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J. Biol. Chem., Vol. 277, Issue 20, 17616-17622, May 17, 2002

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Protein Kinase C  Regulates Function of the DF3/MUC1 Carcinoma Antigen in -Catenin Signaling^*

Jian Ren, Yongqing Li, and Donald Kufe^§

From the Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, January 15, 2002, and in revised form, February 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in most human carcinomas. The MUC1 cytoplasmic domain interacts directly with -catenin, a component of the adherens junction of mammalian epithelial cells. The present results demonstrate that MUC1 associates with protein kinase C (PKC). A TDR sequence adjacent to the -catenin binding motif in the MUC1 cytoplasmic domain functions as a site for PKC phosphorylation. We show that phosphorylation of MUC1 by PKC increases binding of MUC1 and -catenin in vitro and in vivo. The functional significance of the MUC1-PKC interaction is further supported by the demonstration that mutation of the PKC phosphorylation site abrogates MUC1-mediated decreases in binding of -catenin to E-cadherin. We also show that the stimulatory effects of MUC1 on anchorage-independent growth are abrogated by mutation of the PKC phosphorylation site. These findings support a novel role for PKC in regulating the interaction between MUC1 and the -catenin signaling pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The protein kinase C (PKC)¹ family of serine/threonine protein kinases is involved in intracellular signaling pathways that regulate growth, differentiation, and apoptosis. The PKC isoforms have been divided into: (i) the conventional PKCs (cPKCs; , , ) which are dependent on calcium and activated by diacylglycerol or 12-O-tetradecanoylphorbol-13-acetate; (ii) the novel PKCs (nPKCs; , , ) which are calcium-independent and activated by diacylglycerol or 12-O-tetradecanoylphorbol-13-acetate; and (iii) the atypical PKCs (aPKCs; , ) which are calcium-independent and not activated by diacylglycerol or 12-O-tetradecanoylphorbol-13-acetate (1, 2). Of the 12 known PKC isoforms, the ubiquitously expressed PKC is unique as a substrate for tyrosine phosphorylation in response to activation of the epidermal growth factor receptor (EGFR) (3), the platelet-derived growth factor receptor (4), or the insulin-like growth factor I receptor (4). Transformation by Ras (5) or v-Src (6) also results in phosphorylation of PKC on tyrosine. Interactions between PKC and EGFR or c-Src have supported a role for PKC as a tumor suppressor (7, 8). Other studies have provided support for involvement of PKC in the apoptotic response of cells to genotoxic and oxidative stress (9-12). Targeting of PKC to mitochondria induces apoptosis through loss of the mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-3 (12-15).

The human DF3/MUC1 mucin-like transmembrane glycoprotein is expressed on the apical borders of normal secretory epithelial cells and at high levels over the entire surface of carcinoma cells (16). The MUC1 protein consists of an N-terminal ectodomain with variable numbers of 20 amino acid tandem repeats that are extensively modified by O-glycosylation (17, 18). The >250-kDa ectodomain associates with a ~25-kDa C-terminal proteolytic fragment as a heterodimer at the cell surface. The C-terminal subunit includes a transmembrane domain and a 72-amino acid cytoplasmic domain (CD). -Catenin, a component of the adherens junction of mammalian epithelial cells, binds directly to the MUC1/CD at a serine-rich motif that is similar to -catenin-binding sites on E-cadherin and the adenomatous polyposis coli (APC) tumor suppressor (19). MUC1 competes with E-cadherin, but not APC, for binding to -catenin (20). The interaction between MUC1 and -catenin is down-regulated by phosphorylation of the MUC1/CD by glycogen synthase kinase 3 (GSK3) (20). Conversely, phosphorylation of MUC1 by c-Src stimulates binding of MUC1 to -catenin (21). The recent findings that EGFR-mediated phosphorylation of MUC1 regulates the interaction of MUC1 with c-Src and -catenin has suggested that aberrant overexpression of MUC1 in human carcinoma cells could contribute to the transformed phenotype by dysregulation of EGFR signaling (22, 23).

The present studies demonstrate that PKC interacts with MUC1. A T41DR motif in the MUC1/CD has been identified as a PKC phosphorylation site. The results show that PKC regulates binding of MUC1 to -catenin and that the T41A mutant abrogates the effects of MUC1 on anchorage-independent cell growth.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- Human ZR-75-1 breast carcinoma cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Human 293 embryonal kidney (ATCC, Manassas, VA) and HCT116 colon carcinoma (24) cells were cultured in Dulbecco's modified Eagle's medium and Dulbecco's modified Eagle's medium/F-12, respectively, with 10% heat-inactivated fetal bovine serum and antibiotics. Cell growth was assessed by trypan blue staining and counting viable cells.

Cell Transfections-- 293 and HCT116 cells were transiently transfected with control vectors (pIRESpuro2, pEGFP-C1), pIRESpuro2-MUC1 (21), pIRESpuro2-MUC1(T41A), pEGFP-PKC, or pEGFP-PKC(K378R) (14) in the presence of LipofectAMINE (Invitrogen). Transfection efficiency as determined by immunofluorescence microscopy for MUC1 expression (23) and fluorescence microscopy for GFP expression, ranged from 70 to 80% for 293 cells and 25-30% for HCT116 cells. To establish stable lines, HCT116 cells transfected with pIRESpuro2, pIRESpuro2-MUC1, or pIRESpuro2-MUC1(T41A) were selected in the presence of 0.4 mg/ml puromycin (Calbiochem-Novabiochem, San Diego, CA). Two independent transfections were performed for each vector. Single cell clones were isolated by limiting dilution and expanded for analysis.

Generation of Mutants-- The His-MUC1/CD(T41A) mutant was generated on pET28(+)-MUC1/CD (20) using site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA) to change Thr⁴¹ to Ala. pIRESpuro2-MUC1(T41A) was constructed by cloning MUC1(T41A) into pIRESpuro2 as described (21).

Lysate Preparation-- Lysates were prepared from subconfluent cells as described (21).

Immunoprecipitation and Immunoblotting-- Equal amounts of protein from cell lysates were incubated with mAb DF3 (anti-MUC1) (16), anti-PKC (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-E-cadherin (Transduction Laboratories, San Diego, CA), or normal mouse IgG. The immune complexes were prepared as described (21), separated by SDS-PAGE, and transferred to nitrocellulose membranes. The immunoblots were probed with anti-MUC1, anti-PKC, anti--catenin (Zymed Laboratories Inc., San Francisco, CA), or anti-E-cadherin. Reactivity was detected with horseradish peroxidase-conjugated second antibodies and chemiluminescence (PerkinElmer Life Sciences, Boston, MA).

Binding Studies-- Glutathione S-transferase (GST) or GST-MUC1/CD bound to glutathione beads were incubated with 0.3 µg of purified recombinant, kinase-active PKC (PanVera Corp., Madison, WI). The adsorbates were analyzed by immunoblotting with anti-PKC. His-tagged wild-type MUC1/CD and MUC1/CD(T41A) were incubated with 0.3 µg of recombinant PKC (PanVera) for 1 h at 4 °C. Anti-PKC immunoprecipitates were analyzed by immunoblotting with anti-MUC1/CD (20) and anti-PKC. In other experiments, purified His-tagged wild-type and mutant MUC1/CD proteins were incubated with 0.3 µg of PKC (PanVera) in the absence and presence of 200 µM ATP for 20 min at 30 °C. GST--catenin bound to glutathione beads was then added, and the reaction was incubated for 1 h at 4 °C. The precipitated proteins were subjected to immunoblot analysis with anti-MUC1/CD (20).

In Vitro Phosphorylation-- Purified His-tagged, wild-type and mutant MUC1/CD proteins were incubated with 0.3 µg of recombinant PKC (PanVera) in kinase buffer (20 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 5 µM dithiothreitol). The reactions were initiated by addition of 10 µCi of [-³²P]ATP and incubated for 20 min at 30 °C. Phosphorylated proteins were separated by SDS-PAGE and analyzed by autoradiography.

Anchorage-independent Growth-- Cells (4 × 10⁴) were suspended in 1.5 ml of 0.33% Noble agar (DIFCO, Detroit, MI) in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. The cell suspension was layered over 3.5 ml of 0.5% agar/Dulbecco's modified Eagle's medium in 60-mm dishes. One ml of fresh culture medium was added at 2 weeks. Colonies composed of >10 cells were counted at 3 weeks.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

MUC1 Binds Directly to PKC-- To determine whether MUC1 associates with PKC, lysates from human ZR-75-1 cells were subjected to immunoprecipitation with anti-MUC1 and, as a control, IgG. Immunoblot analysis of the precipitates with anti-PKC demonstrated the detection of MUC1-PKC complexes (Fig. 1A, left). In the reciprocal experiment, immunoblot analysis of anti-PKC immunoprecipitates with anti-MUC1 confirmed that MUC1 associates with PKC (Fig. 1A, right). By contrast, there was no detectable interaction between MUC1 and PKCII, PKC or PKCµ (Fig. 1B). To extend these findings, 293 cells, which are negative for MUC1 (20), were transfected to express MUC1 or MUC1+PKC. Immunoblot analysis of anti-MUC1 immunoprecipitates with anti-PKC demonstrated binding of MUC1 with endogenous PKC (Fig. 1C). Moreover, coexpression of MUC1 and PKC resulted in increased formation of MUC1-PKC complexes (Fig. 1C). To assess whether binding is direct, purified GST or a GST fusion protein containing the MUC1/CD (GST-MUC1/CD) was incubated with recombinant PKC. Adsorbates to glutathione beads were subjected to immunoblot analysis with anti-PKC. The finding that PKC binds to GST-MUC1/CD and not to GST alone supported a direct interaction (Fig. 1D).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Binding of MUC1 and PKC in vivo and in vitro. A, lysates from ZR-75-1 cells were subjected to immunoprecipitation with anti-MUC1 (left panel) or anti-PKC (right panel). Mouse IgG was used as a control. The immunoprecipitates and lysates not subjected to immunoprecipitation were analyzed by immunoblotting with anti-PKC (left panel) and anti-MUC1 (right panel). B, anti-MUC1 immunoprecipitates from ZR-75-1 cells were analyzed by immunoblotting with antibodies against PKCII, -, and -µ. As a control, the immunoprecipitates and lysate not subjected to immunoprecipitation were analyzed by immunoblotting with anti-MUC1. C, 293 cells were transfected to express MUC1 or MUC1+PKC. Anti-MUC1 immunoprecipitates were analyzed by immunoblotting with anti-PKC (upper panel) and anti-MUC1 (lower panel). Mouse IgG immunoprecipitates from cells expressing MUC1 and PKC were used as a control (lane 1). Lysate not subjected to immunoprecipitation was also used as a control (lane 4). Note that 293 cells express the 78-kDa PKC and a precursor form of ~115 kDa. D, GST or GST-MUC1/CD bound to glutathione beads was incubated with recombinant PKC. GST-MUC1/CD not incubated with PKC was used as a control. The adsorbates were analyzed by immunoblotting with anti-PKC.

PKC Phosphorylates MUC1 on T41-- To determine whether MUC1/CD is a substrate for PKC, we incubated purified His-MUC1/CD with recombinant PKC and [-³²P]ATP. Analysis of the reaction products by SDS-PAGE and autoradiography demonstrated phosphorylation of MUC1/CD (Fig. 2A). A ST41DRS site in MUC1/CD conforms to the preferred (S/T)X(K/R) motif for PKC phosphorylation. To determine whether ST41DRS is phosphorylated by PKC, we mutated this site in MUC1/CD to SA41DRS (Fig. 2B). PKC-mediated phosphorylation of MUC1/CD(T41A) was attenuated compared with that obtained with wild-type MUC1/CD (Fig. 2C, left). By contrast, phosphorylation of MUC1 by PKC was unaffected by Ser  Ala mutations of either or both of the flanking serines (Fig. 2C, left). The results also demonstrate that autophosphorylation of PKC is decreased in the presence of MUC1/CD(T41A) (Fig. 2C, left). To determine whether the T41A mutation affects binding of PKC, wild-type MUC1/CD and MUC1/CD(T41A) were incubated with recombinant PKC. Immunoblot analysis of anti-PKC immunoprecipitates with anti-MUC1/CD demonstrated that PKC binds equally to wild-type MUC1/CD and MUC1/CD(T41A) (Fig. 2C, right). Previous studies have shown that phosphorylation of the MUC1/CD Ser⁴⁴ site by GSK3 decreases the interaction between MUC1/CD and -catenin (20). To assess the effects of PKC-mediated phosphorylation of MUC1/CD, we incubated MUC1/CD with PKC in the presence and absence of ATP. After phosphorylation of MUC1/CD, GST or GST--catenin was added for 1 h at 4 °C. Proteins precipitated with glutathione beads were analyzed by immunoblotting with anti-MUC1/CD. As shown previously, MUC1/CD binds to GST--catenin and not GST (20). Preincubation of MUC1/CD with PKC and ATP was associated a higher level of MUC1/CD binding to GST--catenin than that obtained in the absence of PKC or ATP (Fig. 2D). By contrast, preincubation of MUC1/CD(T41A) with PKC and ATP had no detectable effect on binding of MUC1/CD(T41A) to -catenin (Fig. 2D). The finding that, in the absence of PKC phosphorylation, less -catenin binds to MUC1/CD(T41A) as compared with that for wild-type MUC1/CD may reflect conformational effects of the T41A mutation on the -catenin-binding site (Fig. 2D). These findings demonstrate that PKC phosphorylates MUC1/CD on Thr⁴¹ and thereby increases binding of MUC1/CD and -catenin.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   PKC phosphorylates MUC1 on Thr41. A, GST or GST-MUC1/CD was incubated with PKC and [-32P]ATP. As a control, GST-MUC1/CD was incubated with [-32P]ATP in the absence of PKC. The reaction products were analyzed by SDS-PAGE and autoradiography. B, schematic representation of wild-type MUC1/CD and the Ser/Thr  Ala mutants. The specified sequences represent mutations made in the entire cytoplasmic domain. Numbers (1-72) reflect amino acids in the CD. TR, tandem repeats. TM, transmembrane region. CD, cytoplasmic domain. C, purified MUC1/CD and the indicated mutants were incubated with PKC and [-32P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography (upper panel, left). Densitometric scanning of the signals obtained with MUC1/CD and the mutants is expressed as fold intensity relative to that for MUC1/CD (WT). Equal loading of the MUC1/CD proteins was assessed by Coomassie Blue staining (middle panel, left). Equal loading of recombinant PKC was confirmed by immunoblot analysis with anti-PKC (lower panel, left). Purified MUC1/CD and MUC1/CD(T41A) were incubated with recombinant PKC. Anti-PKC immunoprecipitates were analyzed by immunoblotting with anti-MUC1/CD (upper panel, right) and anti-PKC (lower panel, right). D, His-MUC1/CD or His-MUC1/CD(T41A) were incubated with PKC and ATP. Controls were performed in the absence of PKC or ATP. After incubation at 37 °C, GST--catenin bound to glutathione beads was added for 1 h at 4 °C. The adsorbates were analyzed by immunoblotting with anti-MUC1/CD and anti--catenin.

PKC Regulates Interaction of MUC1 with -Catenin in Vivo-- To determine whether PKC regulates the interaction between MUC1 and -catenin in vivo, transfection studies were performed in the MUC1-negative 293 cells. After transfection of vectors expressing MUC1 and PKC or the kinase-inactive PKC(K-R) mutant, lysates were subjected to immunoprecipitation with anti-MUC1. Immunoblot analysis of the precipitates with anti--catenin demonstrated that PKC increases the interaction between MUC1 and -catenin as compared with that in cells transfected with PKC(K-R) (Fig. 3A). In concert with these results and involvement of Thr⁴¹, PKC had little if any effect on binding of -catenin to the MUC1(T41A) mutant (Fig. 3A). To extend the analysis, we transfected MUC1-negative HCT116 cells to stably express the empty vector, wild-type MUC1, or MUC1(T41A) (Fig. 3B, left). Anti-MUC1 immunoprecipitates from HCT116/V, HCT116/MUC1, and HCT116/MUC1(T41A) cells were subjected to immunoblotting with anti--catenin. The results demonstrate that MUC1, but not MUC1(T41A), binds to -catenin (Fig. 3B, right). When these cells were transfected to express GFP-PKC, immunoblot analysis of anti-MUC1 immunoprecipitates with anti--catenin demonstrated that PKC induces binding of -catenin to wild-type MUC1 and not the MUC1(T41A) mutant (Fig. 3B, right). Binding of MUC1 to endogenous PKC and ectopically expressed GFP-PKC was also decreased with the MUC1(T41A) mutant as compared with that with wild-type MUC1 (Fig. 3B, right). Previous work has demonstrated that MUC1 and E-cadherin compete for binding to -catenin (20). To determine whether expression of the MUC1(T41A) mutant affects binding of -catenin to E-cadherin, anti-E-cadherin immunoprecipitates were analyzed by immunoblotting with anti--catenin. In concert with previous findings (20), expression of wild-type MUC1 was associated with decreased binding of E-cadherin and -catenin (Fig. 3C). By contrast, expression of MUC1(T41A) had less of an effect on the interaction of E-cadherin and -catenin compared with that in HCT116/MUC1 cells (Fig. 3C). Overexpression of GFP-PKC was associated with decreases in binding of -catenin to E-cadherin in HCT116/V cells (Fig. 3C). Moreover, while GFP-PKC had little additional effect on the interaction of E-cadherin and -catenin in HCT116/MUC1 cells, expression of both GFP-PKC and the MUC1(T41A) mutant resulted in an increase in E-cadherin--catenin complexes (Fig. 3C). These findings demonstrate that PKC regulates the interaction between MUC1 and -catenin in cells and thereby binding of E-cadherin with -catenin.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Effects of PKC-mediated phosphorylation of MUC1/CD on the interaction of MUC1 and -catenin. A, 293 cells were transfected to express MUC1 and GFP-PKC or GFP-PKC(K-R). Cells were also transfected to express MUC1(T41A) and PKC. Anti-MUC1 immunoprecipitates were analyzed by immunoblotting with anti--catenin (upper panel), anti-PKC (middle panel), and anti-MUC1 (lower panel). B, lysates from HCT116/V, HCT116/MUC1, and HCT116/MUC1(T41A) cells were subjected to immunoblot analysis with anti-MUC1 and anti--catenin (left). Anti-MUC1 immunoprecipitates from HCT116/V, HCT116/MUC1, and HCT116/MUC1(T41A) cells were analyzed by immunoblotting with anti--catenin (upper panel), anti-PKC (middle panel), or anti-MUC1 (lower panel) (right, lanes 2-4). Lysate not subjected to immunoprecipitation was used as a control (right, lane 1). Similar studies were performed on cells transfected to express GFP-PKC (right, lanes 5-7). C, anti-E-cadherin immunoprecipitates from HCT116/V, HCT116/MUC1, and HCT116/MUC1(T41A) were analyzed by immunoblotting with anti--catenin (upper panel) and anti-E-cadherin (lower panel) (lanes 1-3). Similar studies were performed on cells transfected to express GFP-PKC (lanes 4-6).

Effects of MUC1 on Anchorage-independent Growth Are Abrogated by the T41A Mutation-- To assess the functional significance of the interaction between MUC1 and PKC, HCT116/V, HCT116/MUC1, and HCT116/MUC1(T41A) cell lines were analyzed for anchorage-dependent and -independent growth. Expression of wild-type MUC1 or the MUC1(T41A) mutant had no apparent effect on growth in tissue culture compared with that for HCT116/V cells (Fig. 4A). Similar results were obtained with clones selected from separate transfections (Fig. 4A). In soft agar, the wild-type MUC1 transfectants formed colonies that were substantially larger than those obtained with HCT116/V cells (Fig. 4B). By contrast, expression of MUC1(T41A) was associated with the formation of colonies that were similar to those found with HCT116/V cells (Fig. 4B). Similar results were obtained with the independently selected clones (Fig. 4B). The number of colonies obtained for HCT116/MUC1 cells was also higher than those found for HCT116/V and HCT116/MUC1(T41A) cells (Fig. 4C). These findings demonstrate that expression of wild-type MUC1 contributes to anchorage-independent growth and that mutation of the PKC phosphorylation site abrogates this effect.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Effects of wild-type MUC1 and the MUC1(T41A) mutant on anchorage-dependent and -indendent cell growth. A, two independently selected clones of HCT116/V, HCT116/MUC1, and HCT116/MUC1(T41A) cells were seeded at 1 × 104 per well of 6-well culture plates. Cell counts were determined at 24 (), 48 (), 72 (), and 96 () h. The results are expressed as the mean of three replicates (S.E. was <15% of the mean). Immunoblot analysis of HCT116/V-1, HCT116/MUC1-1 and HCT116/MUC1(T41A)-1 is shown in Fig. 3B. Similar results for MUC1 expression were obtained for the 2 clones. B, the indicated cells were suspended in soft agar and incubated for 3 weeks. Photomicrographs are shown for independently selected clones (left and right panels). C, colonies >10 cells were counted from 3 plates for each clone of the indicated cells. The results are expressed as the mean ± S.E. number of colonies per plate. D, amino acid sequence of the MUC1 cytoplasmic tail. The PKC phosphorylation site (Thr41), the GSK3 phosphorylation site (Ser44), the EGFR and c-Src phosphorylation site (Tyr46), and the -catenin-binding sites (SAGNGGSSLS) are highlighted.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PKC has been implicated in the response of cells to activation of the EGFR, platelet-derived growth factor receptor, and insulin-like growth factor I receptors (3, 4). Other findings have supported a role for PKC in the apoptotic response of cells to stress (9-15). Functional involvement of PKC at the cell membrane has also been shown through regulation of phospholipid scramblase activity during both cell activation and apoptosis (25). The present studies demonstrate that PKC interacts with the transmembrane MUC1 protein. The results show that PKC phosphorylates the MUC1 cytoplasmic domain at Thr⁴¹ (Fig. 4D). Previous work has shown that GSK3 phosphorylates Ser⁴⁴ in the TDRSPYE domain of MUC1/CD (Fig. 4D) (20). In addition, the Tyr⁴⁶ site is phosphorylated by EGFR (23) and c-Src (21) (Fig. 4D). Phosphorylation of Ser⁴⁴ by GSK3 decreases -catenin binding, while phosphorylation of Tyr⁴⁶ increases the interaction of MUC1 and -catenin. The present results demonstrate that PKC also regulates the formation of MUC1--catenin complexes. In vitro binding of PKC was similar with wild-type MUC1/CD and the MUC1/CD(T41A) mutant. By contrast, binding of PKC to MUC1(T41A) in vivo was decreased compared with that found for wild-type MUC1. These findings suggest that, in the presence of other binding proteins, such as c-Src and GSK3, the T41A mutation disrupts the interaction between MUC1 and PKC. Other work has shown that c-Src disrupts binding of MUC1 and GSK3 (20). Thus, given the proximity of the MUC1 sites for interactions with c-Src, GSK3, and PKC (Fig. 4D), modification of Thr⁴¹, Ser⁴⁴, and/or Tyr⁴⁶ by phosphorylation or mutation may affect the integration of MUC1 signaling with diverse pathways.

The available evidence indicates that MUC1 and E-cadherin compete for binding to the same pool of -catenin (20). E-cadherin functions in homotypic recognition and the regulation of cell mobility (26). The interaction between E-cadherin and -catenin is essential for cell adhesion by connecting E-cadherin to -catenin and thereby the cytoskeleton (27-30). Importantly, disruption of E-cadherin function and cell adhesion has been associated with tumor development (29, 31-33). Other studies have demonstrated that MUC1 affects E-cadherin-mediated cell adhesion (34). The present results demonstrate that PKC increases the interaction between MUC1 and -catenin in vitro and in cells. Moreover, mutation of the MUC1 Thr⁴¹ phosphorylation site abrogates the effects of PKC on formation of MUC1--catenin complexes. The in vitro binding studies indicate that the T41A mutation may also affect the MUC1/CD--catenin interaction, perhaps by causing changes in the tertiary structure of the -catenin-binding site. Nonetheless, while MUC1 decreased binding of E-cadherin and -catenin in cells, expression of the MUC1(T41A) mutant in part reversed this effect. Whereas enforced expression of PKC in HCT116/MUC1(T41A) cells was also associated with increased formation of E-cadherin--catenin complexes, these experimental conditions could result in interactions not found with endogenous PKC. The findings thus support a signaling pathway in which the interaction between MUC1 and PKC increases binding of MUC1 and -catenin, while decreasing the formation of E-cadherin--catenin complexes.

MUC1 is normally expressed at the apical borders of secretory epithelial cells (16). In carcinoma cells, polarization of MUC1 is lost with high levels of expression over the entire cell surface (16, 35). The apical border of the normal glandular epithelium is devoid of cell-cell interactions at the surface lining secretory ducts. Overexpression of MUC1 by carcinoma cells, however, could confer an anti-adhesive function to the entire cell surface by disrupting E-cadherin-mediated homotypic recognition. To address the function of MUC1 on cell growth, MUC1-negative HCT116 cells were stably transfected to express wild-type MUC1 or the MUC1(T41A) mutant. Expression of MUC1 or MUC1(T41A) had no significant effect on adherent growth in tissue culture. By contrast, wild-type MUC1 conferred a substantial increase in anchorage-independent growth in soft agar. These effects could not be attributed to the heavily glycosylated ectodomain because anchorage-independent growth of HCT116 cells expressing similar levels of MUC1(T41A) was comparable with that found with control HCT116/V cells. Rather, the distinct patterns of anchorage-independent growth for HCT116 cells expressing wild-type MUC1 or MUC1(T41A) support direct involvement of the PKC phosphorylation site. These findings and the demonstration that PKC-mediated phosphorylation of MUC1 regulates binding of MUC1 and -catenin are in concert with a model in which PKC subverts E-cadherin function by titrating binding of -catenin to MUC1. Conversely, in a simplified model, mutation of MUC1 at Thr⁴¹ restores binding of -catenin to E-cadherin and decreases anchorage-independent growth.

In addition to interactions with E-cadherin and MUC1 at the cell membrane, -catenin binds directly to the APC gene product in the cytosol (36, 37). The interaction between adenomatous polyposis coli and -catenin regulates -catenin turnover (38) and alters cell adhesion (39). -Catenin also forms nuclear complexes with members of the T-cell factor/lymphoid enhancing factor 1 family of transcription factors (40, 41). Loss of APC-mediated regulation of -catenin in transformed cells is associated with constitutive activation of -catenin-T-cell factor/lymphoid enhancing factor 1 transcriptional complexes (42-44). The present findings contribute to a role for MUC1 in regulating -catenin at the cell membrane. While regulation of -catenin may also occur as a result of binding to MUC1 that accumulates in the cytosol of carcinoma cells (16), there are presently no known functions for MUC1 in control of -catenin signaling in the nucleus.

	ACKNOWLEDGEMENT

We appreciate Kamal Chauhan for his excellent technical support.

	FOOTNOTES

^* This work was supported by Grant CA87421 from the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^§ To whom correspondence should be addressed. E-mail: donald_kufe@dfei.harvard.edu.

Published, JBC Papers in Press, March 4, 2002, DOI 10.1074/jbc.M200436200

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; CD, cytoplasmic domain; GSK3, glycogen synthase kinase 3; APC, adenomatous polyposis coli; GST, glutathione S-transferase; EGFR, epidermal growth factor receptor.

	REFERENCES

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				Q. Shen, J. J. Rahn, J. Zhang, N. Gunasekera, X. Sun, A. R.E. Shaw, M. J. Hendzel, P. Hoffman, A. Bernier, and J. C. Hugh MUC1 Initiates Src-CrkL-Rac1/Cdc42-Mediated Actin Cytoskeletal Protrusive Motility after Ligating Intercellular Adhesion Molecule-1 Mol. Cancer Res., April 1, 2008; 6(4): 555 - 567. [Abstract] [Full Text] [PDF]

				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]

				P. K. Singh, Y. Wen, B. J. Swanson, K. Shanmugam, A. Kazlauskas, R. L. Cerny, S. J. Gendler, and M. A. Hollingsworth Platelet-Derived Growth Factor Receptor {beta}-Mediated Phosphorylation of MUC1 Enhances Invasiveness in Pancreatic Adenocarcinoma Cells Cancer Res., June 1, 2007; 67(11): 5201 - 5210. [Abstract] [Full Text] [PDF]

				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]

				C. L. Russo, S. Spurr-Michaud, A. Tisdale, J. Pudney, D. Anderson, and I. K. Gipson Mucin gene expression in human male urogenital tract epithelia Hum. Reprod., November 1, 2006; 21(11): 2783 - 2793. [Abstract] [Full Text] [PDF]

				E. K. Malmberg, K. A. Noaksson, M. Phillipson, M. E. V. Johansson, M. Hinojosa-Kurtzberg, L. Holm, S. J. Gendler, and G. C. Hansson Increased levels of mucins in the cystic fibrosis mouse small intestine, and modulator effects of the Muc1 mucin expression Am J Physiol Gastrointest Liver Physiol, August 1, 2006; 291(2): G203 - G210. [Abstract] [Full Text] [PDF]