-2α DNA binding domain is located in the C terminus and includes basic and helix-span-helix dimerization regions, whereas the transactivation domain is in the N-terminal half of the protein and involves glutamine- and proline-rich segments (27). The basic region is identical among human, mouse, and rat species, and a high level of conservation (~96%) is observed for this region among different AP-2 isoforms (28). To identify the APC binding motif in AP-2α, GST-tagged AP-2α fragments were generated and used to pull down 35S-labeled APC2 (i.e. the APC fragment shown previously to contain the AP-2α binding region) (Fig. 6). Full-length AP-2α and the fragment AP-2α-3 interacted strongly with APC2, whereas all other AP-2α fragments had signals similar to the GST background control (Fig. 7B). These data indicated that APC binds to the highly conserved basic region of AP-2α.

Fig. 7. APC binds to the basic region of AP-2α.

Fig. 7

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A, schematic presentation of AP-2α and AP-2α fragments containing different functional domains. TA, trans-activation; HSH, helix-span-helix. B, GST-tagged AP-2α and AP-2α fragments were expressed in E. coli, and the purified proteins were used in pull-down assays with L-[35S]methionine-labeled APC2. Negative control was pull-down using GST alone (left lane); positive control was full-length AP-2α (adjacent to the input control containing 50% of the total radioactive APC2; right lane). Note that among the various AP-2α fragments, AP-2α-3 alone interacted with APC2, indicating that APC binds to the basic region in AP-2α.

Finally, to demonstrate that the ability of AP-2α to inhibit β-catenin/TCF/LEF signaling is dependent upon its interaction with APC, we generated constructs expressing truncated forms of AP-2α, namely AP-2α-1, AP-2α-12, and AP-2α-123; (Fig. 8A). In cells transiently transfected with S33Y β-catenin (Fig. 8B) or in cells expressing endogenous β-catenin alone (Fig. 8C), full-length AP-2α inhibited TOPflash activity, as expected, whereas AP-2α-1 and AP-2α-12 lacking the basic region had no effect. Interestingly, AP-2α-123 also failed to suppress TOP-flash activity, despite the presence of the APC-binding basic region.

Fig. 8. Truncated forms of AP-2α fail to inhibit TOPflash reporter activity.

Fig. 8

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A, schematic presentation of full-length and truncated forms of AP-2α transfected into HEK293 cells with TOPflash, β-galactosidase, and S33Y β-catenin (B) or no exogenous β-catenin (C). Lysates were prepared and analyzed for luciferase and β-galactosidase activities 48 h post-transfection. Luciferase activity was normalized to the corresponding β-galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D., n = 3.

DISCUSSION

To our knowledge, this is the first report to show that AP-2α binds directly to APC, stabilizing APC/β-catenin interactions in the nucleus, attenuating β-catenin/TCF-4 interactions, and inhibiting TOPflash reporter activity in human colorectal cancer cells. Thus, AP-2α·APC·β-catenin complex formation shifted the pool of nuclear β-catenin to a more inactive form, effectively sequestering β-catenin away from TCF/LEF transcription factors and lowering the transactivation potential of β-catenin within the nucleus. The results are noteworthy, because they suggest a role for AP-2α in modulating the Wnt signaling pathway as a tumor suppressor that negatively regulates own-stream targets of β-catenin/TCF/LEF (1–4).

This is not the first study to ascribe a tumor suppressor function to AP-2α. Immunohistochemical studies revealed lower than normal expression levels of AP-2α in high grade colorectal carcinomas (13), whereas in colorectal cancer lines, forced overexpression of AP-2 resulted in the inhibition of cell growth (11). Normal melanocytes and nonmetastatic melanoma cell lines had high levels of AP-2, whereas highly metastatic melanoma cell lines expressed little or no AP-2 (14), and forced AP-2 expression suppressed tumorigenicity and metastatic potential of human melanoma cells by down-regulating MCAM/MUC18 (29). AP-2 regulates several genes involved in the progression of human melanoma, such as c-KIT, E-cadherin, MMP-2, and p21, and AP-2α recently was confirmed as a negative regulator of chondrocyte differentiation (30).

We provide here the first evidence that APC is a direct binding partner of AP-2α, but several other AP-2α-interacting proteins have been identified, including retinoblastoma protein (16, 31), Myc (17, 19), SV-40 large T antigen (32), human T-cell leukemia virus type 1 (33), PC4 (34), poly(ADP-ribose) polymerase (18), KLF9, and KLF12 (35, 36). Many of these AP-2α-binding proteins are well established regulators of gene expression. APC is a tumor suppressor that normally promotes the destruction of β-catenin by forming a cytoplasmic complex with axin, glycogen kinase-3β, and casein kinase I (37). However, APC also has been implicated in developmental processes; mouse embryos with truncated Apc do not complete gastrulation, and Apc mutations in zebra fish result in heart malformations (37–40).

Recently, APC was found to shuttle in and out of the nucleus (41). Somewhat surprisingly, truncated forms of mutant APC lacking one or more nuclear localization signals had a nuclear distribution pattern similar to that of full-length APC (42). These findings have stimulated a debate about the nuclear role of APC and the residual function(s) of truncated APC fragments found commonly in tumor cells with APC mutations. Our findings provide possible insight into this question by showing that full-length as well as truncated forms of APC interact with AP-2α in the nucleus.

The AP-2α-binding domain was localized in the N-terminal heptad/Arm repeat region of APC. This is noteworthy, because human colon cancers frequently contain truncated forms of APC. We postulate that severely truncated forms of mutant APC containing only the heptad/Arm repeats are capable of forming a functional AP-2α·APC complex in the nucleus, thereby interfering with the transcriptional activities of AP-2α and thus its tumor suppressor functions. It is noteworthy that the most common germ line mutations in APC occur between codons 1061 and 1309, and over 60% of all somatic mutations in APC are localized between codons 1286 and 1513 (43), leaving one or more 3-catenin binding sites in the truncated APC protein. Some of the less severely truncated forms of APC, which fail to down-regulate β-catenin via the Axin/GSK-3β pathway, retain partial β-catenin binding capability and are predicted to form a functional AP-2α·APC·β-catenin complex in the nucleus. This hypothesis was supported by immunoprecipitation studies in HT29 cells, in which two forms of truncated APC were seen in the AP-2α immunoprecipitation complex (Fig. 4D), an AP-2α·APC·β-catenin complex was detected (Fig. 4B), and β-catenin/TCF-4 interactions were attenuated in the nucleus of HT29 cells (Fig. 5). This led to reduced TOPflash reporter activity in HT29 cells, albeit less marked that in HCT116 and HEK cells containing full-length APC (Fig. 2).

We also identified the basic region of AP-2α as the APC binding domain. This region is highly conserved in different AP-2 isoforms and in different species. The basic region of AP-2α shares 97 and 99% homology with those of AP-2α and AP-2α, respectively, and AP-2α from human, mouse, and rat species has an identical basic region. It has been shown that AP-2 requires a basic region and an adjacent dimerization domain to achieve a sequence-specific protein/DNA interaction (44). Hence, all full-length AP-2 proteins should be able to associate with APC, implying that APC, especially truncated APC, may broadly affect functions of AP-2 family members, and conversely, different AP-2 proteins may interfere with oncogenic functions of mutant APC. Truncated forms of AP-2α failed to inhibit β-catenin/TCF-dependent reporter activity, including fragment AP-2α-123, which contained the APC-binding basic region but not the helix-span-helix domain (Fig. 8). We speculate that the functional complex between AP-2α, APC and β-catenin requires dimerization of AP-2α and possibly interaction with DNA, and follow up studies are in progress to address these questions.

Taken together, our findings underscore the important role of AP-2α as a tumor suppressor; they link AP-2α to the Wnt signaling pathway for the first time and suggest possible crosstalk between the Wnt signaling pathway and other pathways of development and differentiation known to be associated with AP-2α. It is predicted that specific inducers of AP-2α will act as novel agents for chemoprevention and chemotherapy in colo-rectal and other cancers, and further studies in this direction are clearly warranted.

Footnotes

*

This work was supported by National Institutes of Health (NIH) Grants CA65525, CA80176, and CA90890, and by NIEHS Center, NIH, Grant P30 ES00210.

1

The abbreviations used are: APC, adenomatous polyposis coli; GST, glutathione S-transferase; TCF, T-cell factor; LEF, lymphoid enhancer factor; AP, activator protein; HEK, human embryonic kidney; HA, hemagglutinin; Arm, armadillo; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

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