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Originally published In Press as doi:10.1074/jbc.M600831200 on May 3, 2006

J. Biol. Chem., Vol. 281, Issue 26, 17751-17757, June 30, 2006
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Adenomatous Polyposis Coli (APC) Differentially Regulates beta-Catenin Phosphorylation and Ubiquitination in Colon Cancer Cells*

Jun Yang{ddagger}, Wen Zhang{ddagger}, Paul M. Evans{ddagger}, Xi Chen{ddagger}, Xi He§, and Chunming Liu{ddagger}1

From the {ddagger}Sealy Center for Cancer Cell Biology and Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555 and the §Division of Neuroscience, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, January 26, 2006 , and in revised form, April 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Most colorectal cancers have mutations of the adenomatous polyposis coli (APC) gene or the beta-catenin gene that stabilize beta-catenin and activate beta-catenin target genes, leading ultimately to cancer. The molecular mechanisms of APC function in beta-catenin degradation are not completely known. APC binds beta-catenin and is involved in the Axin complex, suggesting that APC regulates beta-catenin phosphorylation. Some evidence also suggests that APC regulates beta-catenin nuclear export. Here, we examine the effects of APC mutations on beta-catenin phosphorylation, ubiquitination, and degradation in the colon cancer cell lines SW480, DLD-1, and HT29, each of which contains a different APC truncation. Although the current models suggest that beta-catenin phosphorylation should be inhibited by APC mutations, we detected significant beta-catenin phosphorylation in these cells. However, beta-catenin ubiquitination and degradation were inhibited in SW480 but not in DLD-1 and HT29 cells. The ubiquitination ofbeta-catenin in SW480 cells can be rescued by exogenous expression of APC. The APC domains required for beta-catenin ubiquitination were analyzed. Our results suggest that APC regulates beta-catenin phosphorylation and ubiquitination by distinct domains and by separate molecular mechanisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal cancer is the second leading cause of cancer-related death in the United States, and the incidence of colorectal cancer is rising in developing countries (1). APC2 is an important tumor suppressor for colorectal cancer. APC mutations are responsible for familial adenomatous polyposis and up to 85% of sporadic colorectal cancers (2). APC is an essential component of the Wnt/beta-catenin signaling pathway, which plays a key role in intestinal homeostasis and colorectal cancers (3, 4).

beta-Catenin is a multifunctional protein. It binds E-cadherin on the cell membrane, regulating cell adhesion. It also binds T-cell factor (TCF) in the nucleus and controls gene expression (3–6). In normal cells, beta-catenin degradation is initiated by serine/threonine (Ser/Thr) phosphorylation at its amino-terminal region (7, 8). Without Wnt signaling, beta-catenin forms a complex with casein kinase I{alpha} (CKI{alpha}), glycogen synthase kinase-3 (GSK-3), and a scaffold protein, Axin, resulting in CKI{alpha}-mediated phosphorylation of beta-catenin at Ser-45. Ser-45 phosphorylation provides a recognition site for GSK-3 and allows GSK-3 to phosphorylate beta-catenin at positions Thr-41, Ser-37, and Ser-33 (9–11). Phosphorylated beta-catenin is recognized by beta-Trcp and targeted for ubiquitination and degradation (12–15). Wnt signaling inhibits GSK-3 phosphorylation of beta-catenin but not CKI{alpha} phosphorylation of beta-catenin (10). However, it is still unknown whether APC regulates beta-catenin phosphorylation or whether APC regulates GSK-3 or CKI{alpha} phosphorylation of beta-catenin. Furthermore, it is unclear how beta-catenin shifts from a complex with Axin to a separate complex with beta-Trcp.

APC has two types of beta-catenin binding domains, three 15-aa repeat domains and seven 20-aa repeat domains (3–6). APC also has three Axin binding domains, which are called SAMP motifs (16). Most colorectal cancers have an APC truncation in one allele and loss of heterozygosity in the other allele (2, 17). APC mutations associated with cancer are consistently truncated before amino acid 1638 and lack the Axin binding domains (18). Since APC has both beta-catenin and Axin binding domains, it has been suggested that APC is an essential component in the Axin complex (16, 19, 20). It would be expected that, in these colon cancer cells, APC mutations prevent the assembly of Axin complex and thus prevent beta-catenin phosphorylation. Recently, crystal structures of beta-catenin/APC and beta-catenin/Axin complex suggest that APC may compete with Axin for beta-catenin binding, thus releasing phosphorylated beta-catenin from the Axin complex for degradation (21–23). APC also has a nuclear export signal and can shuttle between the cytoplasm and nucleus (24–26). It has been suggested that APC may transport beta-catenin out of the nucleus. According to this model, APC mutations should prevent the nuclear export of beta-catenin and thus prevent beta-catenin phosphorylation and degradation in colon cancer cells.

To understand APC function in vivo and to understand why APC mutations inhibit beta-catenin degradation in colon cancer cells, we analyzed beta-catenin phosphorylation in the colon cancer cell lines containing different APC truncations. We found that these APC truncations did not prevent beta-catenin phosphorylation, suggesting that truncated APC retains at least partial activity for beta-catenin phosphorylation. However, beta-catenin ubiquitination and degradation was inhibited in SW480 cells. We demonstrated that the defect in beta-catenin ubiquitination and degradation in SW480 cells is due to an APC truncation. The truncated APC only inhibits beta-catenin ubiquitination but not beta-catenin phosphorylation. These results indicate that the mechanisms of APC function in beta-catenin regulation are more complicated than originally thought.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines—The colon cancer cell lines, HEK293T, HCT116, SW480, DLD-1, and HT29, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% of fetal bovine serum, at 37 °C, 5% CO2. HEK293T cells were transfected using the calcium phosphate method. SW480 cells were transfected with Lipofectamine 2000 (Invitrogen) or infected with adenovirus. For proteasome inhibition, cells were treated with MG132 (2.5 µM) for 6 h.


Figure 1
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  		FIGURE 1.
beta-Catenin phosphorylation in colon cancer cells. A, linear representation of different APC truncations in the colon cancer cell lines, SW480, DLD-1, and HT29. The colon cancer cell line, HCT116, contains wild type APC. B, the cytoplasmic lysates from SW480, DLD-1, HT29, and HCT116 cells were analyzed by Western blot using antibodies against beta-catenin and phosphorylated beta-catenin. Ab45 recognizes Ser-45 phosphorylation, and Ab(33 + 37)/41 recognizes Ser-33/Ser-37/Thr-41 phosphorylation of beta-catenin. C, the beta-catenin immunoprecipitation products from HT29 and HCT116 cell lysates were treated with or without CIP and analyzed with antibodies against beta-catenin and phosphorylated beta-catenin.

 
Constructs—Truncated APC fragments were amplified by PCR and cloned into the pCS2MT vector. DNA sequences were confirmed by automatic sequencing. Adenovirus Ad-CBR was a gift from Dr. B. Vogelstein (27). Adenoviruses that express different APC fragments were constructed using the Ad-Easy kit from ATCC. GST-APC2,3 was a gift from Dr. W. Xu. The putative APC phosphorylation sites Ser-1385, Thr-1388, Ser-1389, Ser-1391, Ser-1501, Ser-1504, Ser-1505, Ser-1507, and Ser-1510 (according to Ha et al. (21)) were changed to alanine in GST-APC2,3 to make an S->A mutant. This site-directed mutagenesis was designed using the computer program SiteFind (28). CKI kinase was purchased from New England Biolabs (Beverly, MA).

Western Blot—Cells were lysed in 1% Triton lysis buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.4), 1.5 mM EDTA, 150 mM NaCl, 10% glycerol, 10 mM NaF, 1 mM Na3VO4, 0.5 mM dithiothreitol, and a mixture of protease inhibitors. GST pulldown assays and Western blots were performed as described previously (13). Cell fractions were isolated using a cell fractionation kit from Sigma. Phospho-specific antibodies for beta-catenin were described previously (10). Mouse anti-beta-Trcp antibody was purchased from Zymed Laboratories Inc. (San Francisco, CA).

Immunohistochemistry—Cells grown on coverslips were fixed for 10 min with 4% paraformaldehyde. The cells were permeabilized with phosphate-buffered saline containing 0.2% (w/v) Triton X-100 for 20 min and then blocked by serum-free protein blocking buffer (DakoCytomation) for 1 h. Anti-beta-catenin antibody (1:100, Upstate Biotechnology, Charlottesville, VA) was diluted in antibody diluent (DakoCytomation) and incubated with cells overnight. The cells were washed three times with Tris-buffered saline with Tween, incubated with Alexa Fluor 594-labeled anti-mouse IgG (1:800), and then diluted in Tris-buffered saline with Tween for 1 h. The coverslips were washed, mounted on glass slides, and photographed with a Zeiss LSM510 confocal microscope (Germany).

Enzymatic Assay—Luciferase activity was analyzed by the Dual-Luciferase reporter assay system (Promega, Madison WI). In vitro kinase assays were performed as described previously (13). The alkaline phosphatase assay was performed by incubating the immunoprecipitated beta-catenin with calf-intestinal alkaline phosphatase (CIP) at 37 °C for 30 min.


Figure 2
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  		FIGURE 2.
beta-Catenin ubiquitination/degradation in colon cancer cells. A, SW480, DLD-1, HT29, and HCT116 cells were treated with MG132 and analyzed with antibodies against beta-catenin and phosphorylated beta-catenin. The upper bands are ubiquitinated beta-catenin (28, 32). B, HEK293T cells were transfected by FLAG-tagged wild type (WT) beta-catenin and mutant beta-catenin, S33A (serine 33 changed to alanine), and S->A (Ser-33, Ser-37, Thr-41, and Ser-45 were changed to alanine), and then treated with MG132. Transfected beta-catenin was detected by anti-FLAG antibody. C, SW480 cells were infected with the APC adenovirus Ad-CBR or the control adenovirus, Ad-GFP, and then treated with MG132. Cytoplasmic fractions were analyzed with antibodies against beta-catenin and phosphorylated beta-catenin.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
beta-Catenin Phosphorylation Was Not Inhibited in Colon Cancer Cells Containing APC Mutations—The majority of colon cancer cells have APC mutations; therefore, it was expected that these cells would not have phosphorylated beta-catenin. We analyzed beta-catenin phosphorylation in APC mutant colon cancer cell lines SW480, DLD-1, and HT29 using phospho-specific antibodies against beta-catenin. APC is deleted at the carboxyl terminus at residue 1338 in SW480 cells, residue 1427 in DLD-1 cells, and residue 1555 in HT29 cells (29–31) (Fig. 1A). The colon cancer cell line HCT116, which has wild type APC, was used as a control. As described in our previous study, antibody Ab45 only recognizes beta-catenin phosphorylation at residue Ser-45, and Ab(33 + 37)/41 only recognizes Ser-33/Ser-37/Thr-41 phosphorylation of beta-catenin (10). Unexpectedly, we found that both Ser-33/Ser-37/Thr-41 and Ser-45 were phosphorylated in these cells, suggesting that APC truncations in these cell lines do not inhibit GSK-3 or CKI{alpha} phosphorylation of beta-catenin (Fig. 1B). To further confirm that these antibodies only recognize phosphorylated beta-catenin, beta-catenin protein was immunoprecipitated with a beta-catenin antibody from HT29 and HCT116 cell lysates and treated with CIP. The levels of beta-catenin protein remained the same as the control samples without CIP treatment. However, beta-catenin phosphorylation could not be detected in the CIP-treated samples (Fig. 1C). These data confirmed the specificity of the phospho-specific antibodies, further suggesting that, in human colon cancer cell lines SW480, DLD-1, and HT29, APC mutations do not prevent beta-catenin phosphorylation. HCT116 cells have one allele of wild type beta-catenin and one allele of mutated beta-catenin that has a deletion at Ser-45 (32). Since the Ser-45 deletion mutant is stabilized relative to the wild type protein, it comprises most of the beta-catenin within the cell. We observed a lesser degree of Ser-45 phosphorylation in these cells.


Figure 3
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  		FIGURE 3.
APC domains required for beta-catenin ubiquitination. A, schematic representation of various truncated APC fragments incorporated into CS2MT as well as the Ad-Easy adenovirus vectors. The 15-amino acid repeats (A–C) and 20-amino acid repeats (1–7) are beta-catenin binding domains, and the SAMP repeats (SAMP1–3) are Axin binding domains. B, the expression of APC fragments in HEK293T cells was analyzed by Western blot using anti-Myc antibody. C, the APC fragments were transfected into SW480 cells with both the TopFlash reporter and the Renilla reporter. The luciferase reporter was analyzed and normalized with Renilla reporter. D, SW480 cells were infected with various APC adenoviruses and then treated with MG132. beta-Catenin phosphorylation and ubiquitination were analyzed by Western blot using beta-catenin antibody and phospho-specific antibodies against beta-catenin.

 
APC Mutation Inhibits beta-Catenin Ubiquitination and Degradation in the SW480 Colon Cancer Cells—To further study how APC regulates beta-catenin degradation, several colon cancer cell lines were treated with MG132, a proteasome inhibitor, and analyzed with beta-catenin antibodies. HCT116 cells were used as a control. It is known that proteasome inhibitors can stabilize ubiquitinated beta-catenin. After MG132 treatment, two major bands were detected by Western blot. The lower band is beta-catenin without ubiquitination; the upper band is beta-catenin with mono-ubiquitination (30, 33). As expected, after MG132 treatment, ubiquitinated beta-catenin was detected in HCT116 cell lysates (Fig. 2A). The ubiquitinated bands of beta-catenin were also detected in DLD-1 and HT29 cells. In HCT116, DLD-1, and HT29 cells treated with MG132, beta-catenin levels increased, suggesting that beta-catenin degradation was not completely inhibited in these cells. In contrast, SW480 cells demonstrated strong beta-catenin phosphorylation, almost undetectable ubiquitination, and no increase in beta-catenin levels after MG132 treatment. This suggests that beta-catenin ubiquitination and degradation were inhibited in SW480 cells (Fig. 2A). Similar results have been reported by Sadot et al. (34). However, it is not known whether the defect of beta-catenin ubiquitination in SW480 cells was caused by an APC mutation or by other mutations. To confirm that beta-catenin phosphorylation is required for ubiquitination, we transfected wild type beta-catenin and mutant beta-catenin into HEK293T cells. The cells were treated with or without MG132. We found that wild type beta-catenin, but not mutant beta-catenin, was modified by ubiquitination (Fig. 2B). We infected SW480 cells with Ad-CBR, which carries a functional APC fragment (27). This fragment contains all of the 15-aa repeats and 20-aa repeats, in addition to the Axin binding domains. We found that Ad-CBR can rescue beta-catenin ubiquitination and degradation in SW480 cells, indicating that APC is required for the ubiquitination and degradation of phosphorylated beta-catenin (Fig. 2C). beta-Catenin phosphorylation was stronger in SW480 cells, most likely because phosphorylated beta-catenin is degraded more slowly and accumulates in SW480 cells. MG132 treatment also stabilized phosphorylated beta-catenin. Ad-CBR increased beta-catenin phosphorylation, indicating that APC is required for the efficient phosphorylation of beta-catenin.

APC Domains Required for beta-Catenin Ubiquitination—Notably, the APC mutations in these colon cancer cells are truncated mutations and not null mutations. These APC proteins still have the 15-aa repeat beta-catenin binding domains and at least one 20-aa beta-catenin binding domain. beta-Catenin phosphorylation may be regulated by the beta-catenin binding domains that remain in the APC mutants. The CBR fragment of APC can rescue beta-catenin ubiquitination in SW480 cells. To determine which APC domains are required for beta-catenin ubiquitination, we cloned truncated CBR fragments into the CS2MT vector as well as an adenovirus vector (Fig. 3A). Except for the APC fragment containing the first two 20-aa repeats (APC-I2), the expression of these APC fragments can be detected by Western blot using anti-Myc antibody (Fig. 3B). We have made several constructs for APC-I2 but were unable to detect this protein. The effects of these APC fragments on a TCF reporter were analyzed in SW480 cells. APC fragments with SAMP domains strongly inhibited the TCF reporter. An APC fragment with one or three 20-aa repeats modestly inhibited TCF reporter activity (Fig. 3C). SW480 cells were infected with several adenoviruses that express each of these APC fragments and then treated with MG132. We found that the APC fragment with the first three 20-aa repeats (APC-I3) can sufficiently rescue beta-catenin ubiquitination (Fig. 3D). Control virus (GFP) or a virus containing the first 20-aa repeat (APC-I1) did not rescue beta-catenin ubiquitination in SW480 cells. These data demonstrate that the second and third 20-aa repeats are required for beta-catenin ubiquitination, whereas the SAMP domains are not required for beta-catenin ubiquitination. It is also possible that any combination of two 20-aa repeats may be sufficient for beta-catenin ubiquitination.


Figure 4
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  		FIGURE 4.
beta-Catenin localization in various APC adenovirus infected SW480 cells. SW480 cells were infected with various APC adenoviruses or a control adenovirus. Cells were immunostained with a beta-catenin antibody, and the nucleus was counterstained with 4',6-diamidino-2-phenylindole (DAPI). GFP indicates the adenovirus-infected cells. Arrows indicate beta-catenin staining in the adenovirus-infected cells.

 
beta-Catenin Localization in SW480 Cells—Since APC has a nuclear export signal, APC may regulate beta-catenin subcellular localization. It has been suggested that a mutation in APC might inhibit the nuclear export of beta-catenin and thus inhibit beta-catenin degradation, resulting in beta-catenin accumulation in the nucleus of colon cancer cells. To test whether beta-catenin ubiquitination is regulated by beta-catenin localization, the SW480 cells were infected with APC adenoviruses, which co-express the GFP protein, and then stained with anti-beta-catenin antibody. Control virus (GFP) or an APC virus with the first 20-aa repeat (APC-I1) had no effect on the cytoplasmic levels of beta-catenin (Fig. 4). beta-Catenin was detected in both the nucleus and the cytoplasm, with or without infection. APC viruses with 3–5 20-aa repeats (APC-I3, APC-I4, and APC-I5) significantly reduced the cytoplasmic beta-catenin levels (Fig. 4). These data confirm that the second and third 20-aa repeats of APC are required for beta-catenin degradation. We did not find a clear correlation between beta-catenin localization, degradation, and APC status. Since beta-catenin can be detected in both the nucleus and the cytoplasm of SW480 cells, the inhibition of beta-catenin ubiquitination is not due to the effects on beta-catenin localization. Although the SAMP domains are not absolutely required for beta-catenin degradation, APC-I4 and APC-I5 are more efficient in beta-catenin degradation.


Figure 5
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  		FIGURE 5.
The function of 15-aa repeats of APC. A, schematic representation of APC fragments without 15-aa repeats (APC-II5). B, top, luciferase reporter activity was analyzed in SW480 cells transfected with APC-II5. Bottom, the expression of APC-II5 detected by an anti-Myc antibody. C, SW480 cells were infected with the APC-II5 adenovirus and then treated with MG132. Cells were analyzed with antibodies against both beta-catenin and phosphorylated beta-catenin. D, immunofluorescent staining of beta-catenin in SW480 cells infected with APC-II5 adenovirus. The arrows indicate beta-catenin staining in the adenovirus-infected cells. DAPI,4',6-diamidino-2-phenylindole

 
The Function of 15-aa Repeats of APC—The 15-aa repeats also bind beta-catenin. To determine whether the 15-aa repeats are required for beta-catenin ubiquitination and degradation, we deleted the 15-aa repeats from the CBR fragment (Fig. 5A). We found that the CBR fragment, without 15-aa repeats (APC-II5), can sufficiently inhibit the TCF reporter and rescue beta-catenin ubiquitination in SW480 cells (Fig. 5, B and C). This construct also reduced cytoplasmic beta-catenin levels, suggesting that the 15-aa repeats are not required for beta-catenin ubiquitination (Fig. 5D). Whether these 15-aa repeats are required for beta-catenin phosphorylation is not known.

Mechanisms for APC-mediated beta-Catenin Ubiquitination—To confirm our results from immunostaining, nuclear and cytoplasmic proteins were isolated from SW480 and HT29 cells (Fig. 6A). Strong beta-catenin signals were detected in both nuclear and cytoplasmic fractions. These data further confirm that beta-catenin ubiquitination but not phosphorylation or nuclear export is inhibited in SW480 cells. Since beta-catenin ubiquitination was inhibited in SW480 but not DLD-1 and HT29 cells, and beta-Trcp couples beta-catenin phosphorylation and ubiquitination, we examined the interaction between beta-catenin and beta-Trcp in these cells. The interaction was detected in all three cell lines, and the interaction in SW480 cells was not dependent on functional APC (Fig. 6B). A similar result has been reported using a Myc-tagged beta-Trcp (12). beta-Catenin ubiquitination in SW480 cells was rescued by APC fragments containing three 20-aa repeats but not an APC fragment containing the first 20-aa repeat, suggesting that the second and third 20-aa repeats of APC may play a critical role in beta-catenin ubiquitination. The 20-aa repeats of APC have several putative phosphorylation sites, which are similar to beta-catenin phosphorylation sites (Fig. 6C). X-ray crystallographic studies suggest that phosphorylated 20-aa repeats of APC can compete with Axin for beta-catenin binding and release phosphorylated beta-catenin from the Axin complex for degradation (21–23). We previously identified a dual-kinase mechanism for beta-catenin phosphorylation (10). To examine whether APC can be phosphorylated by a similar mechanism, we used a GST fusion protein containing the second and third 20-aa repeats of APC (GST-APC2,3). GST-APC2,3 was phosphorylated in vitro by purified CKI and then incubated with HCT116 cell lysate. GST-APC2,3 was pulled down using GST beads, and bound beta-catenin was analyzed by Western blot. CKI induced a band shift of GST-APC2,3, as detected by an anti-GST antibody. Phosphorylation of APC2,3 indeed increased beta-catenin binding affinity (Fig. 6D). As a control, mutations in the putative phosphorylation sites of the second and third 20-aa repeats inhibited CKI phosphorylation and abolished binding with beta-catenin (Fig. 6D). Several quantitative studies have demonstrated the ability of APC phosphorylation to enhance its binding affinity with beta-catenin (21, 23). These data further support our hypothesis that APC regulates beta-catenin phosphorylation and ubiquitination by distinct molecular mechanisms.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
As a tumor suppressor for colorectal cancers, the major function of APC is to regulate beta-catenin (3–6). It is generally believed that APC regulates beta-catenin degradation by regulating beta-catenin phosphorylation and localization. We found that APC also regulates beta-catenin ubiquitination. The APC mutation in SW480 cells inhibits beta-catenin ubiquitination regardless of the status of beta-catenin phosphorylation and localization. Our data suggest that APC regulates beta-catenin degradation by distinct domains and separate molecular mechanisms.


Figure 6
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  		FIGURE 6.
Mechanisms for APC-mediated beta-catenin ubiquitination. A, the nuclear and cytoplasmic fractions were isolated from SW480 and HT29 cells treated with or without MG132. beta-Catenin was analyzed by Western blot. Lamin B and HSP 90 were used as markers for the nuclear (N) and cytoplasmic (C) fractions, respectively. B, the interaction between beta-Trcp and beta-catenin was detected in HT29, DLD-1, and SW480 cells. IP, immunoprecipitation. C, the putative phosphorylation sites in the second and third 20-aa repeats of APC were aligned with beta-catenin phosphorylation sites Thr-41 and Ser-45. D, top, GST-APC2,3, a GST fusion protein containing the second and third 20-aa repeats of APC, was in vitro phosphorylated by purified CKI. The band shift of phosphorylated GST-APC2,3 was detected by anti-GST antibody (W T). When the putative phosphorylation sites in the second and third 20-aa repeats (according to Ha et al. (21)) were mutated to alanine (S -> A), the band shift was greatly reduced. Bottom, phosphorylated GST-APC2,3 (W T) and its mutant (S -> A) were incubated with HCT116 cell lysates and pulled down using GST beads, and beta-catenin was analyzed by Western blot. CKI phosphorylation significantly enhanced the binding between beta-catenin and APC2,3 (W T). S->A mutations of APC inhibited its ability to interact with beta-catenin.

 
beta-Catenin phosphorylation is carried out by the Axin complex containing CKI{alpha}, GSK-3, and APC (16, 19, 20). APC binds both beta-catenin and Axin, and deleting the APC binding domain from Axin blocks beta-catenin/Axin binding (20). A simple model to explain these findings is that APC regulates the assembly of the Axin complex, thus regulating beta-catenin phosphorylation. The colon cancer cell lines HT29, DLD-1, and SW480 all contain a truncated APC protein lacking the Axin binding domains. Despite the fact that beta-catenin degradation was inhibited, we found that beta-catenin can still be phosphorylated in these cells. We hypothesize that APC is required for beta-catenin phosphorylation in normal cells. In tumor cells with high levels of beta-catenin, beta-catenin can be phosphorylated independent of the presence of full-length APC. The rate of both beta-catenin protein synthesis and its degradation are tightly controlled in normal cells to maintain a low level of beta-catenin in both the nuclear and the cytoplasmic compartments. In APC mutant cells, however, the rate of beta-catenin phosphorylation and degradation is lower than the rate of beta-catenin synthesis, resulting in an accumulation of beta-catenin in the nucleus, contributing to tumorigenesis. High levels of beta-catenin phosphorylation in SW480 cells were also observed by Sadot et al. (34), who proposed that the difference between beta-catenin phosphorylation in different APC truncated cells may be the result of a loss in a phosphatase binding site.

We observed beta-catenin ubiquitination in DLD-1 and HT29 cells, which have the first two or first three 20-aa repeats, but not in the SW480 cell line, which only has the first 20-aa repeat, suggesting that the second and third 20-aa repeats are required for beta-catenin ubiquitination. It appears that the first 20-aa repeat of APC in SW480 cells is not sufficient for regulating beta-catenin ubiquitination. Given that APC in DLD-1 cells is truncated between the second and third 20-aa repeats and the fact that beta-catenin ubiquitination can be detected in DLD-1 cells, the first two 20-aa repeats should be sufficient for beta-catenin ubiquitination. Whether each of these seven 20-aa repeats has a similar function is not known. It is possible that any combination of two 20-aa repeats is sufficient for beta-catenin ubiquitination. In our TCF reporter assay, APC fragments with one 20-aa repeat, which cannot rescue beta-catenin ubiquitination, also inhibited the reporter activity. This may be because the APC fragment binds beta-catenin and inhibits the activity of the beta-catenin/TCF complex. It is also possible that the constructs with multiple 20-aa repeats sequestered more beta-catenin from the reporter.

Although the Axin binding domains are not required for beta-catenin ubiquitination, APC fragments containing at least one SAMP repeat are indeed more efficient at promoting beta-catenin degradation. APC protein in both HT29 and DLD-1 cells lacks SAMP domains. Although these cells demonstrate a limited degree of beta-catenin ubiquitination and degradation, beta-catenin levels are dramatically increased when compared with normal cells. Again, this may be due to the fact that loss of SAMP domain decreases the efficiency of APC-mediated degradation, disrupting the balance of beta-catenin synthesis and degradation.

After phosphorylation, beta-catenin must be released from the Axin complex for ubiquitination and degradation. beta-Catenin phosphorylation, but not ubiquitination, is noted in SW480 cells, suggesting that mutations in APC prevent the proper assembly of the ubiquitin ligase complex. A possible explanation for this phenomenon is that truncated APC cannot release phosphorylated beta-catenin from the Axin complex, thus preventing the proper assembly of beta-catenin/beta-Trcp ubiquitin ligase complex. The interaction between beta-catenin and beta-Trcp was detected in SW480 cells, suggesting that other steps of beta-catenin ubiquitination were inhibited by APC truncation. However, we cannot rule out the possibility that beta-Trcp does not bind beta-catenin in vivo in SW480 cells but interacts with beta-catenin during immunoprecipitation. The second and third 20-aa repeats are involved in beta-catenin ubiquitination. We have confirmed that the 20-aa repeats can be phosphorylated in vitro and that phosphorylated APC binds beta-catenin more strongly. Our data support the model proposed from crystallographic studies. However, it is still unknown which kinase phosphorylates which sites of APC in vivo. To address these questions, we are currently generating phospho-specific antibodies against these putative phosphorylation sites.

It has been reported that the localization of beta-catenin is regulated by the cell density in SW480 cells and that several antibodies used for APC immunostaining detect nonspecific bands in the nucleus, suggesting that the nuclear function of APC should be interpreted with caution (29, 35). Since APC protein in these colon cancer cell lines still has beta-catenin binding domains, these domains may be sufficient for beta-catenin nuclear export. However, in at least the colon cancer cell line SW480, APC truncation did not completely block beta-catenin nuclear export and phosphorylation but did inhibit beta-catenin ubiquitination and degradation. Recently, Krieghoff et al. (36) reported that APC regulates beta-catenin subcellular localization by retention rather than by active transport.

Both 15-aa and 20-aa repeats bind to the conserved beta-catenin surface made by Armadillo repeats 5–9. The 20-aa repeats have additional interactions when they are phosphorylated (21). We have shown that the 15-aa repeats are not required for beta-catenin ubiquitination. In the colon cancer cell lines used in this study, truncated APC contains three 15-aa repeats, but beta-catenin can still be phosphorylated. If APC is required for beta-catenin phosphorylation, it is possible that the 15-aa repeats are required for beta-catenin phosphorylation and the 20-aa repeats are required for beta-catenin ubiquitination.

APC-mediated regulation of beta-catenin degradation by different domains and separate steps has important clinical implications. For example, different APC truncations correspond to different levels of tumor progression (4, 37). Truncations that still retain the three 15-aa repeats and one or two of the 20-aa repeats provide the strongest selective advantage and demonstrate a more aggressive tumor phenotype. Truncations that have lost all of the beta-catenin regulating domains or retain both beta-catenin and Axin binding domains produce more attenuated tumors (37). Our results suggest that different APC truncations have different molecular consequences on beta-catenin regulation, thus providing novel insights into the correlation between APC truncations and colon cancer phenotypes.


   FOOTNOTES
 
* 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. Back

1 Supported by a John Sealy Memorial Fund recruitment award and by Grant R21 CA112007 from the NCI, National Institutes of Health. To whom correspondence should be addressed. Tel.: 409-747-1909; Fax: 409-747-1938; E-mail: chliu{at}utmb.edu.

2 The abbreviations used are: APC, adenomatous polyposis coli; aa, amino acids; TCF, T-cell factor; CKI, casein kinase I; GSK-3, glycogen synthase kinase-3; CIP, calf-intestinal alkaline phosphatase; WT, wild type. CBR, catenin-binding region; GFP, green fluorescent protein; SAMP, Ser-Ala-Met-Pro. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Bert Vogelstein for the Ad-CBR adenovirus, to Dr. Wenqing Xu for GST-APC2,3 construct, and to Dr. B. Mark Evers for advice and critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. American Cancer Society. (2005) Cancer Facts and Figures 2005, pp. 10–12, American Cancer Society, Washington, D. C.
  2. Kinzler, K. W., and Vogelstein, B. (1996) Cell 87, 159–170[CrossRef][Medline] [Order article via Infotrieve]
  3. Giles, R. H., van Es, J. H., and Clevers, H. (2003) Biochim. Biophys. Acta 1653, 1–24[Medline] [Order article via Infotrieve]
  4. Polakis, P. (2000) Genes Dev. 14, 1837–1851[Free Full Text]
  5. Moon, R. T., Brown, J. D., and Torres, M. (1997) Trends Genet. 13, 157–162[CrossRef][Medline] [Order article via Infotrieve]
  6. Wodarz, A., and Nusse, R. (1998) Annu. Rev. Cell Dev. Biol. 14, 59–88[CrossRef][Medline] [Order article via Infotrieve]
  7. Aberle, H., Bauer, A., Stappert, J., Kispert, A., and Kemler, R. (1997) EMBO J. 16, 3797–3804[CrossRef][Medline] [Order article via Infotrieve]
  8. Orford, K., Crockett, C., Jensen, J. P., Weissman, A. M., and Byers, S. W. (1997) J. Biol. Chem. 272, 24735–24738[Abstract/Free Full Text]
  9. Amit, S., Hatzubai, A., Birman, Y., Andersen, J. S., Ben-Shushan, E., Mann, M., Ben-Neriah, Y., and Alkalay, I. (2002) Genes Dev. 16, 1066–1076[Abstract/Free Full Text]
  10. Liu, C., Li, Y., Semenov, M., Han, C., Baeg, G. H., Tan, Y., Zhang, Z., Lin, X., and He, X. (2002) Cell 108, 837–847[CrossRef][Medline] [Order article via Infotrieve]
  11. Yanagawa, S., Matsuda, Y., Lee, J. S., Matsubayashi, H., Sese, S., Kadowaki, T., and Ishimoto, A. (2002) EMBO J. 21, 1733–1742[CrossRef][Medline] [Order article via Infotrieve]
  12. Hart, M., Concordet, J. P., Lassot, I., Albert, I., del los Santos, R., Durand, H., Perret, C., Rubinfeld, B., Margottin, F., Benarous, R., and Polakis, P. (1999) Curr. Biol. 9, 207–210[CrossRef][Medline] [Order article via Infotrieve]
  13. Liu, C., Kato, Y., Zhang, Z., Do, V. M., Yankner, B. A., and He, X. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6273–6278[Abstract/Free Full Text]
  14. Spencer, E., Jiang, J., and Chen, Z. J. (1999) Genes Dev. 13, 284–294[Abstract/Free Full Text]
  15. Winston, J. T., Strack, P., Beer-Romero, P., Chu, C. Y., Elledge, S. J., and Harper, J. W. (1999) Genes Dev. 13, 270–283[Abstract/Free Full Text]
  16. Behrens, J., Jerchow, B. A., Wurtele, M., Grimm, J., Asbrand, C., Wirtz, R., Kuhl, M., Wedlich, D., and Birchmeier, W. (1998) Science 280, 596–599[Abstract/Free Full Text]
  17. Rowan, A. J., Lamlum, H., Ilyas, M., Wheeler, J., Straub, J., Papadopoulou, A., Bicknell, D., Bodmer, W. F., and Tomlinson, I. P. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3352–3357[Abstract/Free Full Text]
  18. Smits, R., Kielman, M. F., Breukel, C., Zurcher, C., Neufeld, K., Jagmohan-Changur, S., Hofland, N., van Dijk, J., White, R., Edelmann, W., Kucherlapati, R., Khan, P. M., and Fodde, R. (1999) Genes Dev. 13, 1309–1321[Abstract/Free Full Text]
  19. Ikeda, S., Kishida, S., Yamamoto, H., Murai, H., Koyama, S., and Kikuchi, A. (1998) EMBO J. 17, 1371–1384[CrossRef][Medline] [Order article via Infotrieve]
  20. Itoh, K., Krupnik, V. E., and Sokol, S. Y. (1998) Curr. Biol. 8, 591–594[CrossRef][Medline] [Order article via Infotrieve]
  21. Ha, N. C., Tonozuka, T., Stamos, J. L., Choi, H. J., and Weis, W. I. (2004) Mol. Cell 15, 511–521[CrossRef][Medline] [Order article via Infotrieve]
  22. Xing, Y., Clements, W. K., Kimelman, D., and Xu, W. (2003) Genes Dev. 17, 2753–2764[Abstract/Free Full Text]
  23. Xing, Y., Clements, W. K., Le Trong, I., Hinds, T. R., Stenkamp, R., Kimelman, D., and Xu, W. (2004) Mol. Cell 15, 523–533[CrossRef][Medline] [Order article via Infotrieve]
  24. Henderson, B. R. (2000) Nat. Cell Biol. 2, 653–660[CrossRef][Medline] [Order article via Infotrieve]
  25. Henderson, B. R., and Fagotto, F. (2002) EMBO Rep. 3, 834–839[CrossRef][Medline] [Order article via Infotrieve]
  26. Rosin-Arbesfeld, R., Townsley, F., and Bienz, M. (2000) Nature 406, 1009–1012[CrossRef][Medline] [Order article via Infotrieve]
  27. Shih, I. M., Yu, J., He, T. C., Vogelstein, B., and Kinzler, K. W. (2000) Cancer Res. 60, 1671–1676[Abstract/Free Full Text]
  28. Evans, P. M., and Liu, C. (2005) BMC Mol. Biol. 6, 22[Medline] [Order article via Infotrieve]
  29. Brocardo, M., Nathke, I. S., and Henderson, B. R. (2005) EMBO Rep. 6, 184–190[CrossRef][Medline] [Order article via Infotrieve]
  30. Hinoi, T., Yamamoto, H., Kishida, M., Takada, S., Kishida, S., and Kikuchi, A. (2000) J. Biol. Chem. 275, 34399–34406[Abstract/Free Full Text]
  31. Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., Horii, A., Koyama, K., Utsunomiya, J., Baba, S., and Hedge, P. (1991) Science 253, 665–669[Abstract/Free Full Text]
  32. Wang, Z., Vogelstein, B., and Kinzler, K. W. (2003) Cancer Res. 63, 5234–5235[Abstract/Free Full Text]
  33. Kitagawa, M., Hatakeyama, S., Shirane, M., Matsumoto, M., Ishida, N., Hattori, K., Nakamichi, I., Kikuchi, A., Nakayama, K., and Nakayama, K. (1999) EMBO J. 18, 2401–2410[CrossRef][Medline] [Order article via Infotrieve]
  34. Sadot, E., Conacci-Sorrell, M., Zhurinsky, J., Shnizer, D., Lando, Z., Zharhary, D., Kam, Z., Ben-Ze'ev, A., and Geiger, B. (2002) J. Cell Sci. 115 (Pt 13), 2771–2780[Abstract/Free Full Text]
  35. Davies, M. L., Roberts, G. T., Spiller, D. G., and Wakeman, J. A. (2004) Oncogene 23, 1412–1419[CrossRef][Medline] [Order article via Infotrieve]
  36. Krieghoff, E., Behrens, J., and Mayr, B. (2006) J. Cell Sci. 119, 1453–1463[Abstract/Free Full Text]
  37. Matsumoto, T., Iida, M., Kobori, Y., Mizuno, M., Nakamura, S., Hizawa, K., and Yao, T. (2002) Gut 50, 402–404[Abstract/Free Full Text]

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