Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention Joint Metastasis Research Society-AACR Conference on Metastasis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huang, L.
Right arrow Articles by Kufe, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, L.
Right arrow Articles by Kufe, D.
[Cancer Research 65, 10413-10422, November 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

MUC1 Oncoprotein Blocks Glycogen Synthase Kinase 3ß–Mediated Phosphorylation and Degradation of ß-Catenin

Lei Huang, Dongshu Chen, Derek Liu, Li Yin, Surender Kharbanda and Donald Kufe

Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts

Requests for reprints: Donald Kufe, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Phone: 617-632-3141; Fax: 617-632-2934; E-mail: donald_kufe{at}dfci.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dysregulation of ß-catenin is of importance to the development of diverse human malignancies. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and associates with ß-catenin. However, the functional significance of the MUC1-ß-catenin interaction is not known. Here, we show that MUC1 increases ß-catenin levels in the cytoplasm and nucleus of carcinoma cells. Previous studies have shown that glycogen synthase kinase 3ß (GSK3ß) phosphorylates ß-catenin and thereby targets it for proteosomal degradation. Consistent with the up-regulation of ß-catenin levels, our results show that MUC1 blocks GSK3ß-mediated phosphorylation and degradation of ß-catenin. To further define the interaction between MUC1 and ß-catenin, we identified a serine-rich motif (SRM) in the MUC1 cytoplasmic domain that binds directly to ß-catenin Armadillo repeats. Mutation of the SRM attenuated binding of MUC1 to ß-catenin and MUC1-mediated inhibition of ß-catenin degradation. Importantly, disruption of the MUC1-ß-catenin interaction with the SRM mutant also attenuated MUC1-induced anchorage-dependent and -independent growth and delayed MUC1-mediated tumorigenicity. These findings indicate that MUC1 promotes transformation, at least in part, by blocking GSK3ß-mediated phosphorylation and thereby degradation of ß-catenin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Wnt/Wingless signaling pathway is of importance to development and tumorigenesis (1). Wnt-1, a member of the Wnt family, regulates cytoplasmic levels of ß-catenin, a component of the adherens junction of mammalian epithelial cells that, through {alpha}-catenin, links cadherin cell-surface adhesion molecules to the actin cytoskeleton. The abundance of ß-catenin in the cytosol is regulated by glycogen synthase kinase 3ß (GSK3ß; ref. 2) in a complex with the adenomatous polyposis coli (APC) protein (3) and axin (4). After priming ß-catenin by casein kinase 1{alpha}–mediated phosphorylation of Ser-45 (5), GSK3ß phosphorylates ß-catenin on Thr-41, Ser-37, and Ser-33 and thereby targets ß-catenin for recognition by ß-Trcp, ubiquitination, and degradation (5, 6). Down-regulation of GSK3ß by Wnt signaling increases levels of ß-catenin, promotes the formation of nuclear ß-catenin complexes with the Tcf/Lef family of DNA-binding proteins, and thereby activates Wnt target genes (7, 8).

ß-Catenin also interacts with the MUC1 transmembrane glycoprotein (9). MUC1 is expressed on the apical borders of normal epithelial cells (10). With transformation and loss of polarity, MUC1 is aberrantly overexpressed in the cytosol and on the entire cell membrane (10, 11). The MUC1 protein is cleaved into NH2- and COOH-terminal subunits in the endoplasmic reticulum and the two subunits form a stable complex which is expressed at the cell membrane (12). The >250-kDa MUC1 NH2-terminal subunit (MUC1-N) consists of variable numbers of highly glycosylated 20-amino-acid tandem repeats (13). The MUC1 COOH-terminal subunit (MUC1-C) includes a 58-amino-acid extracellular domain, a 28-amino-acid transmembrane domain, and a 72-amino-acid cytoplasmic domain (14). MUC1-C, but not MUC1-N, is targeted to the nucleus (15–18) and to mitochondria (19). Importantly, overexpression of MUC1 confers anchorage-independent growth, tumorigenicity, and resistance to stress-induced apoptosis (17, 19–23).

The MUC1 cytoplasmic domain (MUC1-CD) contains a serine-rich motif (SRM) with homology to sequences in E-cadherin and APC protein that function as ß-catenin binding sites (9). GSK3ß phosphorylates MUC1-CD on serine in a SPY site that is just upstream to the SRM (24). Moreover, the tyrosine in the SPY site is phosphorylated by the epidermal growth factor receptor and the Src family kinases c-Src, Lyn, and Lck (15, 25–27). GSK3ß-mediated phosphorylation of MUC1-CD decreases the interaction with ß-catenin (24). Conversely, tyrosine phosphorylation of the SPY site increases binding of MUC1 and ß-catenin (15, 25–27). Importantly, MUC1-C is detectable in the nucleus in a complex with ß-catenin (15–18) and functions as a coactivator of ß-catenin-Tcf–mediated transcription (20).

In the present work, we show that the MUC1 cytoplasmic domain binds directly to the ß-catenin Armadillo (Arm) repeats. We also show that MUC1 blocks GSK3ß-mediated phosphorylation of ß-catenin and thereby stabilizes ß-catenin. The interaction between MUC1 and ß-catenin increases nuclear levels of dephosphorylated ß-catenin and contributes to the malignant phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human ZR-75-1/vector, ZR-75-1/CsiRNA, and ZR-75-1/MUC1siRNA cells (19) were grown in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine. Human HeLa carcinoma cells, rat 3Y1 fibroblasts (17), and human 293 cells were cultured in DMEM with 10% heat-inactivated FBS, penicillin, streptomycin, and L-glutamine. Cell growth was assessed by trypan blue exclusion and counting viable cells.

Plasmids. The glutathione S-transferase (GST)-ß-catenin, Flag-ß-catenin, and GSK3ß vectors were prepared as described (5, 26). Truncations of ß-catenin were generated by deleting regions of the pCS2-ß-catenin(S33A)-VP-16 vector (28). MUC1-CD with the mutated SRM [MUC1-CD(mSRM)] mutant was generated in pIRESpuro2-MUC1-CD and green fluorescent protein-MUC1-CD (20) using site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA) to mutate SAGNGGSSLS to AAGNGGAAAA.

Transfections. 3Y1 cells were transfected with pIRESpuro2, pIRESpuro2-Flag-MUC1-CD, or pIRESpuro2-Flag-MUC1-CD(mSRM) in the presence of FuGENE 6 (Roche Applied Science, Indianapolis, IN). Stable transfectants were selected in 1.6 µg/mL puromycin (Calbiochem-Novabiochem, San Diego, CA). Two independent transfections were done for each vector. Single-cell clones were isolated by limiting dilution and expanded for analysis. 293 cells were transiently transfected in the presence of Lipofectamine (Invitrogen, Carlsbad, CA).

Immunoblotting and immunoprecipitations. Lysates were prepared from subconfluent cells as described (26). Nuclear and cytoplasmic fractions were prepared as described (16, 19). Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti–MUC1-C (Ab-5; NeoMarkers, Fremont, CA), anti-ß-actin (AC-15; Sigma, St. Louis, MO), anti-ß-catenin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti–lamin B (C-20; Santa Cruz Biotechnology), anti-I{kappa}B{alpha} (H4; Santa Cruz Biotechnology), anti-ß-catenin, anti–green fluorescent protein, anti-etoposide (VP-16; BD Biosciences Clontech, Palo Alto, CA), anti-Flag (M2; Sigma), anti-GSK3ß, anti–phospho-ß-catenin (Cell Signaling Technology, Beverly, MA), and anti–dephospho-ß-catenin (8E4; Alexis Biochemicals, San Diego, CA). Lysates were also first subjected to immunoprecipitation with anti-Flag and the immune complexes were analyzed by immunoblotting.

In vitro binding assays. Full-length and truncated ß-catenin proteins were labeled with [35S]methionine using the TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI). GST and GST fusion proteins (1 µg) immobilized to glutathione-Sepharose beads were incubated with in vitro translated proteins for 4 hours at 4°C. The adsorbates were analyzed by SDS-PAGE and autoradiography.

Pulse-chase analysis. Cells were labeled with 200 µCi/mL [35S]methionine/cysteine (NEG-072; Perkin-Elmer Life Science, Boston, MA) for 30 minutes at 37°C and chased with 1 mg/mL cold methionine/cysteine for the indicated times. Cell lysates were subjected to immunoprecipitation with anti-ß-catenin and protein G-Sepharose (Amersham Biosciences, Piscataway, NJ) for 2 hours at 4°C. The immunoprecipitates were separated by SDS-PAGE and autoradiography.

Luciferase assays. Cells were transfected with pcycD1(–22)-Luc or pcycD1(–161)-Luc (20) and ß-gal in the presence of Lipofectamine. Luciferase assays were done with the Luciferase Assay System (Promega) at 48 hours after transfection. The efficiency of transfection was normalized with the ß-gal expression vector.

Confocal microscopy. Subcellular localization of MUC1-CD and ß-catenin was visualized by confocal microscopy as described (17, 19).

Anchorage-independent growth. Cells (1 x 104) were suspended in 1.5 mL of 0.33% Noble agar (Difco, Detroit, MI) in DMEM supplemented with 10% heat-inactivated FBS and antibiotics. The cell suspension was layered over 3.5 mL of 0.5% agar/medium in 60-mm dishes. Colonies composed of >10 cells were counted at 3 weeks.

Tumorigenicity assays. Cells (1 x 107) were injected s.c. in the flanks of 6- to 8-week-old nude mice and tumor volume was determined by bidimensional measurements as described (17).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MUC1 up-regulates ß-catenin levels in human carcinoma cells. To assess the effects of MUC1 on ß-catenin, we first studied human ZR-75-1 breast cancer cells in which endogenous MUC1 was silenced with a MUC1siRNA. Immunoblot analysis of lysates from MUC1-positive ZR-75-1 cells expressing empty vector or scrambled control CsiRNA showed that ß-catenin levels are higher than that found in two separately isolated ZR-75-1 cell clones expressing the MUC1siRNA (Fig. 1A). Subcellular fractionation further showed that MUC1 silencing is associated with decreases in both nuclear and cytoplasmic ß-catenin levels (Fig. 1B). Purity of the nuclear and cytoplasmic fractions was confirmed by immunoblotting with antibodies against the nuclear lamin B and cytoplasmic I{kappa}B{alpha} proteins (Fig. 1B). To assess gain of MUC1 function, HCT116 colon cancer cells, which are negative for MUC1 and express a mutant ß-catenin (8), were stably transfected with an empty vector or MUC1 (25). HCT116/vector and HCT116/MUC1 cells exhibited similar levels of ß-catenin (Fig. 1C). By contrast, HeLa cervical carcinoma cells stably expressing the empty vector or MUC1 showed that expression of exogenous MUC1 is associated with marked up-regulation of ß-catenin levels (Fig. 1C). In HCT116 cells, MUC1 showed no apparent effect on localization of ß-catenin to the nucleus and cytoplasm (Fig. 1D). However, in HeLa cells, MUC1-induced increases in ß-catenin levels were found predominantly in the nucleus (Fig. 1D). These findings indicated that MUC1 increases ß-catenin levels in ZR-75-1 and HeLa cells, which, unlike HCT116 cells, are not known to express a mutant and thereby stable form of ß-catenin.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 1. MUC1 increases cytoplasmic and nuclear levels of ß-catenin in human carcinoma cells. A, lysates of human ZR-75-1 breast cancer cells stably expressing empty vector, control siRNA (CsiRNA), or MUC1siRNA were subjected to immunoblotting with the indicated antibodies. B, nuclear and cytoplasmic fractions from the indicated ZR-75-1 cell clones were analyzed by immunoblotting with antibodies against ß-catenin, MUC1-C, nuclear lamin B, and cytoplasmic I{kappa}B{alpha}. C and D, human HCT116 colon cancer cells (mutant ß-catenin) and HeLa cervical cancer cells were stably transfected to express an empty vector or MUC1. Lysates (C) or nuclear and cytoplasmic fractions (D) were immunoblotted with the indicated antibodies.

 
MUC1 cytoplasmic domain increases ß-catenin levels in 3Y1 cells. MUC1-CD associates with ß-catenin (9). To determine if MUC1-CD is sufficient to up-regulate ß-catenin, we stably transfected 3Y1 cells with empty vector or one expressing Flag-tagged MUC1-CD. Immunoblot analysis of the transfectants showed reactivity with the ~10-kDa Flag-MUC1-CD protein in 3Y1/MUC1-CD, but not in 3Y1/vector, cells (Fig. 2A, left). Two clones for each of the 3Y1/vector and 3Y1/MUC1-CD cells were selected from independent transfections (Fig. 2A, left). Immunoblot analysis of subcellular fractions further showed expression of MUC1-CD in both the nucleus and cytosol (Fig. 2A, right). Notably, coimmunoprecipitation studies showed that MUC1-CD is sufficient for binding to ß-catenin in 3Y1 cells (Fig. 2B). Moreover, consistent with the findings in ZR-75-1 and HeLa cells, ß-catenin levels were increased in 3Y1/MUC1-CD cells as compared with those in 3Y1/vector cells (Fig. 2C, left). Subcellular fractionation also showed that the MUC1-CD–induced increases in ß-catenin are detectable in both the nuclear and cytoplasmic fractions (Fig. 2C, right). Confocal microscopy further showed that MUC1-CD colocalizes with ß-catenin predominantly in the nucleus (Fig. 2D). These findings indicate that MUC1-CD confers binding to ß-catenin and increases ß-catenin levels.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 2. MUC1-CD increases cytoplasmic and nuclear levels of ß-catenin in 3Y1 cells. A, 3Y1 cells were stably transfected to express empty vector or MUC1-CD. Left, whole-cell lysates from the indicated clones were subjected to immunoblotting with anti–MUC1-C and anti-ß-actin. Right, nuclear and cytoplasmic fractions from the indicated 3Y1 cells were subjected to immunoblot analysis with antibodies against MUC1-C, nuclear lamin B, and cytoplasmic I{kappa}B{alpha}. B, anti-Flag immunoprecipitates from the indicated 3Y1 cells were analyzed by immunoblotting with anti-ß-catenin and anti–MUC1-C. C, whole-cell lysates from the indicated cells were subjected to immunoblotting with anti-ß-catenin and anti-ß-actin (left). Nuclear and cytoplasmic fractions from the indicated cells were subjected to immunoblot analysis with anti-ß-catenin, anti–lamin B, and anti-I{kappa}B{alpha} (right). D, 3Y1/vector and 3Y1/MUC1-CD cells were fixed, permeabilized, and incubated with anti–MUC1-C and anti-ß-catenin. Nuclei were stained with TO-PRO-3. The cells were analyzed by confocal microscopy. Similar results were obtained with the separately isolated clones.

 
MUC1-CD binds to the ß-catenin Armadillo repeats. ß-Catenin includes 12 Arm repeats that are flanked by nonrepetitive NH2-terminal (amino acids 1-108) and COOH-terminal (amino acids 623-781) regions (Supplementary Fig. S1A). To identify the region(s) of ß-catenin that interacts with MUC1, we first incubated GST-MUC1-CD with 35S-labeled ß-catenin(1-575) or ß-catenin(552-781). Binding of MUC1-CD was detectable with both ß-catenin(1-575) and ß-catenin(552-781) (Supplementary Fig. S1B). As controls, there was no detectable binding of GST to either ß-catenin fragment (Supplementary Fig. S1B). To further define the binding sites, we first generated deletion mutants of ß-catenin(1-575) (Supplementary Fig. S1A). There was no detectable binding of MUC1-CD to ß-catenin(1-101) or ß-catenin(101-301) (Supplementary Fig. S1C). However, MUC1-CD binding was detectable with ß-catenin(101-575) and ß-catenin(301-575) (Supplementary Fig. S1C). Moreover, there was no detectable binding of MUC1-CD to ß-catenin(101-301/536-575) (data not shown), indicating that MUC1-CD binds to ß-catenin in the region (amino acids 301-536) that includes Arm repeats R6-R10. Deletion mutants of ß-catenin(552-781) were also generated to define binding of MUC1-CD to the ß-catenin COOH terminus. MUC1-CD formed complexes with ß-catenin(552-710) and ß-catenin(624-781), but not with ß-catenin(708-781) (Supplementary Fig. S1D), consistent with binding to ß-catenin between amino acids 624 and 708. To assess binding in vivo, we expressed Flag-MUC1-CD and VP-16-tagged ß-catenin in 293 cells. Immunoblot analysis of anti-Flag immunoprecipitates with anti-VP-16 showed binding of MUC1-CD to ß-catenin(1-781) (Supplementary Fig. S1E). In agreement with the in vitro data, MUC1-CD also formed complexes with ß-catenin(101-575) and ß-catenin(301-575), but not with ß-catenin(1-101) or ß-catenin(101-301) (Supplementary Fig. S1E). In addition, MUC1-CD associated with ß-catenin(552-781), ß-catenin(552-710), and ß-catenin(624-781), but not with ß-catenin(708-781) (Supplementary Fig. S1E). These findings indicate that MUC1 binds to both the ß-catenin Arm repeats and the nonrepetitive COOH-terminal region.

MUC1 serine-rich motif is necessary for binding to ß-catenin. MUC1-CD contains a SRM (SAGNGGSSLS; amino acids 50-59, Supplementary Fig. S2A) with homology to motifs in E-cadherin and APC that confer binding to ß-catenin. Consistent with involvement of the SRM, MUC1-CD(1-59) formed complexes with ß-catenin(1-575) (Supplementary Fig. S2B). In addition, binding of MUC1-CD to ß-catenin(1-575) was substantially decreased with MUC1-CD(1-45) which is devoid of the SRM (Supplementary Fig. S2B). Similar results were obtained when these MUC1-CD deletion mutants were incubated with ß-catenin(552-781) (Supplementary Fig. S2B). To extend this analysis, we assessed binding of MUC1 to ß-catenin after mutating the SRM (SAGNGGSSLS->AAGNGGAAAA) (Supplementary Fig. S2A). MUC1-CD(mSRM) was less effective than wild-type MUC1-CD in forming complexes with ß-catenin (Supplementary Fig. S2C). Consistent with these data, binding of MUC1-CD to both ß-catenin(301-575) and ß-catenin(624-781) was decreased with the mSRM mutant (Supplementary Fig. S2D). In cells, binding of MUC1-CD to ß-catenin(301-575) was also substantially decreased with MUC1-CD(mSRM) (Supplementary Fig. S2E). By contrast, the MUC1-CD(mSRM) mutant had little, if any, effect on binding to ß-catenin(624-781) in cells (Supplementary Fig. S2E). These findings indicate that the SRM motif is necessary for binding of MUC1-CD to ß-catenin(301-575) in vitro and in vivo. The results also indicate that MUC1-CD binds directly to ß-catenin(624-781) in vitro by a mechanism dependent on the SRM motif and that binding to this region can occur indirectly in cells.

MUC1 blocks glycogen synthase kinase 3ß–mediated phosphorylation of ß-catenin. Destruction of ß-catenin is mediated by GSK3ß phosphorylation of the ß-catenin NH2 terminus (1). Phosphorylated ß-catenin is necessary for recognition by ß-Trcp, a component of the E3 ubiquitin ligase. To determine if MUC1 affects GSK3ß-mediated phosphorylation, we probed ß-catenin with an antibody that detects phosphorylated (Ser-33, Ser-37, and Thr-41) residues. We also used an antibody specific for ß-catenin that is not phosphorylated on Ser-33 and Ser-37. The anti–dephospho-ß-catenin antibody is thus different from anti-ß-catenin antibodies that do not distinguish between phosphorylated or dephosphorylated forms. In agreement with stabilization of ß-catenin, MUC1 expression was associated with decreases in phospho-ß-catenin and a corresponding up-regulation of dephospho-ß-catenin (Fig. 3A). In HeLa cells, MUC1-induced up-regulation of ß-catenin was associated with more pronounced increases in dephospho-ß-catenin as compared with that obtained for phospho-ß-catenin (Fig. 3B), consistent with MUC1-mediated attenuation of GSK3ß phosphorylation. To extend these findings, 293 cells were transiently transfected to assess the effects of MUC1-CD on GSK3ß-mediated phosphorylation of ß-catenin. GSK3ß increased ß-catenin phosphorylation and decreased ß-catenin levels (Fig. 3C, lane 2). The results also show that MUC1-CD decreases phospho-ß-catenin and reverses GSK3ß-induced down-regulation of ß-catenin (Fig. 3C, lane 3). By contrast, the effects of MUC1-CD on GSK3ß-mediated phosphorylation and down-regulation of ß-catenin were attenuated with the MUC1-CD(mSRM) mutant (Fig. 3C, lane 4). MUC1-CD functions as a substrate for GSK3ß phosphorylation (24). MUC1 could thus decrease phosphorylation of ß-catenin by competing with ß-catenin as a GSK3ß substrate. Consistent with such an effect, the addition of MUC1-CD to an in vitro GSK3ß kinase reaction attenuated the phosphorylation of ß-catenin (Fig. 3D). These findings indicate that MUC1 blocks GSK3ß-mediated phosphorylation of ß-catenin in vitro and in cells.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 3. MUC1-CD attenuates GSK3ß-mediated phosphorylation. A and B, lysates from the indicated ZR-75-1 (A) or HeLa (B) cells were immunoblotted with antibodies that react with ß-catenin phosphorylated on Ser-33, Ser-37, and Thr-41 (phospho-ß-catenin) or not phosphorylated on Ser-33 and Ser-37 (dephospho-ß-catenin). C, lysates from 293 cells transfected with Flag-ß-catenin, GSK3ß, and/or MUC1-CD were immunoblotted with the indicated antibodies. D, purified GST-ß-catenin (0.25 µg), GSK3ß (500 units; New England Biolabs, Inc., Beverly, MA), and 200 µmol/L ATP were incubated in kinase buffer as indicated in the absence and presence of His-MUC1-CD (0.1 µg) for 1 hour at 30°C. The reaction products were immunoblotted with the indicated antibodies.

 
MUC1-CD stabilizes ß-catenin. Studies done on 3Y1/vector and 3Y1/MUC1-CD cells confirmed that MUC1-CD decreases phosphorylation of ß-catenin (Fig. 4A). Analysis of subcellular fractions further showed that the MUC1-CD–induced increases in dephospho-ß-catenin are found predominantly in the nucleus (Fig. 4B). To determine if MUC1-CD affects ß-catenin stability, we pulsed 3Y1/vector and 3Y1/MUC1-CD cells with [35S]methionine and immunoprecipitated ß-catenin at different times during a chase. Autoradiography of the immunoprecipitates showed that MUC1-CD stabilizes ß-catenin (Fig. 4C). Densitometric scanning of the signals showed that the calculated half-lives of ß-catenin in 3Y1/vector and 3Y1/MUC1-CD cells are <0.5 and >2 hours, respectively (Fig. 4D). These findings indicate that MUC1-CD stabilizes ß-catenin by attenuating GSK3ß-mediated phosphorylation of the ß-catenin NH2-terminal region.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 4. MUC1-CD increases nuclear levels of dephospho-ß-catenin. A, whole-cell lysates from 3Y1/vector and 3Y1/MUC1-CD cells were immunoblotted with the indicated antibodies. B, nuclear and cytoplasmic fractions from the indicated cells were immunoblotted with anti–dephospho-ß-catenin, anti–lamin B, and anti-I{kappa}B{alpha}. C, 3Y1/vector and 3Y1/MUC1-CD cells were pulsed with [35S]methionine, washed, and then chased in complete medium for the indicated times. Anti-ß-catenin immunoprecipitates were subjected to SDS-PAGE and autoradiography. D, intensity of the ß-catenin signals was determined by densitometric scanning and plotted as the percentage of ß-catenin remaining compared with the control (0 hours). Similar results were obtained in two separate experiments.

 
Binding of MUC1-CD to ß-catenin is necessary for increasing dephospho-ß-catenin. To further assess the effects of the interaction between MUC1 and ß-catenin, 3Y1 cells were stably transfected with MUC1-CD(mSRM). Immunoblot analysis of separately isolated MUC1-CD(mSRM) clones showed expression levels higher than that found with wild-type MUC1-CD (Fig. 5A). As expected from the in vitro and transient expression studies (Supplementary Fig. S2), binding of MUC1-CD to ß-catenin was decreased in 3Y1/MUC1-CD(mSRM) cells (Fig. 5B). Moreover, compared with MUC1-CD, MUC1-CD(mSRM) was less effective in increasing dephospho-ß-catenin and ß-catenin levels (Fig. 5C). Dephospho-ß-catenin activates Tcf-mediated transcription (29). To determine if MUC1-CD–induced increases in dephospho-ß-catenin affect Tcf-dependent reporter activity, 3Y1/vector, 3Y1/MUC1-CD, and 3Y1-MUC1-CD(mSRM) cells were studied for activation of pcycD1(–161)-Luc, which contains three Tcf-binding sites, as compared with the minimal pcycD1(–22)-Luc reporter. MUC1-CD stimulated transcription about 9-fold relative to that found in 3Y1/vector cells (Fig. 5D). By contrast, the effects of MUC1-CD on activation of the cyclin D1 promoter were substantially decreased by mutation of the SRM (Fig. 5D). These findings indicate that binding of MUC1-CD to ß-catenin in 3Y1 cells contributes to dephosphorylation of ß-catenin and activation of ß-catenin-Tcf–mediated transcription.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 5. MUC1-CD–induced regulation of ß-catenin is attenuated by the mSRM mutation. A, 3Y1 cells were transfected to stably express MUC1-CD(mSRM). Lysates from 3Y1/vector, 3Y1/MUC1-CD, and two separately isolated clones (A and B) of 3Y1/MUC1-CD(mSRM) cells were immunoblotted with the indicated antibodies. B, lysates from the indicated cells were immunoprecipitated with immunoglobulin G (IgG) or anti-Flag. The precipitates were immunoblotted with the indicated antibodies. C, lysates from the indicated cells were immunoblotted with antibodies against dephospho-ß-catenin, ß-catenin, and ß-actin. D, the indicated cells were transfected with pcycD1(–161)-Luc or pcycD1(–22)-Luc and ß-gal. Luciferase activity was assessed at 48 hours after transfection. Results expressed as the ratio of pcycD1(–161)/pcycD1(–22) activity compared with that obtained in 3Y1/vector cells (assigned value of 1). Columns, mean of three separate experiments; bars, SD.

 
MUC1-CD–induced growth and tumorigenicity of 3Y1 cells is attenuated by mutation of the serine-rich motif. Analysis of cell number after seeding in tissue culture flasks showed that MUC1-CD has a marked stimulatory effect on growth compared with 3Y1/vector cells (Fig. 6A). Notably, however, MUC1-CD(mSRM) had little, if any, growth stimulatory effect (Fig. 6A). In soft agar, there was no detectable growth of 3Y1/vector cells (Fig. 6B). Colonies of 3Y1/MUC1-CD cells were, however, readily apparent by 10 to 14 days (Fig. 6B). Colonies of 3Y1/MUC1-CD(mSRM) cells were also visible but to a significantly lesser extent than those expressing MUC1-CD (Fig. 6B). The plating efficiency was determined by seeding 104 cells and then counting colonies on day 14. The results from three independent experiments showed plating efficiencies of 16% and 4.5% for the 3Y1/MUC1-CD-A and 3Y1/MUC1-CD-B clones, respectively (Fig. 6B). Plating efficiency for the 3Y1/MUC1-CD(mutSRM) cell clones was ≤1% (Fig. 6B). To determine if the interaction between MUC1 and ß-catenin contributes to tumorigenicity, 107 3Y1/vector, 3Y1/MUC1-CD, or 3Y1/MUC1-CD(mSRM) cells were injected s.c. into nude mice. Significantly, tumors were detectable in mice injected with 3Y1/MUC1-CD cells but not in mice injected with 3Y1/vector (Fig. 6C), indicating that MUC1-CD is sufficient for conferring tumorigenesis. Moreover, 3Y1/MUC1-CD(mSRM) cells formed tumors that were delayed compared with that found with the 3Y1/MUC1-CD clones (Fig. 6C). Staining of the 3Y1/MUC1-CD tumors with anti-MUC1 confirmed that the cells maintain expression of MUC1-CD (Fig. 6D). Confocal microscopy of tumor sections further showed that, as found for cultured cells, MUC1-CD colocalizes with ß-catenin in the nucleus (Fig. 6D). These results indicate that MUC1-CD confers increases in anchorage-dependent and anchorage-independent growths and that these effects are attenuated by disrupting the interaction between MUC1 and ß-catenin. The results also indicate that mutation of the SRM attenuates MUC1-CD–induced tumorigenicity.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 6. MUC1-CD confers anchorage-dependent and -independent growths. A, the indicated cells (1 x 104) were seeded in triplicate wells of a 6-well plate and grown in complete medium at 37°C. Cell were stained with trypan blue and counted on the indicated days. B, the indicated cells (1 x 104 per plate) were suspended in soft agar and incubated for 3 weeks. Representative photomicrographs depict colony formation. Colonies larger than 10 cells were counted for each of the indicated cells. Points, mean number of colonies obtained in three independent experiments; bars, SD. C, the indicated cells (1 x 107) were injected s.c. into the posterior flank of nude mice. Tumor volumes were calculated from bidimensional measurements on the indicated days after injection. Points, mean tumor volume obtained from two independent experiments that each included six to eight mice; bars, SD. D, sections of formalin-fixed, paraffin-embedded 3Y1/MUC1-CD tumors were stained with H&E, control IgG, or anti–MUC1-C. Top, photomicrographs obtained at x200 magnification. Fixed sections from a 3Y1/MUC1-CD tumor were permeabilized and incubated with anti–MUC1-C and anti-ß-catenin. Nuclei were stained with TO-PRO-3. Bottom, confocal microscopy was done to assess colocalization of MUC1-CD and ß-catenin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MUC1 increases cytosolic and nuclear ß-catenin levels. GSK3ß-mediated phosphorylation of ß-catenin (5) is essential for recognition by the F-box protein ß-Trcp and thereby targeting of ß-catenin for ubiquitination and degradation (30, 31). The importance of regulating ß-catenin stability is supported by the findings that GSK3ß phosphorylation sites are mutated in human colorectal cancers and other malignancies (7, 8, 32). Loss of APC or axin has also been linked to accumulation of ß-catenin and the development of human carcinomas (7, 33). In addition, ß-catenin is increased in human breast cancers in the absence of APC, axin, or ß-catenin mutations, indicating that other events can contribute to the dysregulation of this protein (34, 35). The present studies show that the interaction between MUC1 and ß-catenin represents another mechanism that regulates ß-catenin stabilization. Silencing of MUC1 in ZR-75-1 breast cancer cells was associated with decreases in ß-catenin levels. Consistent with these results, introduction of MUC1 into HeLa cells increased ß-catenin levels. In addition, stable expression of the MUC1 cytoplasmic domain (MUC1-CD) was sufficient to up-regulate ß-catenin in 3Y1 cells. In these cell models, MUC1 expression was associated with increases in both cytosolic and nuclear ß-catenin levels. Notably, MUC1 had no detectable effect on ß-catenin mRNA levels (data not shown). In addition, MUC1 had no effect on ß-catenin levels in HCT116 cells that express a mutant and thereby stable form of ß-catenin (1). MUC1 associates with ß-catenin (9, 24) and colocalizes with ß-catenin in the nucleus (15, 17, 18, 36). The present work further shows that the MUC1 cytoplasmic domain is sufficient for up-regulation of ß-catenin in cells and for nuclear colocalization with ß-catenin. These findings collectively indicated that binding of the MUC1 cytoplasmic domain to ß-catenin contributes to ß-catenin stabilization and its nuclear accumulation.

MUC1-CD binds directly to the ß-catenin Armadillo repeats and COOH-terminal region. MUC1-CD contains a SRM (SAGNGGSSLS) that is similar to sites in E-cadherin (SEAASLSSLN) and APC (SRXSSLSSLS) that bind to ß-catenin (37–39). Moreover, like E-cadherin and APC (40, 41), we found that MUC1-CD binds to the ß-catenin Arm repeats. E-Cadherin binds to Arm repeats R3 through R8 (42). APC binding to ß-catenin has been mapped to Arm repeats R3 to R4 and R5 to R8 (35, 42). Binding of axin, conductin, Tcf, and Pin1 to ß-catenin has also been mapped to Arm repeats R2 to R7 (43), R3 to R7 (44), R1 to R2 (40), and R3 (35), respectively. Our data indicate that MUC1-CD binds to Arm repeats R6 to R10. How the first 10 Arm repeats bind to all of these different partners is not clear. However, structural analysis of tandem Arm repeats showed that helices in the repeats pack together to form a platform with an elongated surface that can accommodate diverse binding partners (41, 45). E-Cadherin and APC form mutually exclusive complexes with ß-catenin and may compete for binding (42, 46). Moreover, the observation that E-cadherin and MUC1 compete for binding to ß-catenin may also be explained by interactions with overlapping regions within the Arm repeats (9). The results also show that the MUC1-CD binds to the ß-catenin COOH-terminal region, which confers the ß-catenin cotranscription function. As found for the Arm repeats, the MUC1-CD SRM sequences were required for direct binding to ß-catenin(624-781) in vitro. However, in contrast to the Arm repeats, binding of MUC1-CD to ß-catenin(624-781) in cells was independent of the SRM motif, suggesting that this interaction can also be mediated by an indirect mechanism, perhaps through another protein.

Binding of MUC1 to ß-catenin attenuates glycogen synthase kinase 3ß phosphorylation of ß-catenin. The ß-catenin degradation complex includes GSK3ß, APC, axin, and casein kinase 1{alpha}. In addition to its role in regulating ß-catenin, GSK3ß phosphorylates APC and axin and thereby increases their binding to ß-catenin (2, 47, 48). The present results show that MUC1 attenuates GSK3ß-mediated phosphorylation and degradation of ß-catenin. In addition, these effects were attenuated with MUC1 mutated at the SRM in the cytoplasmic domain, lending support to a mechanism involving binding of MUC1-CD to the ß-catenin Arm repeats. Of note, MUC1 had no apparent effect on casein kinase 1{alpha}–mediated phosphorylation of ß-catenin at Ser-45, which primes ß-catenin for GSK3ß phosphorylation (data not shown). As such, our results indicate that MUC1 selectively disrupts GSK3ß-mediated phosphorylation. Other studies have shown that GSK3ß phosphorylates MUC1-CD on Ser-44 (24). Thus, binding of MUC1 to ß-catenin could result in a setting in which MUC1 and ß-catenin compete as substrates for GSK3ß phosphorylation. In this model, overexpression of MUC1, as found in human breast and other carcinomas, could competitively decrease ß-catenin phosphorylation and thereby result in increased ß-catenin levels. The MUC1 Ser-44 site is closely upstream to the SRM that confers binding to ß-catenin (Supplementary Fig. S2A). Moreover, previous work has shown that GSK3ß phosphorylation of MUC1 on Ser-44 decreases the association between MUC1 and ß-catenin (24). These findings collectively support the possibility that, under physiologic conditions in which MUC1 is not overexpressed, the interaction between MUC1 and ß-catenin is tightly regulated by GSK3ß and that MUC1 may transiently stabilize ß-catenin before GSK3ß induces dissociation of the MUC1-ß-catenin complex.

MUC1-induced transformation is mediated by the interaction of MUC1-CD with ß-catenin. MUC1 is aberrantly overexpressed by most human carcinomas (10) and certain hematologic malignancies, such as multiple myeloma (49). Overexpression of MUC1 induces a transformed phenotype and resistance to apoptosis (17, 19–21, 23, 50). The present studies show that the MUC1 cytoplasmic domain is sufficient to increase anchorage-dependent growth. MUC1-CD was also sufficient to confer growth in soft agar and tumorigenicity. As found in cultured cells, confocal microscopy confirmed nuclear colocalization of MUC1-CD and ß-catenin in 3Y1/MUC1-CD tumor cells. Importantly, the results also show that mutation of the MUC1-CD SRM, and thereby disruption of the interaction between MUC1-CD and ß-catenin, attenuates MUC1-CD–induced anchorage-dependent and -independent growths in soft agar and delays the formation of tumors. MUC1-CD also attenuated the apoptotic response of 3Y1 cells to genotoxic stress.1 However, this MUC1-CD–induced phenotype was not reversed with the SRM mutant, consistent with the interaction of MUC1-CD (amino acids 9-45) with p53 and attenuation of p53-induced apoptosis (23). These findings thus support a model in which MUC1 contributes to the malignant phenotype through its interaction with ß-catenin.


   Acknowledgments
 
Grant support: U.S. Army grant BC02215 and National Cancer Institute grants CA29431 and CA97098.

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.

We thank Dr. Xi He for the Flag-ß-catenin plasmid and Kamal Chauhan for technical support.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

1 Unpublished data. Back

Received 7/15/05. Revised 8/25/05. Accepted 9/ 8/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Polakis P. Wnt signaling and cancer. Genes Dev 2000;14:1837–51.[Free Full Text]
  2. Rubinfeld B, Albert I, Porfiri E, et al. Binding of GSK3ß to the APC-ß-catenin complex and regulation of complex assembly. Science 1996;272:1023–6.[Abstract]
  3. Munemitsu S, Albert I, Souza B, Rubinfeld B, Polakis P. Regulation of intracellular ß-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc Natl Acad Sci U S A 1995;92:3046–50.[Abstract/Free Full Text]
  4. Zeng L, Fagotto F, Zhang T, et al. The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell 1997;90:181–92.[CrossRef][Medline]
  5. Liu C, Li Y, Semenov M, et al. Control of ß-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 2002;108:837–47.[CrossRef][Medline]
  6. Orford K, Crockett C, Jensen J, Weissman A, Byers S. Serine phosphorylation-regulated ubiquitination and degradation of ß-catenin. J Biol Chem 1997;272:24735–8.[Abstract/Free Full Text]
  7. Korinek V, Barker N, Morin PJ, et al. Constitutive transcriptional activation by a ß-catenin-Tcf complex in APC–/– colon carcinoma. Science 1997;275:1784–7.[Abstract/Free Full Text]
  8. Morin PJ, Sparks AB, Korinek V, et al. Activation of ß-catenin-Tcf signaling in colon cancer by mutations in ß-catenin or APC. Science 1997;275:1787–90.[Abstract/Free Full Text]
  9. Yamamoto M, Bharti A, Li Y, Kufe D. Interaction of the DF3/MUC1 breast carcinoma-associated antigen and ß-catenin in cell adhesion. J Biol Chem 1997;272:12492–4.[Abstract/Free Full Text]
  10. Kufe D, Inghirami G, Abe M, et al. Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors. Hybridoma 1984;3:223–32.[Medline]
  11. Croce MV, Isla-Larrain MT, Rua CE, et al. Patterns of MUC1 tissue expression defined by an anti-MUC1 cytoplasmic tail monoclonal antibody in breast cancer. J Histochem Cytochem 2003;51:781–8.[Abstract/Free Full Text]
  12. Ligtenberg MJ, Kruijshaar L, Buijs F, et al. Cell-associated episialin is a complex containing two proteins derived from a common precursor. J Biol Chem 1992;267:6171–7.[Abstract/Free Full Text]
  13. Siddiqui J, Abe M, Hayes D, et al. Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen. Proc Natl Acad Sci U S A 1988;85:2320–3.[Abstract/Free Full Text]
  14. Merlo G, Siddiqui J, Cropp C, et al. DF3 tumor-associated antigen gene is located in a region on chromosome 1q frequently altered in primary human breast cancer. Cancer Res 1989;49:6966–71.[Medline]
  15. Li Y, Chen W, Ren J, et al. DF3/MUC1 signaling in multiple myeloma cells is regulated by interleukin-7. Cancer Biol Ther 2003;2:187–93.[Medline]
  16. Li Y, Yu W-H, Ren J, et al. Heregulin targets {gamma}-catenin to the nucleolus by a mechanism dependent on the DF3/MUC1 protein. Mol Cancer Res 2003;1:765–75.[Abstract/Free Full Text]
  17. Li Y, Liu D, Chen D, Kharbanda S, Kufe D. Human DF3/MUC1 carcinoma-associated protein functions as an oncogene. Oncogene 2003;22:6107–10.[CrossRef][Medline]
  18. Wen Y, Caffrey T, Wheelock M, Johnson K, Hollingsworth M. Nuclear association of the cytoplasmic tail of MUC1 and ß-catenin. J Biol Chem 2003;278:38029–39.[Abstract/Free Full Text]
  19. Ren J, Agata N, Chen D, et al. Human MUC1 carcinoma-associated protein confers resistance to genotoxic anti-cancer agents. Cancer Cell 2004;5:163–75.[CrossRef][Medline]
  20. Huang L, Ren J, Chen D, et al. MUC1 cytoplasmic domain coactivates Wnt target gene transcription and confers transformation. Cancer Biol Ther 2003;2:702–6.[Medline]
  21. Raina D, Kharbanda S, Kufe D. The MUC1 oncoprotein activates the anti-apoptotic PI3K/Akt and Bcl-xL pathways in rat 3Y1 fibroblasts. J Biol Chem 2004;279:20607–12.[Abstract/Free Full Text]
  22. Yin L, Huang L, Kufe D. MUC1 oncoprotein activates the FOXO3a transcription factor in a survival response to oxidative stress. J Biol Chem 2004;279:45721–7.[Abstract/Free Full Text]
  23. Wei X, Xu H, Kufe D. Human MUC1 oncoprotein regulates p53-responsive gene transcription in the genotoxic stress response. Cancer Cell 2005;7:167–78.[CrossRef][Medline]
  24. Li Y, Bharti A, Chen D, Gong J, Kufe D. Interaction of glycogen synthase kinase 3ß with the DF3/MUC1 carcinoma-associated antigen and ß-catenin. Mol Cell Biol 1998;18:7216–24.[Abstract/Free Full Text]
  25. Li Y, Ren J, Yu W-H, et al. The EGF receptor regulates interaction of the human DF3/MUC1 carcinoma antigen with c-Src and ß-catenin. J Biol Chem 2001;276:35239–42.[Abstract/Free Full Text]
  26. Li Y, Kuwahara H, Ren J, Wen G, Kufe D. The c-Src tyrosine kinase regulates signaling of the human DF3/MUC1 carcinoma-associated antigen with GSK3ß and ß-catenin. J Biol Chem 2001;276:6061–4.[Abstract/Free Full Text]
  27. Li Q, Ren J, Kufe D. Interaction of human MUC1 and ß-catenin is regulated by Lck and ZAP-70 in activated Jurkat T cells. Biochem Biophys Res Commun 2004;315:471–6.[CrossRef][Medline]
  28. Hecht A, Vleminckx K, Stemmler MP, van Roy F, Kemler R. The p300/CBP acetyltransferases function as transcriptional coactivators of ß-catenin in vertebrates. EMBO J 2000;19:1839–50.[CrossRef][Medline]
  29. Staal FJ, Noort Mv M, Strous GJ, Clevers HC. Wnt signals are transmitted through N-terminally dephosphorylated ß-catenin. EMBO Rep 2002;3:63–8.[CrossRef][Medline]
  30. Hart M, Concordet JP, Lassot I, et al. The F-box protein ß-TrCP associates with phosphorylated ß-catenin and regulates its activity in the cell. Curr Biol 1999;9:207–10.[CrossRef][Medline]
  31. Winston JT, Strack P, Beer-Romero P, et al. The SCFß-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in I{kappa}B{alpha} and ß-catenin and stimulates I{kappa}B{alpha} ubiquitination in vitro. Genes Dev 1999;13:270–83.[Abstract/Free Full Text]
  32. Rubinfeld B, Robbins P, El-Gamil M, et al. Stabilization of ß-catenin by genetic defects in melanoma cell lines. Science 1997;275:1790–2.[Abstract/Free Full Text]
  33. Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell 2000;103:311–20.[CrossRef][Medline]
  34. Lin SY, Xia W, Wang JC, et al. ß-Catenin, a novel prognostic marker for breast cancer: its roles in cyclin D1 expression and cancer progression. Proc Natl Acad Sci U S A 2000;97:4262–6.[Abstract/Free Full Text]
  35. Ryo A, Nakamura M, Wulf G, Liou YC, Lu KP. Pin1 regulates turnover and subcellular localization of ß-catenin by inhibiting its interaction with APC. Nat Cell Biol 2001;3:793–801.[CrossRef][Medline]
  36. Baldus SE, Monig SP, Huxel S, et al. MUC1 and nuclear ß-catenin are coexpressed at the invasion front of colorectal carcinomas and are both correlated with tumor prognosis. Clin Cancer Res 2004;10:2790–6.[Abstract/Free Full Text]
  37. Su L-K, Vogelstein B, Kinzler KW. Association of the APC tumor suppressor protein with catenins. Science 1993;262:1734–7.[Abstract/Free Full Text]
  38. Rubinfeld B, Souza B, Albert I, et al. Association of the APC gene product with ß-catenin. Science 1993;262:1731–4.[Abstract/Free Full Text]
  39. Hoschuetzky H, Aberle H, Kemler R. ß-Catenin mediates the interaction of the cadherin-catenin complex with epidermal growth factor receptor. J Cell Biol 1994;127:1375–80.[Abstract/Free Full Text]
  40. Graham TA, Weaver C, Mao F, Kimelman D, Xu W. Crystal structure of a ß-catenin/Tcf complex. Cell 2000;103:885–96.[CrossRef][Medline]
  41. Huber AH, Nelson WJ, Weis WI. Three-dimensional structure of the armadillo repeat region of ß-catenin. Cell 1997;90:871–82.[CrossRef][Medline]
  42. Hulsken J, Birchmeier W, Behrens J. E-cadherin and APC compete for the interaction with ß-catenin and the cytoskeleton. J Cell Biol 1994;127:2061–9.[Abstract/Free Full Text]
  43. Xing Y, Clements WK, Kimelman D, Xu W. Crystal structure of a ß-catenin/axin complex suggests a mechanism for the ß-catenin destruction complex. Genes Dev 2003;17:2753–64.[Abstract/Free Full Text]
  44. Behrens J, Jerchow BA, Wurtele M, et al. Functional interaction of an axin homolog, conductin, with ß-catenin, APC, and GSK3ß. Science 1998;280:596–9.[Abstract/Free Full Text]
  45. Shapiro L. ß-Catenin and its multiple partners: promiscuity explained. Nat Struct Biol 2001;8:484–7.[CrossRef][Medline]
  46. Rubinfeld B, Souza B, Albert I, Munemitsu S, Polakis P. The APC protein and E-cadherin form similar but independent complexes with {alpha}-catenin, ß-catenin, and plakoglobin. J Biol Chem 1995;270:5549–55.[Abstract/Free Full Text]
  47. Yamamoto H, Kishida S, Kishida M, et al. Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3ß regulates its stability. J Biol Chem 1999;274:10681–4.[Abstract/Free Full Text]
  48. Willert K, Shibamoto S, Nusse R. Wnt-induced dephosphorylation of axin releases ß-catenin from the axin complex. Genes Dev 1999;13:1768–73.[Abstract/Free Full Text]
  49. Takahashi T, Makiguchi Y, Hinoda Y, et al. Expression of MUC-1 on myeloma cells and induction of HLA-unrestricted CTL against MUC1 from a multiple myeloma patient. J Immunol 1994;153:2102–9.[Abstract]
  50. Schroeder JA, Masri AA, Adriance MC, et al. MUC1 overexpression results in mammary gland tumorigenesis and prolonged alveolar differentiation. Oncogene 2004;23:5739–47.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
T. Kawano, M. Ito, D. Raina, Z. Wu, J. Rosenblatt, D. Avigan, R. Stone, and D. Kufe
MUC1 Oncoprotein Regulates Bcr-Abl Stability and Pathogenesis in Chronic Myelogenous Leukemia Cells
Cancer Res., December 15, 2007; 67(24): 11576 - 11584.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
R. Sangha and C. Butts
L-BLP25: A Peptide Vaccine Strategy in Non Small Cell Lung Cancer
Clin. Cancer Res., August 1, 2007; 13(15): 4652s - 4654s.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. L. Beatty, S. E. Plevy, A. R. Sepulveda, and O. J. Finn
Cutting Edge: Transgenic Expression of Human MUC1 in IL-10-/- Mice Accelerates Inflammatory Bowel Disease and Progression to Colon Cancer
J. Immunol., July 15, 2007; 179(2): 735 - 739.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Leng, C. Cao, J. Ren, L. Huang, D. Chen, M. Ito, and D. Kufe
Nuclear Import of the MUC1-C Oncoprotein Is Mediated by Nucleoporin Nup62
J. Biol. Chem., July 6, 2007; 282(27): 19321 - 19330.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
X. Wei, H. Xu, and D. Kufe
Human Mucin 1 Oncoprotein Represses Transcription of the p53 Tumor Suppressor Gene
Cancer Res., February 15, 2007; 67(4): 1853 - 1858.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Yin, S. Kharbanda, and D. Kufe
Mucin 1 Oncoprotein Blocks Hypoxia-inducible Factor 1{alpha} Activation in a Survival Response to Hypoxia
J. Biol. Chem., January 5, 2007; 282(1): 257 - 266.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
J. Ren, D. Raina, W. Chen, G. Li, L. Huang, and D. Kufe
MUC1 Oncoprotein Functions in Activation of Fibroblast Growth Factor Receptor Signaling
Mol. Cancer Res., November 1, 2006; 4(11): 873 - 883.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huang, L.
Right arrow Articles by Kufe, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, L.
Right arrow Articles by Kufe, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online