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Distinct Activities of the -Catenin Family, -Catulin and -Catenin, on ß-Catenin-Mediated Signaling

Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

Received 3 September 2003/ Returned for modification 7 October 2003/ Accepted 17 December 2003

	ABSTRACT

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-Catenin, an integral part of cadherin-catenin adhesion complexes, is a major binding partner of ß-catenin, a key component of the Wnt pathway, which activates T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription and is often upregulated in cancers. Recently, we identified an -catenin-related protein, -catulin, whose function is poorly understood, as part of a Rho GTPase signaling complex. Here, based on evidence suggesting that -catulin may associate with a ß-catenin fraction, we investigated the role of -catenin family members in ß-catenin-mediated signals. Expression of the full length or a 103-residue region of -catenin strongly inhibits the induction of the TCF/LEF-responsive TOPFLASH reporter in HEK293T cells expressing activated ß-catenin or in cancer cells with constitutively upregulated Wnt signaling, whereas -catulin expression had no effect. Interestingly, -catulin expression attenuates the activation of the cyclin D1 promoter, a target of Wnt pathway signals. -Catulin appears to inhibit Ras-mediated signals to the cyclin D1 promoter, rather than ß-catenin signals, and the synergy between Ras and ß-catenin required to fully activate this promoter. Data suggesting the involvement of Rho in this response are presented and discussed. These results suggest a novel function for -catulin and imply that -catenin and -catulin have distinct activities that downregulate, respectively, ß-catenin and Ras signals converging on the cyclin D1 promoter.

	INTRODUCTION

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The Wnt signaling pathway is a key pathway in the mediation of cell growth and development (4). Stimulation of this pathway via Wnt ligand binding to the cell surface seven-transmembrane receptor Frizzled leads to the activation of a series of complex intracellular signals which are still not completely understood (20). This potentially oncogenic pathway is activated by mutations in several of its signaling components, notably the adenomatous polyposis coli (APC) tumor suppressor and ß-catenin, which result in the stabilization of cytoplasmic ß-catenin (21, 27). The accumulation of cytoplasmic ß-catenin allows ß-catenin to complex with, and translocate to the nucleus with, members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors (26). This process subsequently results in the transcription of multiple oncogenes, including c-myc (12) and cyclin D1 (39, 43). Activating mutations in components of the pathway, such as ß-catenin, are thought to lead to constitutive Wnt signaling and subsequent tumorigenesis and are frequently associated with many types of cancer, including colon cancer, desmoid and gastric cancers, hepatocarcinoma, medulloblastoma, melanoma, ovarian cancer, pancreatic cancer, and prostate cancer (21).

Cyclin D1, the regulatory subunit for cdk4 and cdk6, plays a key role in the progression of cells through the cell cycle and past the G₁ checkpoint (14, 38). The importance of cyclin D1 in G₁-phase progression is supported by the finding that the induction of cyclin D1 in rat fibroblasts can shorten G₁ duration (35, 36). Importantly, cyclin D1 is upregulated in many types of human cancer, including B-cell lymphoma and breast, gastric, esophageal, and colon carcinomas (14, 42).

Prior to its discovery as a critical component of the Wnt signaling pathway, ß-catenin was originally identified as a key component of the cadherin-catenin complex, which mediates cell-cell adhesion via the cadherin transmembrane receptor. Within this complex, ß-catenin links the cytoplasmic portion of cadherin to -catenin, a distinct protein (6, 18, 19). -Catenin links cadherin complexes to the actin cytoskeleton and is necessary for functional cell-cell adhesion by these complexes, which requires this connection with the internal actin cytoskeleton (16). The region of -catenin necessary for binding to ß-catenin has been mapped to within the N-terminal 163 amino acids of the protein (3, 13, 30, 31). Loss-of-function mutations in -catenin can lead to tumorigenesis, and the expression of wild-type protein in cells containing these mutations leads to tumor suppression and a restoration of growth control (3). Tumor progression associated with loss of -catenin has been reported in human cancers (44). The phenotypic effects in these tumors are generally thought to be due to loss of adhesion, but it is also conceivable that aberrations of intracellular growth control pathways may contribute to the effect. In support of this idea, evidence increasingly indicates that -catenin plays a role in cell signaling. An early study showed that -catenin strongly inhibits Wnt pathway-mediated developmental processes in Xenopus embryos (37). Moreover, -catenin expression inhibits ß-catenin-dependent activation of TCF-mediated transcription in various cell types (7, 8, 40). The N-terminal 210 amino acids of the protein, which contain the ß-catenin binding site, are required for these functions (37, 40). Recently, it has been proposed that the mechanism by which -catenin downregulates ß-catenin-dependent transcriptional activation is via -catenin interference with the interaction of the ß-catenin-TCF/LEF complex with its target DNA in the nucleus (7).

We recently described an -catenin-related protein, -catulin, as part of a Rho GTPase signaling complex. Our findings indicated that -catulin interacts with the Rho GTP/GDP exchange factor (GEF), Lbc, and may be a putative scaffold for Lbc (32). Little else is known about the function(s) of the recently described -catulin, which shares sequence similarity with -catenin and vinculin, members of the vinculin superfamily. -Catenin is a well-characterized binding partner of ß-catenin (6, 18, 19), and vinculin has also been suggested to bind ß-catenin (11). On the basis of these findings, here we analyzed the potential role of -catulin in ß-catenin-mediated signaling pathways.

	MATERIALS AND METHOD

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Plasmids Flag epitope-tagged pcDNA:ß-catenin 45 (28) was a gift from H. Clevers (University Medical Center, Utrecht, The Netherlands). Hemagglutinin (HA)-tagged pCGN:-catenin and mutant constructs (3) were gifts from L. Bullions (Wyeth). The pGFP:vinculin construct was a gift from B. Geiger (Weizmann Institute). The pTOPFLASH reporter construct (28) was a gift from B. Vogelstein (Johns Hopkins School of Medicine). Cyclin D1 reporter constructs were gifts from F. McCormick (University of California, San Francisco). pEXV:RasV12 and pEXV:RhoL63 constructs were gifts from C. Marshall and A. Hall (Institute of Cancer Research, University College London). pcDNA and pEGFPN3 backbone vectors were obtained from Clonetech Laboratories, and pRenilla was obtained from Promega.

The Myc epitope-tagged -catenin 46-149 expression plasmid (pcDNA:-catenin 46-149), containing an N-terminal KpnI site and a C-terminal SacII site on the insert, was created as follows. The -catenin 46-149 insert was generated by PCR with Platinum Pfx DNA polymerase (Invitrogen) and full-length -catenin in the pCGN vector (3) as a template with the primers 5'-CGGGGTACCATGGGGCCCTCTAATAAGAAG (forward) and 5'-TCCCCGCGGTTTGTAGACATCTGCCAT (reverse). The PCR product was then subcloned into the pcDNA4/Myc-His B vector (Invitrogen) between the KpnI and SacII restriction sites with T4 DNA ligase (Invitrogen) for 16 h at 14°C.

Cell culture HEK293T cells (a human kidney epithelial cell line), Caco2A colon carcinoma cells, and A427 lung carcinoma cells were obtained from the American Type Culture Collection. HEK293T and Caco2A cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum. A427 cells were maintained in minimal essential medium (Life Technologies) supplemented with nonessential amino acids and 10% heat-inactivated fetal bovine serum. Cells were maintained in a humidified 5% CO₂ incubator at 37°C. Where indicated, cells were treated with 15 mM lithium chloride (Sigma) for the indicated times immediately preceding lysis.

Cell transfection Cells at 80 to 90% confluence were transfected by using the Lipofectamine reagent (Invitrogen) as recommended by the manufacturer. Six hours posttransfection, medium was replaced with fresh medium containing serum, and cells were harvested 24 to 36 h posttransfection. In all cases, backbone vector was added to the transfection mixture to ensure that all groups were transfected with equal amounts of total plasmids, and experiments were performed in triplicate.

Cell selection Selection of transfected cells was performed by using the MACS K^k.II system (Miltenyi Biotec) according to the manufacturer's instructions 24 h posttransfection. Cells were cotransfected with 2 µg of the pMACSK^k.II vector to select for transfected cells. Where indicated, cells were treated for 3 h with 15 mM LiCl (Sigma) immediately prior to the cell selection process. Selected cells were then immediately lysed in cell nucleus lysis buffer (50 mM Tris-Cl [pH 8.0], 250 mM NaCl, 2 mM EDTA, 50 mM NaF, 10 mM Na₃VO₄, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], 1x Protease Arrest [Calbiochem]).

Transcriptional reporter assays Cells were transfected in 6-well dishes at 90% confluence with either the TOPFLASH, the FOPFLASH, or the cyclin D1 luciferase reporter constructs and the indicated expression plasmids. Each well was also cotransfected with the pRenilla luciferase expression plasmid to provide an internal control for the normalization of all data for transfection efficiency, cell viability, and cell number. Backbone vector was used to ensure the addition of equal amounts of total DNA to all groups during transfections. Thirty-six hours posttransfection, cells were washed with phosphate-buffered saline and 500 µl of Promega reporter lysis buffer was added to each well. Lysates were then collected and assayed for luciferase activity by using the dual luciferase reporter assay system (Promega) according to the manufacturer's instructions. Each experiment was performed in triplicate and repeated at least two times, for a total of six or more experiments.

Immunoprecipitation Cells were lysed in a solution of 1% Triton X-100, 1% Nonidet P-40, 50 mM Tris-Cl [pH 7.5], and 150 mM NaCl containing 1 mM phenylmethylsulfonyl fluoride and 10 µg of aprotinin/ml. Lysates were then cleared of insoluble material by centrifugation at 10,000 x g for 10 min and precleared with 1 µg of mouse immunoglobulin G (IgG) (Santa Cruz Biotechnology) and 20 µl of protein A-agarose conjugate (Santa Cruz Biotechnology) for 1 h at 4°C. For immunoprecipitations, 2 µg of either mouse anti-c-Myc (9E10; Santa Cruz Biotechnology), goat anti-p120 (M-19; Santa Cruz Biotechnology), mouse anti-ß-catenin (E-5; Santa Cruz Biotechnology), rabbit anti--catenin (H-297; Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) antibody was used as indicated. Immunoprecipitation was carried out as described previously (32).

Nucleus isolation Nuclei were isolated essentially as described previously (10). Briefly, about 10⁷ to 10⁸ cells were resuspended in 5 ml of buffer A (60 mM KCl, 15 mM NaCl, 15 mM HEPES [pH 7.8], 1 mM phenylmethylsulfonyl fluoride, 10 µg of aprotinin/ml)-0.3 M sucrose-0.5% NP-40 (Calbiochem). The cell suspension was then disrupted with 50 strokes in a Dounce homogenizer and layered onto 2.5 ml of buffer A plus 0.9 M sucrose. Samples were centrifuged at 3,100 rpm (2,000 x g) in a Beckman J-6B centrifuge for 10 min, and the supernatant was removed. The pellet was resuspended in 5 ml of buffer A plus 0.3 M sucrose plus 0.2% NP-40 and centrifuged over 2.5 ml of buffer A plus 0.9 M sucrose. The supernatant was then removed, and the pellet was resuspended in the cell nucleus lysis buffer described above.

Immunoblotting Immunoblotting was carried out as described previously (32) and was visualized with the Perkin-Elmer enhanced chemiluminescence detection system.

Antibodies The following primary antibodies were used: anti-c-Myc (9E10; Santa Cruz Biotechnology), anti-Flag (M2; Sigma), anti-HA tag (HA-7; Sigma), anti-ß-catenin (E-5; Santa Cruz Biotechnology), anti--catenin (H-297; Santa Cruz Biotechnology), anti-cyclin D1 (HD11; Santa Cruz Biotechnology), anti--tubulin (B-7; Santa Cruz Biotechnology), and rabbit polyclonal anti--catulin (32). Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse IgG (goat; NEN) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Promega).

Statistical analysis Data were analyzed with Student's t test; a P value of <0.05 was considered to indicate significance.

	RESULT

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-Catulin, a member of the vinculin superfamily, associates with a ß-catenin fraction in vivo Previously (32), the -catulin protein, which has 50% overall similarity to, and 29% identity to, -catenin, was identified. -Catulin is a member of the vinculin superfamily of proteins, which includes both -catenin and vinculin, and previous analysis of the overall amino acid sequences and genomic structures of these proteins indicated that -catulin is more closely related to -catenin than to vinculin (17). Previously, multiple studies (3, 13, 30, 31) mapped the ß-catenin binding site on -catenin to an N-terminal region of the protein. Based on these reports, the smallest consensus region of -catenin suggested to bind ß-catenin comprises residues 46 to 149, shown in Fig. 1A, and BLAST analysis (National Center for Biotechnology Information) indicates that this region is more similar to the corresponding region of -catulin (31% identity, 52% similarity) than to that of vinculin (28% identity, 45% similarity). On this basis, we tested whether -catulin, like -catenin, associates with ß-catenin in vivo by using coimmunoprecipitation. As a control, we initially determined complex formation between endogenous -catenin and ß-catenin by immunoprecipitating ß-catenin from HEK293T cells with an anti-ß-catenin antibody. Following separation by SDS-polyacrylamide gel electrophoresis, immunoblotting of the immunoprecipitated material with anti--catenin antibody showed coimmunoprecipitation of substantial amounts of endogenous -catenin, as represented by the 95-kDa band of the expected size in Fig. 1B (top panel, third lane), in keeping with previous reports (1, 18). The reciprocal experiment was also done, and following immunoprecipitation of endogenous -catenin and immunoblotting for ß-catenin, substantial amounts of ß-catenin were observed to coimmunoprecipitate with -catenin (middle panel, second lane). Next, complex formation between endogenous -catulin and ß-catenin was evaluated. Upon immunoprecipitation of endogenous ß-catenin with anti-ß-catenin antibody, immunoblotting of immunoprecipitated material with anti--catulin antiserum (32) showed coimmunoprecipitation of endogenous -catulin (bottom panel, third lane), albeit at a level lower than that of -catenin coimmunoprecipitation (top panel, third lane). As a control for specificity, no -catulin coimmunoprecipitated with p120 catenin (bottom panel, fourth lane) or with mouse IgG (bottom panel, second lane). Since the previously described anti--catulin antiserum (32) may not efficiently immunoprecipitate endogenous -catulin, to test the reciprocal coimmunoprecipitation of ß-catenin with -catulin, Myc-tagged -catulin expression plasmid (pcDNA:Myc--catulin) was transiently transfected into HEK293T cells and Myc--catulin was immunoprecipitated with anti-Myc antibody. Following immunoblotting with anti-ß-catenin antibody, we observed coimmunoprecipitation of the 90-kDa ß-catenin with Myc--catulin precipitate (middle panel, fourth lane). In contrast, this band was not observed upon immunoprecipitation with the mouse IgG control (middle panel, third lane), and -catenin did not coimmunoprecipitate with Myc--catulin (top panel, fourth lane), indicating specificity of the coimmunoprecipitate. In keeping with this finding, a larger amount of ß-catenin coimmunoprecipitated with endogenous -catenin (middle panel, second lane) than with transfected Myc--catulin (middle panel, fourth lane), although it should be noted that these two coimmunoprecipitates cannot be directly compared, since exogenously expressed -catulin is presumably expressed in only a fraction of the cells. Together, these results suggest that endogenous ß-catenin associates with -catulin, although apparently not as extensively as it associates with -catenin.

View larger version (29K): [in this window] [in a new window]   FIG. 1. -Catulin, a vinculin superfamily member, associates with a ß-catenin fraction. (A) Alignment of the conserved ß-catenin binding region of -catenin (residues 46 to 149) to corresponding -catulin and vinculin sequences by BLASTp (National Center for Biotechnology Information) analysis, showing 31% identity and 52% similarity between -catenin and -catulin and 28% identity and 45% similarity between -catenin and vinculin. (B) In the top panel, HEK293T whole-cell lysate from untransfected cells was immunoprecipitated with mouse IgG control antibody (second lane) or anti-ß-catenin antibody (third lane), and whole-cell lysate from HEK293T cells transfected with 1 µg of pcDNA:Myc--catulin was immunoprecipitated with anti-Myc antibody (fourth lane). Whole-cell lysate from transfected cells (first lane) and immunoprecipitates (IP) (second, third, and fourth lanes) were then immunoblotted (IB) with an anti--catenin antibody to detect coimmunoprecipitation of -catenin. In the middle panel, whole-cell lysate from HEK293T cells was immunoprecipitated with anti--catenin antibody (second lane), and lysate from HEK293T cells transfected with 1 µg of pcDNA:Myc--catulin was immunoprecipitated with mouse IgG control antibody (third lane) or anti-Myc antibody (fourth lane), and whole-cell lysate (first lane) and immunoprecipitates (second, third, and fourth lanes) were immunoblotted with an anti-ß-catenin antibody to detect coimmunoprecipitation of ß-catenin. In the bottom panel, HEK293T whole-cell lysate from untransfected cells was immunoprecipitated with mouse IgG control antibody (second lane), anti-ß-catenin antibody (third lane), or anti-p120 catenin antibody (fourth lane), and whole-cell lysate (first lane) and immunoprecipitates (second, third, and fourth lanes) were immunoblotted with an anti--catulin antibody to detect coimmunoprecipitation of -catulin.

-Catenin, but not -catulin or vinculin, inhibits TCF/LEF-mediated transcription

View larger version (21K): [in this window] [in a new window]   FIG. 2. -Catenin, but not -catulin or vinculin, inhibits ß-catenin-induced pTOPFLASH reporter activity. (A) Diagram of the composition of the TOPFLASH reporter used to measure ß-catenin-induced transcriptional activation showing the encoded TCF/LEF binding sites. (B) HEK293T cells were cotransfected with increasing amounts of either pCGN:HA--catenin (-catenin) (100 to 800 ng), pcDNA:Myc--catulin (-catulin) (0.5 to 4 µg), or pGFP:vinculin (vinculin) (0.5 to 2 µg), as indicated, and 500 ng of pcDNA:Flag ß-catenin 45 (ß-catenin 45) (except in lane 1) to induce TCF-mediated transcription. (C) Caco2A and A427 cells were cotransfected with 1 to 4 µg of pcDNA:Myc--catulin (-catulin), pCGN:HA--catenin (-catenin), pcDNA:Myc--catenin 46-149 (-catenin 46-149), or pCGN:HA--catenin 129-907 (-catenin 129-907), as indicated. (D) HEK293T cells were cotransfected with a constant amount of pCGN:HA--catenin (-catenin) (100 ng) and increasing amounts of pcDNA:Myc--catulin plasmid (-catulin) (0.5 to 4 µg). For all panels, backbone vector was used to ensure that cells were transfected with equal amounts of plasmid, all groups contained 20 ng of pRenilla, and each group was tested again with either the TOPFLASH or the FOPFLASH reporter. Experiments were performed in triplicate and repeated at least twice. Immunoblots show expression levels of -catenin, -catulin, and vinculin proteins. In panels B to D, the average pTOPFLASH and pFOPFLASH luciferase activities were normalized against Renilla luciferase activities, and the ratios of the normalized pTOPFLASH:pFOPFLASH luciferase activities were calculated for each group. In panels B and D, the pTOPFLASH:pFOPFLASH ratios are plotted as percentages of the ratio obtained from cells transfected with pcDNA:Flag ß-catenin 45 plus pcDNA (lane 2), which was set to 100%, and in panel C, these ratios are plotted as percentages of the ratio obtained from cells transfected with pcDNA alone (lane 1), which was set to 100%. Error bars represent standard deviations.

Given the complex formation between ß-catenin and -catulin, we next tested whether -catulin could compete with -catenin's activity in this system. As shown in Fig. 2D, coexpression of increasing amounts of -catulin (lanes 5 to 9) with a constant amount of -catenin which yields a submaximal level of inhibition (lane 4) did not lessen this inhibition of pTOPFLASH activation mediated by -catenin, suggesting that -catulin is unable to compete with -catenin in this inhibitory effect.

-Catenin, but not -catulin, cotranslocates with ß-catenin into the nucleus after LiCl treatment Based on previous reports suggesting that -catenin can cotranslocate to the nucleus with ß-catenin (7, 8), we evaluated the subcellular localization of -catenin and -catulin in response to LiCl-induced ß-catenin accumulation and translocation to the nucleus by isolating nuclear fractions from LiCl-stimulated and unstimulated cells, and immunoblotting for the endogenous proteins. Figure 3 shows the presence of ß-catenin in unstimulated (0 h) HEK293T whole-cell lysate, while no ß-catenin was detected in the nuclear fraction of unstimulated cells. Similarly, no -catenin or -catulin was detected in the nuclear fraction of unstimulated cells. Following stimulation of the cells with 15 mM LiCl treatment, a time-dependent increase in total cellular levels of ß-catenin was observed, while levels of -catenin and -catulin did not change. Moreover, ß-catenin was detected in the nuclear fraction within 1 h of treatment, and its level was further increased by 3 h. In addition, an accompanying appearance of endogenous -catenin in the nuclear fraction was observed, in agreement with Giannini et al.'s reports (7, 8). Interestingly, unlike -catenin, -catulin showed no nuclear accumulation in response to LiCl treatment. To monitor fraction contents, -tubulin was used as a cytosolic marker and histone H1 was used as a nuclear marker. As shown by the immunoblots in Fig. 3, -tubulin was absent from the nuclear fraction, whereas histone H1 was enriched in this fraction, validating the purity of the fractionations and the results obtained.

View larger version (29K): [in this window] [in a new window]   FIG. 3. -Catenin and ß-catenin, but not -catulin, translocate into the nucleus in response to LiCl treatment. HEK293T cells at 75% confluence were treated with 15 mM LiCl for the indicated times. Cells were collected by scraping, and nuclei were isolated as described in Materials and Methods. A small fraction of the collected cells were lysed in cell-nucleus lysis buffer prior to nucleus isolation to yield the whole-cell lysate fractions. Cell fractions were resolved by SDS-polyacrylamide gel electrophoresis and separately immunoblotted with antibodies against ß-catenin, -catenin, -catulin, -tubulin, and histone H1.

-Catulin partially inhibits ß-catenin-induced activation of the cyclin D1 promoter

View larger version (26K): [in this window] [in a new window]   FIG. 4. -Catulin inhibits ß-catenin-induced cyclin D1 reporter activation. (A) In a diagram adapted from Tetsu and McCormick (43), the different transcription factor binding sites (boxes) for the -1748 CD1, the wild-type, and two mutant -962 CD1 luciferase reporter constructs are shown. (B) HEK293T cells were cotransfected with the -1748 CD1 reporter (500 ng), pRenilla (20 ng) as an internal control, and pcDNA:Flag-ß-catenin 45 expression plasmid (500 ng) to induce TCF-mediated cyclin D1 promoter transcription with either 500 ng of pCGN:HA--catenin (-catenin) or 1 to 4 µg of pcDNA:Myc--catulin (-catulin), as indicated. (C) Caco2A cells were cotransfected with 1 µg of the -1748 CD1 reporter (lanes 2 to 4) or pGL3 Basic (lane 1), pRenilla (20 ng), and either pCGN:HA--catenin (-catenin) (4 µg) or pcDNA:Myc--catulin (-catulin) (4 µg), as indicated. (D) HEK293T cells were cotransfected with one of the cyclin D1 reporters (500 ng) described in panel A, pRenilla (20 ng), and either pcDNA vector alone (4.5 µg) or pcDNA:Flag-ß-catenin 45 (beta-catenin) (500 ng) along with either pcDNA vector (4 µg), pCGN:HA--catenin (alpha-catenin) (2 µg), or pcDNA:Myc--catulin (alpha-catulin) (4 µg), as indicated. Backbone vector was used to equalize the total amounts of transfected plasmid. For panels B to D, luciferase activity was measured as an indication of promoter activation. The means of the ratios of the reporter/Renilla luciferase readings from at least two independent experiments performed in triplicate were calculated and are presented as percentages of the value for cells transfected with reporter plus pcDNA:Flag-ß-catenin 45 in panels B and D and as percentages of the value for cells transfected with the -1748 CD1 reporter alone in panel C. For panel D, background levels from cells transfected with reporter plus pcDNA alone were subtracted from all values prior to calculating percentages and plotting. Error bars show standard deviations. In panels B and D, asterisks indicate statistically significant (P < 0.005) decreases in reporter activation compared to that in cells transfected with ß-catenin 45 plus pcDNA vector (panel B, lane 2; panel D, dark shaded bars).

-Catulin inhibits Ras-induced activation of the cyclin D1 promoter and synergistic activation of this reporter by Ras and ß-catenin To provide further support for the above findings, the effect of -catulin on cyclin D1 transcription induced by activated Ras was tested (Fig. 5A). The expression of activated RasV12 in HEK293T cells resulted in activation of the -1748 CD1 reporter (lane 2) by approximately 2.5-fold over the background stimulation observed in cells transfected with the vector alone (lane 1). Coexpression of -catulin inhibited Ras-induced activation of the -1748 CD1 reporter (lanes 3 to 4), in contrast to -catenin, which had no effect on the induction of this reporter by RasV12 (lane 5). In fact, -catulin decreased cyclin D1 reporter activation to below the background activation level (lane 1) of the reporter; a possible explanation for this finding is discussed later.

View larger version (13K): [in this window] [in a new window]   FIG. 5. -Catulin inhibits Ras signaling to the cyclin D1 promoter and its synergy with ß-catenin signaling to the same promoter. The effect of -catulin and -catenin on -1748 CD1 activation was observed in HEK293T cells transfected with pEXV:RasV12 (1 µg) (RasV12) (A) or with both pEXV:RasV12 (1 µg) and pcDNA:Flag-ß-catenin 45 (500 ng) (ß-catenin) (B). For each panel, cells were also transfected with the -1748 CD1 reporter (500 ng), pRenilla (20 ng) as an internal control, and pCGN:HA--catenin (2 µg) (-catenin) or pcDNA:Myc--catulin (1 to 4 µg) (-catulin), as indicated. Backbone vector was used to equalize total amounts of plasmid transfected. Luciferase activity was measured as an indicator of promoter activation, and the means of the ratios of the -1748 CD1/Renilla luciferase readings were calculated. Values were normalized to the reading for cells transfected with reporter plus vector alone (lane 1), which was set to a value of 1. Experiments were performed in triplicate and repeated at least twice, and data shown are those from a representative experiment. Error bars represent standard deviations.

-Catulin inhibits cyclin D1 reporter activation induced by activated RhoA Recently, Liberto et al. (24) showed that Rho can activate cyclin D1 transcription and that Rho function is required for epidermal growth factor (EGF)-induced activation of the cyclin D1 promoter and for both EGF- and activated Ras-induced S-phase entry. These results suggest that Rho may mediate Ras-induced proliferation and cell cycle progression by regulating cyclin D1 expression. Hence, we evaluated whether -catulin may modulate cyclin D1 promoter activation induced by activated RhoA. As shown in Fig. 6, coexpression of -catulin (lane 3) potently inhibited activation of the -1748 CD1 reporter induced by activated RhoL63 (lane 2), whereas the expression of -catenin had no effect (lane 4), similar to its lack of effect on Ras-induced cyclin D1 transcription.

View larger version (13K): [in this window] [in a new window]   FIG. 6. -Catulin inhibits cyclin D1 reporter activation induced by activated RhoA. HEK293T cells were cotransfected with the -1748 CD1 luciferase reporter construct (500 ng), pRenilla (20 ng), and pEXV:RhoL63 (RhoL63) (1 µg), pcDNA:Myc--catulin (-catulin) (4 µg), and pCGN:HA--catenin (-catenin) (2 µg), as indicated. Backbone vector was used to ensure that all cells were transfected with equal amounts of plasmid. Ratios of -1748 CD1/Renilla luciferase readings were calculated, and the data shown represent the means of the values from three independent experiments performed in triplicate and are presented as percentages of the activity from cells transfected with the reporter and RhoL63 alone (lane 2). Error bars represent standard deviations. An asterisk indicates statistical significance (P < 0.005) for the comparison of group 3 (RhoL63 plus -catulin) with group 2 (RhoL63 alone).

Cyclin D1 protein expression and Caco2A colony formation are partially inhibited by -catulin expression

View larger version (28K): [in this window] [in a new window]   FIG.7. -Catulin attenuates LiCl-induced cyclin D1 protein expression and colony formation in Caco2A cancer cells. (A) HEK293T cells were cotransfected with the pMACS Kk.II plasmid (2 µg) and either pcDNA vector (5 µg), pcDNA:Myc--catulin (2.5 µg) plus pcDNA (2.5 µg) (pcDNA:-catulin), pCGN:HA--catenin (2.5 µg) plus pcDNA (2.5 µg) (pCGN:-catenin), or pcDNA:Myc--catulin (2.5 µg) plus pCGN:HA--catenin (2.5 µg) (pcDNA:-catulin + pCGN:-catenin), as indicated. Twenty-four hours posttransfection, transfected cells were treated with 15 mM LiCl for 3 h where indicated and isolated by using the pMACSKk.II magnetic cell selection system. Isolated cells were lysed in cell nucleus lysis buffer, and 100 µg of lysate from each group was immunoblotted for cyclin D1 and -tubulin as described in Materials and Methods. Relative band densities were quantified by using an Alpha Innotech IS-2200 Digital Imaging System and software. The ratios of cyclin D1 band densities to -tubulin band densities were calculated for each group and normalized to the ratio obtained for LiCl-treated cells transfected with pcDNA vector alone (lane 2), which was set to a value of 1. These values are presented in the graph below the immunoblots. (B) Caco2A cells were transfected with equimolar (0.25-pmol) amounts of pcDNA vector or pcDNA:Myc--catulin (pcDNA: -catulin), each of which confers neomycin resistance, and grown in the presence of Geneticin (1.75 µg/ml) for 3 weeks, with the media being changed every third day. Colonies were then stained with crystal violet and counted. The average number of resistant colonies for three experiments relative to the average number for cells transfected with pcDNA vector alone are shown for each group in the graph. Error bars represent standard deviations.

	DISCUSSION

Top Abstract Introduction Materials and Method Result Discussion Reference

 
Despite the fact that ß-catenin is a key component of the Wnt pathway, much remains to be understood about ß-catenin and its binding partners. While we consistently detected an -catulin:ß-catenin complex in vivo upon immunoprecipitation of ß-catenin, the amount of -catulin associated with ß-catenin appeared to be smaller than that of -catenin associated with ß-catenin. Given that the majority of ß-catenin is normally bound to -catenin, this result was not altogether unexpected. ß-Catenin has several other binding partners, including APC and axin (20), which also show much lower stoichiometric association than -catenin, yet these partners are biologically relevant inhibitors. Hence, the stoichiometry of a physical association as determined by immunoprecipitation may not necessarily be a true indicator of activity, and this was the rationale for the subsequent experiments.

The dramatic inhibition of pTOPFLASH activation that was observed upon -catenin expression in HEK293T and cancer cells is consistent with data from other reports (7, 8, 40) and represents an intriguing activity for a protein that is generally viewed as a cytoskeletal linker protein. The effect of full-length -catenin on pTOPFLASH activation was significantly stronger in HEK293T cells than in the Caco2A and A427 cells, a finding we presently attribute to lower transfection efficiencies in these cancer cell lines. Since activated ß-catenin does not need to be exogenously expressed in these two cell types to activate TCF/LEF-mediated transcription due to endogenously upregulated ß-catenin levels, lower expression of the transfected -catenin would result in a weaker inhibition of pTOPFLASH activation than that observed in HEK293T cells.

In addition, we were able to further define an -catenin core bioactive region to a 103-residue stretch (residues 46 to 149), a finding which may be useful in potential therapeutic approaches to treating cancers with upregulated Wnt signaling. This region represents the smallest consensus region necessary for ß-catenin binding, as mapped by four previous studies (3, 13, 30, 31), although the signaling activity of this domain was not determined. While two of those studies suggest that smaller portions of this region (residues 97 to 148 [30] and residues 117 to 143 [13]) are sufficient to bind ß-catenin, we observed that residues 84 to 907 did not inhibit ß-catenin-mediated activation of pTOPFLASH in HEK293T cells (data not shown), suggesting that residues 46 to 149 span the smallest region presently determined to both bind ß-catenin (3) and inhibit its signaling function.

The contrasting finding that -catulin did not inhibit ß-catenin-induced pTOPFLASH activity indicates a different functional role for -catulin, despite its sequence homology with -catenin. Similarly, vinculin had no inhibitory effect, although this finding was not as unexpected, as cDNA and genomic analyses indicate that vinculin is not as closely related to -catenin as -catulin is (17, 46).

LiCl treatment led to a robust increase in total cellular levels of ß-catenin, although levels of the other proteins assayed did not change. In addition, the appearance of nuclear ß-catenin was observed, reflecting its required function in TCF/LEF transcription, which is activated by LiCl treatment, as indicated by activation of the TOPFLASH reporter (data not shown). Two mechanisms have been proposed for the inhibitory effect of -catenin on ß-catenin-mediated transcription. One proposed mechanism involves sequestration of ß-catenin in the cytosol through its binding to -catenin (40). In contrast, Giannini et al. (7) proposed that the association of -catenin with ß-catenin in the nucleus interferes with protein-DNA interactions required for TCF-mediated transcription. Our finding that LiCl treatment of cells correlates with nuclear translocation of endogenous -catenin is in keeping with Giannini et al.'s proposal and their corresponding finding, by use of immunofluorescent staining, of endogenous -catenin in the nuclei of two colon cancer cell lines with upregulated Wnt signaling (7). Since we did not observe nuclear translocation of -catulin upon treatment with LiCl, it is unlikely that -catulin can interfere with these protein-DNA interactions, and this improbability may, at least partially, explain the different effects of -catenin and -catulin on ß-catenin-mediated transcription that we observed.

The inability of excess -catulin to compete out, or reduce, -catenin's inhibition of ß-catenin-induced pTOPFLASH activation again suggests that -catulin may associate with a different ß-catenin fraction than -catenin associates with. Thus, it is possible that -catulin associates with one of the multiple, distinct ß-catenin pools proposed to exist other than the major pool associated with cadherin-catenin adhesion complexes (9) or with distinct forms of modified ß-catenin, such as tyrosine phosphorylated ß-catenin, whose protein interactions are altered (15, 22). Hence, it is conceivable that the -catulin-associated ß-catenin fraction has a function other than transcriptional activation.

The finding that -catulin inhibits up to 50% of the ß-catenin-induced activation of the -1748 cyclin D1 promoter in both HEK293T and Caco2A cells provided the first clue for an activity for -catulin in transcriptional responses. One possible explanation for why -catulin inhibits ß-catenin-mediated activation of the cyclin D1 promoter but not that of the TOPFLASH reporter may be the difference in the number of TCF sites on the two promoters. Since pTOPFLASH contains three TCF sites, the inhibition of the ß-catenin-induced activation of this reporter by -catulin may be more difficult to detect. However, since -catulin does not inhibit ß-catenin-induced activation of the -962 EtsB mt cyclin D1 reporter but does inhibit the activation of the wild-type -962 cyclin D1 reporter, both of which have the same number of TCF sites, we think this is an unlikely possibility, although it cannot presently be ruled out.

As depicted in the model in Fig. 8, the cyclin D1 promoter is also activated by Ras, which signals to this promoter through the EtsB binding site (43). The use of cyclin D1 promoter mutants suggests that -catulin attenuates cyclin D1 activation by endogenous Ras rather than by ß-catenin signals. The inhibition of RasV12-induced -1748 CD1 activity by -catulin in HEK293T cells and of the synergistic activation of this reporter by RasV12 and ß-catenin 45 further supports these findings. Since Ras and ß-catenin synergistically stimulate cyclin D1 transcription to a level higher than that induced by either protein alone (43), the total activation of the cyclin D1 promoter by ß-catenin 45 likely results from synergy of this activated ß-catenin with endogenous Ras signals induced by the presence of serum in the media used in the assays described here. Thus, the inhibition of such endogenous Ras signals to the cyclin D1 promoter by -catulin would result in the partial inhibition (up to 50%) of ß-catenin-induced cyclin D1 promoter activation which we observed. In keeping with this idea, -catulin not only completely inhibited RasV12-induced -1748 CD1 activation but also further lowered the reporter activity to below background levels when expressed in the highest amounts, also likely due to its effect on serum-induced Ras signals. Whether -catulin can attenuate Ras signals in contexts other than the cyclin D1 promoter remains to be determined.

View larger version (9K): [in this window] [in a new window]   FIG. 8. Proposed model for the observed inhibitory effects of -catulin and -catenin on the activation of cyclin D1 transcription. The cyclin D1 promoter is activated by serum via a Ras-mediated pathway which signals to the EtsB binding site and by a ß-catenin-mediated signaling pathway (inducible by LiCl) which signals to the TCF-1 binding site. -Catenin likely inhibits ß-catenin-mediated activation through its direct interaction with ß-catenin, as proposed by others (7). We propose that -catulin may inhibit Ras-induced cyclin D1 promoter activation via attenuation of Rho-mediated signaling downstream of Ras.

We propose an antiproliferative role for -catulin based on its attenuation of cyclin D1 transcription. Previous studies (32) did not detect an effect of -catulin on a different Rho-dependent transcriptional pathway mediated by the SRF, although -catulin coexpression enhanced Lbc-induced SRF activity. While the basis for this difference is not precisely understood, a number of possibilities exist. The activation of different Rho-dependent transcriptional pathways is likely associated with distinct Rho complexes containing particular scaffolds and Rho effectors. On this basis, the presence of a putative scaffold, such as -catulin, in a Rho complex may disrupt a particular downstream Rho effector that is required for cyclin D1 transcription but not for SRF-mediated responses. Alternatively, the presence of -catulin may limit the availability of common Rho effectors for a Rho complex required for cyclin D1 transcription by sequestering them into a distinct Rho complex associated with SRF activation. While at this point these and other possibilities remain to be explored, the identification of other binding partners of -catulin may yield further clues concerning its mechanisms of signal modulation.

Our finding that -catulin expression downregulates cyclin D1 protein levels is in keeping with the results obtained by using cyclin D1 transcriptional reporter assays and, together with our finding that -catulin expression attenuates colony formation in Caco2A cells, supports the notion that -catulin can modulate endogenous growth signaling pathways. -Catenin expression was also previously shown to lead to reduced colony formation in cells containing mutant -catenin (3), suggesting a possible function for this protein family in cell growth control. While our observations are based on the exogenous expression of -catenin and -catulin, it is certainly conceivable that levels of the endogenous proteins are modulated. The vinculin gene contains SRF-responsive elements which respond to serum stimulation (25), and whether the -catenin and -catulin genes contain SRF binding elements remains to be determined. Such a possibility would potentially allow for the downregulation of Wnt-associated signaling by increased endogenous levels of these two proteins in response to serum, thus establishing regulatory loops between different growth pathways and providing tight, time-dependent control over the expression of genes involved in specific stages of inductive cell growth.

The Wnt pathway is upregulated in many human cancers, including colon cancer (2), the second most commonly occurring cancer, and activating Ras mutations are present in approximately 20% of all cancers (5). At least in the context described here, our findings indicate that -catenin and -catulin may down-modulate the activity of dominantly active oncogenes such as ß-catenin 45 and RasV12, respectively, along with their endogenously activated signals found in transformed cells, and thus potentially provide a basis for future cancer therapies.

	ACKNOWLEDGMENTS

 
We thank L. Bullions, H. Clevers, B. Geiger, A. Hall, C. Marshall, F. McCormick, and B. Vogelstein for plasmids.

Funding for this study was provided by grant NCI CA62029, a Howard Hughes Medical Institute bridge support grant, the Hershey Family Fund for Prostate Cancer Research, grant NIHT32 DK07542, and in part by the Center for Gastroenterology Research on Absorptive and Secretory Processes (GRASP) (grant NIDDK 1 P30 DK39428).

	FOOTNOTES

 
* Corresponding author. Mailing address: Tufts University School of Medicine, Department of Cellular and Molecular Physiology, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6914. Fax: (617) 636-0445. E-mail: keith.merdek{at}tufts.edu.

	REFERENCE

Top Abstract Introduction Materials and Method Result Discussion Reference

 
1. Aberle, H., S. Butz, J. Stappert, H. Weissig, R. Kemler, and H. Hoschuetzky. 1994. Assembly of the cadherin-catenin complex in vitro with recombinant proteins. J. Cell Sci. 107:3655-3663.[Abstract]

2. Bienz, M., and H. Clevers. 2000. Linking colorectal cancer to Wnt signaling. Cell 103:311-320.[CrossRef][Medline]

3. Bullions, L. C., D. A. Notterman, L. S. Chung, and A. J. Levine. 1997. Expression of wild-type alpha-catenin protein in cells with a mutant alpha-catenin gene restores both growth regulation and tumor suppressor activities. Mol. Cell. Biol. 17:4501-4508.[Abstract]

4. Cadigan, K. M., and R. Nusse. 1997. Wnt signaling: a common theme in animal development. Genes Dev. 11:3286-3305.[Free Full Text]

5. Downward, J. 2003. Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 3:11-22.[CrossRef][Medline]

6. Geiger, B., S. Yehuda-Levenberg, and A. D. Bershadsky. 1995. Molecular interactions in the submembrane plaque of cell-cell and cell-matrix adhesions. Acta Anat. 154:46-62.[Medline]

7. Giannini, A. L., M. Vivanco, and R. M. Kypta. 2000. Alpha-catenin inhibits beta-catenin signaling by preventing formation of a beta-catenin*T-cell factor*DNA complex. J. Biol. Chem. 275:21883-21888.[Abstract/Free Full Text]

8. Giannini, A. L., M. M. Vivanco, and R. M. Kypta. 2000. Analysis of beta-catenin aggregation and localization using GFP fusion proteins: nuclear import of alpha-catenin by the beta-catenin/Tcf complex. Exp. Cell Res. 255:207-220.[CrossRef][Medline]

9. Gottardi, C. J., and B. M. Gumbiner. 2001. Adhesion signaling: how beta-catenin interacts with its partners. Curr. Biol. 11:R792-R794.[CrossRef][Medline]

10. Hagenbuchle, O., and P. K. Wellauer. 1992. A rapid method for the isolation of DNA-binding proteins from purified nuclei of tissues and cells in culture. Nucleic Acids Res. 20:3555-3559.[Abstract/Free Full Text]

11. Hazan, R. B., L. Kang, S. Roe, P. I. Borgen, and D. L. Rimm. 1997. Vinculin is associated with the E-cadherin adhesion complex. J. Biol. Chem. 272:32448-32453.[Abstract/Free Full Text]

12. He, T. C., A. B. Sparks, C. Rago, H. Hermeking, L. Zawel, L. T. da Costa, P. J. Morin, B. Vogelstein, and K. W. Kinzler. 1998. Identification of c-MYC as a target of the APC pathway. Science 281:1509-1512.[Abstract/Free Full Text]

13. Huber, O., M. Krohn, and R. Kemler. 1997. A specific domain in alpha-catenin mediates binding to beta-catenin or plakoglobin. J. Cell Sci. 110:1759-1765.[Abstract]

14. Hunter, T., and J. Pines. 1994. Cyclins and cancer. II. Cyclin D and CDK inhibitors come of age. Cell 79:573-582.[CrossRef][Medline]

15. Ilan, N., S. Mahooti, D. L. Rimm, and J. A. Madri. 1999. PECAM-1 (CD31) functions as a reservoir for and a modulator of tyrosine-phosphorylated beta-catenin. J. Cell Sci. 112:3005-3014.[Abstract]

16. Imamura, Y., M. Itoh, Y. Maeno, S. Tsukita, and A. Nagafuchi. 1999. Functional domains of alpha-catenin required for the strong state of cadherin-based cell adhesion. J. Cell Biol. 144:1311-1322.[Abstract/Free Full Text]

17. Janssens, B., K. Staes, and F. van Roy. 1999. Human alpha-catulin, a novel alpha-catenin-like molecule with conserved genomic structure, but deviating alternative splicing. Biochim. Biophys. Acta 1447:341-347.[Medline]

18. Jou, T. S., D. B. Stewart, J. Stappert, W. J. Nelson, and J. A. Marrs. 1995. Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex. Proc. Natl. Acad. Sci. USA 92:5067-5071.[Abstract/Free Full Text]

19. Kemler, R. 1993. From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet. 9:317-321.[CrossRef][Medline]

20. Kikuchi, A. 2000. Regulation of beta-catenin signaling in the Wnt pathway. Biochem. Biophys. Res. Commun. 268:243-248.[CrossRef][Medline]

21. Kikuchi, A. 2003. Tumor formation by genetic mutations in the components of the Wnt signaling pathway. Cancer Sci. 94:225-229.[CrossRef][Medline]

22. Kim, K., and K. Y. Lee. 2001. Tyrosine phosphorylation translocates beta-catenin from cellcell interface to the cytoplasm, but does not significantly enhance the LEF-1-dependent transactivating function. Cell Biol. Int. 25:421-427.[CrossRef][Medline]

23. Klein, P. S., and D. A. Melton. 1996. A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. USA 93:8455-8459.[Abstract/Free Full Text]

24. Liberto, M., D. Cobrinik, and A. Minden. 2002. Rho regulates p21(CIP1), cyclin D1, and checkpoint control in mammary epithelial cells. Oncogene 21:1590-1599.[CrossRef][Medline]

25. Moiseyeva, E. P., P. A. Weller, N. I. Zhidkova, E. B. Corben, B. Patel, I. Jasinska, V. E. Koteliansky, and D. R. Critchley. 1993. Organization of the human gene encoding the cytoskeletal protein vinculin and the sequence of the vinculin promoter. J. Biol. Chem. 268:4318-4325.[Abstract/Free Full Text]

26. Molenaar, M., M. van de Wetering, M. Oosterwegel, J. Peterson-Maduro, S. Godsave, V. Korinek, J. Roose, O. Destree, and H. Clevers. 1996. XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86:391-399.[CrossRef][Medline]

27. Morin, P. J. 1999. Beta-catenin signaling and cancer