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. Author manuscript; available in PMC: 2015 Oct 7.
Published in final edited form as: Int J Cancer. 2012 Mar 28;131(10):2223–2233. doi: 10.1002/ijc.27519

Regulation of the DLG tumour suppressor by β-catenin

Vanitha Krishna Subbaiah 1, Nisha Narayan 1, Paola Massimi 1, Lawrence Banks 1,*
PMCID: PMC4596009  EMSID: EMS65090  PMID: 22392736

Abstract

The Discs Large (DLG) tumor suppressor plays essential roles in regulating cell polarity and proliferation. It localizes at sites of cell-cell contact where it acts as a scaffold for multiple protein interactions, including with the APC tumor suppressor, which in turn regulates β-catenin. Furthermore, many tumor types including breast and colon have increased levels of β-catenin activity with correspondingly low levels of DLG expression. Here we provide evidence of a direct functional link between these apparently separate phenomena. We show that overexpressed β-catenin can enhance the turnover of DLG in a proteosome dependent manner. This effect is specific to DLG, and is not seen with two other PDZ domain-containing targets of β-catenin, MAGI-1 and Scribble. Furthermore, siRNA mediated ablation of endogenous β-catenin expression also enhances DLG stability. β-catenin induced degradation of DLG appears to be a consequence of a direct association between the two proteins, and requires β-catenin PDZ binding potential. In contrast, the enhanced turnover of DLG requires the unique N terminal sequences and its PDZ domains. Finally we also show that the capacity of DLG to inhibit transformed cell growth in an oncogene cooperation assay is inhibited by β-catenin. Taken together these studies suggest that one mechanism by which deregulated β-catenin can contribute to tumorigenesis is through enhancing DLG degradation.

Keywords: DLG, β -catenin, proteasome

INTRODUCTION

The formation of adhesion structures between adjacent cells called adherens junctions is crucial for a variety of biological processes, both during embryonic development and in the adult (1). The establishment and stabilization of adherens junctions in epithelial cells is a highly regulated process, controlled by transmembrane protein complexes and cell-cell junctional proteins. β-catenin is a multifunctional protein crucial for two distinct cellular processes, namely E-cadherin mediated cell adhesion and regulation of Wnt signaling, deregulation of the latter being a major cause of colorectal cancers (2). β-catenin is cytoplasmic and binds to the intracellular cytoplasmic domain of E-cadherin and is also complexed with α-catenin bound to actin. This adhesive pool of β-catenin bound to the E- cadherin complex ultimately links the cytoskeletal networks of adjacent cells, contributing to the maintainance of normal tissue architecture and morphogenesis (3, 4).

Loss of intercellular junctions is a characteristic feature of many malignant tumors and experimental evidence has shown that perturbations in the E-Cadherin/catenin complex are characterized by loss of epithelial differentiation and the onset of invasion (5). Furthermore, deregulated expression of β-catenin and mutations in the regulatory region of β-catenin have been reported in many diseases and cancers (6; 7), including prostate cancer (8), endometrial carcinomas (9) and squamous cell carcinomas of the esophagus (10). A high percentage of colon cancers are caused by inactivation of the APC gene, resulting in the inhibition of ubiquitin-mediated degradation of β-catenin, (11) while a small percentage of colon cancers also harbour β-catenin stabilizing mutations (12). In both cancers the common feature being increased levels of β-catenin expression.

Interestingly, various structural components of the intercellular junctions have characteristics associated with tumor suppressor gene products. For example, the APC tumor suppressor has been reported to bind to E-cadherin (13; 14), to the tight junction protein ZO-1 and to the PDZ (PSD-95/DLG/ZO-1) domain containing tumor suppressor Discs Large (DLG) (15). DLG is a prototypic member of the MAGUK family of proteins and was originally identified as a tumor suppressor gene in Drosophila, since inactivation of this gene resulted in neoplastic overgrowth of the imaginal disc epithelium. These epithelial cells were characterized by a loss of apico-basal polarity, lack of differentiation and loss of cell adhesion (16;17). Consistent with its role as a potential tumor suppressor, DLG is also the target of a number of viral oncoproteins, including HTLV-1 Tax (18), the adenovirus 9 E4ORF1 oncoprotein (19) and the cancer-causing HPV E6 oncoprotein (20). In addition, numerous studies have also shown the loss of DLG expression in a variety of different late-stage tumours including oesophageal, gastric and, colon cancers (21; 22; 20), and in cervical cancer (23; 24). Interestingly, loss of the anti-proliferative functions of DLG has been linked to loss of cell polarity, which may affect specific receptor-mediated signaling (25), and possibly affect its interaction with APC and thereby contribute to the regulation of β-catenin signaling (15; 26). A number of studies have shown that other PDZ domain-containing proteins, including NE-DLG (27), MAGI-1 (28), LIN-7 (29), Erbin (30), lp-DLG/KIAA0583 (31), and Scribble (32), have the propensity to associate with β-catenin, most often through its carboxy terminal class I PDZ binding motif (PBM) (28).

Although much is known about the role of DLG as a tumor suppressor and β-catenin as a proto-oncogene, no studies have been done to investigate any potential direct effects of β-catenin on DLG expression. This seems particularly relevant since deregulation of β-catenin and loss of regulation of cell-cell adhesion is a common feature of cancer development, and the corresponding loss of DLG also appears frequent in diverse epithelial tumours, including colon and cervical tumors (15,26, 23). In this study, we aimed to determine whether β-catenin could impact directly upon the levels of DLG expression and tumour suppressor function.

MATERIALS AND METHODS

Cell culture and transfections

HEK293 (Human embryonic kidney), Caco-2 (colorectal cancer derived) and C33A (cervical cancer derived) cells were grown in DMEM supplemented with 10% FBS. Cells were transfected using calcium phosphate precipitation or Lipofectamine 2000 (Invitrogen). MG132-proteosome inhibitor (SIGMA) was used at a concentration of 100 μM for 3 hours. Primary BRK cell transformatin assays were done as previously described (33)

Plasmid constructs

The wild type and mutant HA-tagged DLG expression constructs have been described previously (33) as have HA-tagged Scribble (34) and Flag-tagged MAGI-1 (35). The mutant DLG constructs used in this study were: HA-DLG-NTPDZ1 (aa 1–276); HA-DLG-NTPDZ1-2 (aa 1–382); HA-DLG-3PDZ (aa 186–511); HA-DLG-ΔNT (Δ1–222) and DLG-ΔSH3 (Δ549–617), HA-CT (aa 549-904). The wild type HA- β-Catenin plasmid was a kind gift from Prof. Claudio Brancolini. The PDZ mutant of β-Catenin was generated using the Gene Tailor site-directed mutagenesis kit following the manufacturer’s instructions (Invitrogen). The primers used were: forward primer 5′-GACAGCAATCAGCTGGCCTGGTTTTGAGATACTGAC -3′ and reverse primer 5′-AAACCAGGCCAGCTGATTGCTGTCACCTGG-3′. The mutation was confirmed by sequencing. Flag tagged, His tagged and untagged wt- β-Catenin (16828, 17198 and 16518) and the Flag tagged and untagged β-Catenin S33Y mutant (19286 and 16519) were purchased from Addgene.

Expression and purification of His-tagged β-catenin, GST tagged Dlg and interaction assays

His-tagged β-catenin was expressed in E.coli BL21 by inducion with IPTG for 5hrs. The cell lysates were suspended in lysis buffer containing 10mM Tris (pH8.0), 300mM NaCl, 100mM NaH2PO4 and then clarified by centrifugation. The supernatant was incubated with Ni-NTA agarose beads (Qiagen, Milano, Italy) for 4hrs at 4°C. The beads were then pelleted and bound proteins washed extensively with 50mM imidazole containing lysis buffer. Purified protein was then eluted using 300mM imidazole. GST.Dlg or GST alone were induced and purified as described previously (34). For the interaction assays purified GST fusion proteins bound to glutathione agarose beads were incubated with purified His-tagged β-catenin for 3-4 hrs at 4°C. The beads were then extensively washed and bound proteins detected by western blotting.

Western blot

Cells were lysed in extraction buffer (25 mM HEPES pH 7.0, 0.1% NP-40, 150 mM NaCl, protease inhibitor cocktail I (Calbiochem,Milano Italy)), and then subjected to (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel Germany). Primary antibodies used were mouse anti-HA monoclonal antibody (Roche, Milano Italy), mouse anti- β-Catenin monoclonal antibody (Santa Cruz, Heidelberg Germany) and mouse anti-β-galactosidase monoclonal antibody (Promega, Milano Italy) and secondary anti-mouse antibodies were conjugated to horseradish peroxidase (Dako, Milano Italy). The proteins were visualized by enhanced chemiluminescence (GE Healthcare, Milano Italy).

Immunoprecipitation assays and antibodies

For coimmunoprecipitation of endogenous DLG, the protein was immunoprecipitated from HEK293 cells extracted in extraction buffer and incubated with anti-DLG antibody (Santa Cruz) for 4h. Protein-A-Sepharose beads (GE Healthcare) were then added for an additional 50 minutes and then washed with extraction buffer and precipitated proteins were analysed by Western blot. Endogenous DLG was detected using anti-DLG monoclonal antibody (2D11, Santa Cruz) and HA-tagged DLG was detected using anti-HA monoclonal antibody (Roche). Tubulin was detected using anti-tubulin monoclonal antibody (Sigma, Milano Italy).

siRNA transfections

Cells were seeded on 10 cm dishes and transfected using Lipofectamine 2000 (Invitrogen) with control siRNA against luciferase (siLuc) or siRNA against β-Catenin (Dharmacon, Milano Italy). Seventy-two hours after transfection, the cells were harvested and analysed by Western blot.

Half life determinations

HEK293 cells were seeded in 6cm dishes and transfected with 1.5 μg HA-DLG either alone or in combination with 1.5 μg wt-β-catenin, and 0.1 μg of the β-gal plasmid as a transfection control. After 24hrs, cells were treated with cycloheximide (50 μg/ml in dimethyl sulfoxide) for different time points to block protein synthesis. Total cellular extracts were prepared and the residual DLG was analysed by Western blot and quantitated using the ImageJ software.

RESULTS

The oncogenic activity of β-catenin has been largely attributed to its role in regulating cell-cell adhesion and in nuclear signalling, as a key effector molecule of the Wnt signaling pathway (36). An important feature of this is a de-regulation of β-catenin turnover, which can occur either through loss of APC or through mutation of the β-catenin regulatory phospho-acceptor sites. There is still limited information, however, on the potential relevance of its PDZ binding motif in these activities, although previous studies have reported association with Scribble, NE-DLG and MAGI-1 (27; 28, 32, 37). Considering that DLG belongs to the same pathway of polarity control as Scribble (25), and that it is also frequently lost in colon cancers where β-catenin is overexpressed (12), we were interested in determining whether there was any direct causal relationship between these observations. To investigate this, we first analysed the effects of β-catenin overexpression upon DLG. HEK293 cells were transfected with a HA-tagged DLG expression plasmid in the presence and absence of ectopically overexpressed β-catenin and the levels of DLG expression were ascertained by Western blot using anti-HA antibodies. The results in Figure 1a show that β-catenin induces a marked down-regulation in the levels of DLG expression. We then analysed whether this was due to enhanced proteasomal degradation of DLG by inclusion of the proteasome inhibitor MG132. The results in Figure 1b demonstrate that the increased degradation of DLG in the presence of high levels of β-catenin expression is indeed proteasome-mediated.

Figure 1. β-catenin degrades DLG but not Scribble or MAGI-1 through the proteosome pathway.

Figure 1

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(a) HA-tagged DLG was transfected into HEK293 cells in the absence and presence of ectopically expressed β-catenin or (b) in the presence of wt β-catenin and the proteosome inhibitor (MG132) as indicated. Cell extracts were analysed by western blot and DLG was detected by probing with anti-HA antibody and β-catenin was detected by probing with anti- β-catenin antibody. Transfection efficiency was controlled by probing the blots with anti-β-galactosidase antibody (βgal). (c) HA-tagged Scribble and (d) Flag-tagged MAGI-1 were transfected into HEK293 cells in the absence and presence of ectopically expressed β-catenin and either treated or untreated with the proteosome inhibitor (MG132) as indicated. Cell extracts were analysed by Western blot and Scribble and MAGI-1 was detected by probing with anti-HA antibody and anti-Flag antibodies respectively. The blots were also probed for βgal to control for transfection efficiency.

Since Scribble and MAGI-1 have also been reported to be interacting partners of β-catenin (28, 32, 37) we then proceeded to investigate whether they were also subject to enhanced degradation in the presence of over-expressed β-catenin. HEK293 cells were transfected with HA-tagged Scribble and Flag-tagged MAGI-1 expression plasmids, together with ectopically expressed β-catenin. The levels of MAGI-1 and Scribble were then ascertained by Western blot and the results in Figure 1c and Figure 1d show that the levels of both Scribble and MAGI-1 expression are unaffected by increased levels of β-catenin. These results demonstrate that enhanced degradation of DLG in the presence of high levels of β-catenin is not a common feature of proteins that possess PDZ domains, but is instead specific to DLG.

We next wanted to ascertain whether the enhanced proteasomal degradation of DLG in the presence of ectopically expressed β-catenin was due to enhanced turnover of DLG. To do this, HEK293 cells were co-transfected with the β-catenin and HA-tagged DLG expression plasmids. After 24hrs the cells were treated with cycloheximide for different periods of time to block further protein synthesis. The residual levels of DLG expression were then determined by Western blot and the results obtained are shown in Figure 2a, together with the quantifications from multiple assays in Figure 2b. As can be seen, under normal circumstances DLG has a half-life of more than 12hrs. However in the presence of ectopically expressed β-catenin the DLG half-life is reduced to between 4 and 6hrs. These results demonstrate a dramatic increase in the rate of DLG turnover in the presence of high levels of β-catenin.

Figure 2. β-catenin reduces the half-life of ectopically expressed DLG in HEK293 cells.

Figure 2

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(a) HEK293 cells were transfected with HA-tagged DLG in the absence and presence of wtβ-catenin as indicated. After 24hrs, cycloheximide was added to the dishes every 2hrs up to 12hrs and the cellular extracts were analysed for residual DLG expression using anti-HA antibody and the blots were also probed for β-catenin and β-gal. (b) Shows the cumulative data from three independent experiments, analyzed using the ImageJ program to quantify the intensity of bands The graph shows the percentage of DLG remaining relative to the first time point. The error bars represent ±SD. (c) (i) HEK293 (ii) Caco-2 (iii) C33A cells were transfected with siluc or si β-catenin and the cell lysates were analysed for expression of endogenous DLG using anti-SAP97 (2D11) and anti-β-catenin antibodies. Blots were probed for gamma-tubulin as loading control.

Having shown that β-catenin can increase the rate of proteasomal degradation of DLG, we then proceeded to investigate whether loss of endogenous β-catenin expression could also result in an increase in the levels of DLG expression. To do this, HEK293, Caco-2 colorectal adenocarcinoma cells and HPV-negative C33A cervical cancer cells were transfected with a β-catenin siRNA and si-Luc as a control. After 72hrs the cells were harvested and the levels of DLG expression assessed by Western blot. The results in Figure 2c show in all three cell lines a clear increase in the levels of DLG expression in the absence of β-catenin, further supporting a direct role for β-catenin in the regulation of DLG protein expression levels.

Previous studies have shown that MAGI-1 and Lin-7 can both associate with β-catenin through their PDZ domains (28; 29). Since DLG has three such domains we next wanted to determine whether β-catenin and DLG have the potential to form a complex in vivo and, if so, whether this is PDZ dependent. To do this, endogenous DLG was immunoprecipitated from HEK293 cells using anti DLG antibody and co-precipitating β-catenin detected by Western blot using anti β-catenin antibody. The results obtained are shown in Figure 3a where it can be seen that both DLG and β-catenin form a complex in vivo. To determine if this interaction was PDZ dependent, HEK293 cells were transfected with either wild type HA-tagged β-catenin or a mutant HA-tagged β-catenin which was generated by deleting the PBM at the extreme C-terminus. After 24hrs the cells were harvested and cell extracts immunoprecipitated using anti-HA antibody, and any co-immunoprecipitated DLG was detected by Western blot. The results in Figure 3b show that wild type β-catenin can form a complex with DLG. Furthermore, this interaction appears to be PDZ specific since the mutant β-catenin that lacks the PBM interacts only very weakly with DLG. We then repeated the co-immunoprecipitation assay using endogenous wild type β-catenin and overexpressed HA-tagged deletion constructs of DLG. A schematic representation of the various deletion mutants of DLG used in these studies is shown in Figure 3c. HEK293 cells were transfected with wild type DLG, a DLG mutant lacking the N terminal 222 amino acids but retaining intact PDZ domains (ΔNT-DLG) and a mutant encompassing the N terminus and the first two PDZ domains (NT PDZ1/2). After 24hrs the cells were harvested and immunoprecipitated with anti-HA antibody, and bound β-catenin was detected by Western blot analysis with anti β-catenin antibody. The results in Figure 3d demonstrate a number of interesting features. Again, a clear co-immunoprecipitation is obtained with the wild type DLG and β-catenin. However, DLG that lacks the N terminal 222 amino acids is reduced in its ability to interact with β-catenin, whilst a mutant DLG that lacks the carboxy terminus and PDZ domain 3 retains a capacity to interact with β-catenin, albeit not as strongly as the wild type. These results demonstrate that sequences within both the PDZ domains and the DLG N terminal region are important for DLG’s capacity to interact with β-catenin. In contrast, the principal interaction site on β-catenin would appear to reside within its PDZ binding motif. This suggests that sequences within the N terminus of DLG can potentially influence accessibility of ligands to the downstream PDZ domains. We then proceeded to investigate whether the interaction between Dlg and β-catenin was direct, since in the above analyses we cannot exclude the possibility that the association was indirect. To do this Dlg and β-catenin were expressed in E.coli as GST and Histidine (His)-tagged fusion proteins respectively and purified by affinity chromatography. The glutathione bound GST-Dlg or GST alone, was then incubated with purified His-β-catenin and bound β-catenin detected by western blot analysis using anti-β-catenin antibody. The results in Figure 3e show a clear direct interaction between Dlg and β-catenin.

Figure 3. DLG and β-catenin interact in vivo.

Figure 3

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(a) Endogenous DLG was immunoprecipitated from HEK293 cell extracts with anti-DLG monoclonal antibody, and mouse monoclonal anti-Flag antibody was used as a negative control. Co-immunoprecipated β-catenin was then detected by western blotting with anti-β-catenin antibody. (b) HEK293 cells were transfected with vector or HA-tagged wt- β-catenin or the HA-tagged ΔPBM mutant of β-catenin and equal amounts of the cellular proteins was used to immunoprecipitate the ectopically over expressed β-catenin. Samples were analysed by Western blot and the amount of endogenous DLG bound to wt-β-catenin or the ΔPBM mutant was detected using anti-DLG (2D11) antibody. Overexpressed β-catenin was detected using anti-HA antibody. 30% of the input is used to show that equal amount of protein was used in the immunoprecipitations. The blots for input samples were probed for endogenous DLG and overexpressed β-catenin using anti-DLG antibody and anti-HA antibodies respectively. (c) Schematic representation of deletion constructs of DLG used in this study (d) HEK293 cells were transfected with vector, HA-tagged wt DLG, a mutant lacking the N terminal 222aa (ΔNT-DLG) or a mutant retaining just the N terminus along with the PDZ1 and PDZ2 domains. Equal amounts of the cell extract was used to immunoprecipitate the HA-tagged proteins (left hand panel). Samples were analysed by Western blot and the amount of endogenous β-catenin bound to wt-DLG or the mutants were detected using anti- β-catenin antibody. The immunoprecipitated DLG was detected using the anti-HA antibody. Thirty percent of the input is used to show that an equal amount of protein was used in the immunoprecipitations (right hand panel) and were probed for endogenous β-catenin and overexpressed DLG (HA) and tubulin. The arrows indicate the bands corresponding to HA-tagged wt DLG, the mutant lacking the N terminal 222aa (ΔNT-DLG) or the mutant retaining just the N terminus along with the PDZ1 and PDZ2 domains respectively. (e) Purified GST.Dlg or GST alone was incubated with purified His-tagged β-catenin and after extensive washing the bound β-catenin detected by westrn blotting using anti- β-catenin antibody (upper panel). The lower panel shows the Ponceau stain of the nitrocellulose membrane confirming similar levels of protein loading.

Having found that the optimal interaction between β-catenin and DLG requires the PBM on β-catenin and sequences involving the PDZ domains and the N terminal region of DLG, we were then interested in determining whether these were similarly required for the effects of β-catenin on the turnover of DLG. To do this we co-expressed wild type DLG and a panel of DLG mutants with the wild type β-catenin in HEK293 cells and analysed the levels of DLG expression by Western blot. The results in Figure 4 show that sequences within the N-terminal 222 amino acids of DLG appear to be essential for the ability of β-catenin to induce optimal degradation of DLG, with the ΔNT mutant being largely unaffected. However, the DLG PDZ domains would also appear to be required. Thus degradation is seen with wild type DLG, and with the ΔSH3, NT1 and NT1-2 mutants and, interestingly, a much weaker but consistent degradation is also seen with the 3PDZ mutant, which lacks the N terminal region of DLG. This suggests that when in isolation, the DLG PDZ domains are susceptible to β-catenin targeting. No change in expression levels of DLG were seen with a mutant (DLGCT) that expresses only the carboxy terminal third of the protein.

Figure 4. DLG N-terminal sequences are required for β-catenin induced degradation.

Figure 4

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HEK293 cells were transfected with HA-tagged wt-DLG and a panel of DLG mutants in the presence and absence of wt- β-catenin. Western blot analysis was performed on the cell extracts and DLG was detected by probing the blots for anti-HA antibody, Overexpressed β-catenin was detected with anti- β-catenin antibody and βgal is also shown as a control for the transfection efficiency.

We then analysed whether an intact PBM was required on β-catenin for it to be able to enhance DLG turnover. Wild type DLG and the 3PDZ and ΔNT mutants were co-expressed with the wild type (Figure 5a, lanes 4-6) and the ΔPBM mutant of β-catenin (lanes 7-9) and the residual levels of DLG were ascertained by Western blot. The results again confirm the critical importance of the N terminal domain in DLG for β-catenin enhanced degradation (compare lanes 2, 5 and 8). Furthermore, they also confirm that the 3PDZ mutant of DLG is also still susceptible to β-catenin targeting (compare lanes 3,6 and 9). Most strikingly however these data also show that an intact PBM on β-catenin is critical for its ability to enhance degradation of DLG (compare lanes 1 and 7). Quantification of protein bands from three independant experiments is also shown in the Figure 5b. As a further confirmation, we also found that a C-terminally tagged β-catenin (which also blocks the PBM) was also unable to degrade DLG (Figure 5c). In many tumors β-catenin is constitutively activated by mutation of the regulatory serines (2). To investigate if cancer derived mutant β-catenin could also enhance DLG degradation we analysed the S33Y β-catenin mutant. Cells were transfected with DLG together with the wild type and S33Y mutant β-catenin and residual levels of DLG ascertained by western blotting. The results in Figure 5d show increased levels of DLG degradation by the mutant β-catenin. Taken together these results demonstrate that β-catenin can recognize DLG through a PBM, which in turn can result in enhanced degradation of DLG protein. However, sequences within the N terminal region of DLG also play a critical role both in β-catenin recognition and in subsequent proteolytic degradation.

Figure 5. Removal of the PBM in β-catenin impairs its ability to degrade DLG.

Figure 5

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(a) HEK293 cells were transfected with either HA-tagged wt-DLG or the HA-tagged mutants (Lanes 1-3) in the presence of the wt- β-catenin (Lanes 4-6) or the ΔPBM mutant of β-catenin as indicated (Lanes 7-9). Cell extracts were analysed by Western blot for DLG by probing the blots with anti-HA antibody, β-catenin was detected using anti- β-catenin antibody and anti-βgal was used to control for the trasnfection efficiency. (b) Graph showing the quatification of bands from multiple experiments. Error bars represent ±SD of three independent experiments (c) Western blot of DLG following co-transfection with carboxy-terminal flag tagged wild type and S33Y mutant β-Catenin (d) Western blot of DLG following co-transfection with untagged wild type and S33Y mutant β-Catenin.

To determine whether the degradation of DLG by β-catenin was physiologically relevant, we analysed the effects of the wild-type β-catenin and the ΔPBM β-catenin mutant construct in an oncogene cooperation assay. Primary baby rat kidney (BRK) cells were transfected with human papillomavirus-16 E7 plus EJ-ras, in the presence or absence of either the DLG, wt- β-catenin or the β-catenin mutant expression plasmids. After 2 weeks, the cells were fixed and stained and the numbers of colonies counted. The results shown in Figure 6a and Figure 6b demonstrate that E7 and EJ-ras induced transformation is inhibited by DLG, and this is in agreement with previous studies (38). However, the inclusion of wt- β-catenin readily overcomes this inhibition. In contrast, inclusion of the ΔPBM mutant of β-catenin is unable to overcome the suppressive effects of DLG. These results demonstrate that one of the functional consequence of the ability of β-catenin to bind DLG and to enhance DLG turnover is an abolition of DLG’s tumour suppressor activity.

Figure 6. β-catenin inhibits DLG tumour suppressor activity.

Figure 6

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BRK cells were transfected with HPV-16 E7 and activated ras together with DLG and either wt-β-catenin or the β-catenin ΔPBM mutant, as indicated. After 2 weeks under G418 selection, the cells were fixed and stained and the colonies were counted. (a) Representative dishes from one such assay. (b) Histogram showing the average number of colonies obtained from four independent assays and the error bars represent ±SD of four independent assays.

DISCUSSION

In this study, we have investigated the potential influence of a proto-oncogene β-catenin on the DLG tumor suppressor. We show that β-catenin significantly downregulates DLG protein levels through a mechanism that involves the proteosome pathway. Moreover, this effect of β-catenin is specific to DLG since two other PDZ-containing proteins, Scribble and MAGI-1, were not affected. We also provide evidence for an interaction between wt-β-catenin and wt-DLG in vivo. The residues required for interaction are also those required for enhanced β-catenin induced degradation of DLG.

β-catenin is an oncogene implicated in the development of several human tumors (39). Its role in tumourigenesis is surmised from the fact that it is a major adherens junction component and an effector molecule of the Wnt signaling pathway, mutations in which are linked to the development of several human cancers (39). Three lines of evidence point to a direct role for β-catenin in tumourigenesis. This can be achieved through activating mutations in β-catenin’s regulatory regions (40), mutations within APC which result in a loss of its ability to bind β-catenin, or finally, aberrant activation of the Wnt signaling pathway itself, thereby leading to the constitutive activation of β-catenin. The net result in all three cases is increased levels of β-catenin expression and increased transcriptional activation of its downstream target genes (36). In addition to its role in the regulation of transcription, β-catenin also possesses a class I PDZ binding motif (28) which confers the capacity to interact with diverse cellular proteins that possess class I PDZ domains (41). Indeed, several studies have also shown that the interaction of β-catenin with PDZ domain-containing scaffolding proteins such as LIN-7 and lp-DLG/KIAA0583 (29; 31), may play a role in the formation of proper cellular junctions and mediate signal transduction from the membrane to the cytoskeleton (30). We now show that the DLG tumour suppressor protein, which is itself a PDZ domain-containing scaffolding protein, is also a target of β-catenin.

There are several lines of evidence suggesting that β-catenin can directly contribute to the regulation of DLG expression levels. We first found that overexpressed β-catenin can downregulate DLG protein levels in a proteasome dependent manner. Furthermore we also demonstrated that the negative effect of β-catenin on DLG levels is caused by β-catenin enhancing DLG protein turnover, resulting in a greatly shortened DLG half-life. Finally, we also showed that ablation of endogenous β-catenin expression using siRNA, also resulted in a significant increase in the levels of endogenous DLG expression. Most importantly, these effects of β-catenin on DLG expression levels are also highly specific. Thus, previous studies have shown that the interaction between MAGI-1 and β-catenin in neuronal cells supports the increased targeting of the non-neuronal isoform of MAGI-I (S-SCAM) to synapses (42). The interaction between MAGI-1 and β-catenin has also been demonstrated in epithelial cells (28) suggesting that this interaction can occur in multiple cell types. In our study, overexpression of β-catenin did not have any influence on MAGI protein levels, which is in contrast to its effects on DLG. Similarly, we did not observe any significant alteration in the levels of Scribble expression in the presence of overexpressed of β-catenin. These data are in agreement with previous studies (37) that showed mislocalization of Scribble in β-catenin overexpressing cells, but no change in the levels of Scribble expression. The selective influence of β-catenin on DLG stability rather than on MAGI-1 or Scribble, which are also both PDZ domain-containing junctional proteins, highlights the specificity of the effects of β-catenin on DLG.

Previous studies have shown that DLG is subject to regulation through the proteasome by different mechanisms. This is in part determined by the degree of cell-cell contact, with DLG being stabilized upon increased levels of cell contact (43). The ubiquitin ligase, β-TrCP has also been implicated in contributing to the control of DLG expression levels in a phosphorylation dependent manner (44). Other targeting pathways involve the HPV E6 oncoprotein and recruitment of cellular ubiquitin ligases to enhance DLG degradation (20; 45). At present the mechanisms by which β-catenin can enhance DLG turnover is unclear. Certainly there is no evidence to suggest that β-catenin is itself directly targeting DLG in a way equivalent to a ubiquitin ligase, suggesting that the effects are indirect, but which nonetheless correlate perfectly with its ability to bind DLG. It is important to note that this is not due to a simple PDZ domain-PDZ ligand interaction. Clearly DLG and β-catenin can form a complex in vivo and downregulation of DLG expression correlates with the capacity of the two proteins to bind, which is in turn dependent upon an intact PBM on β-catenin. However, the N terminal region of DLG also appears to play a critical role both in the capacity of β-catenin to bind to DLG, and also in determining the susceptibility of DLG to β-catenin induced proteasome-mediated degradation. This is very reminiscent of studies with HPV E6, in which although the viral oncoprotein binds to DLG through a classic PDZ domain-ligand interaction, sequences within the DLG N terminal region are required for optimal levels of E6-induced degradation (33). Since this region of DLG contains a plethora of known (46) and potential phospho-acceptor sites, some of which are already known to contribute to the regulation of DLG turnover in modes that are both HPV E6 dependent and independent (47; 48; 49), it is conceivable that these sites may also be important in β-catenin induced degradation of DLG. In many tumours β-catenin is constitutively activated as a consequence of a variety of activating mutations. One such mutant S33Y was also found to exhibit an increased capacity to enhance DLG degradation in comparison to the wild type protein. Furthermore, we also found that a consequence of the ability of β-catenin to downregulate DLG expression levels is an inhibition of DLG’s tumour suppressive activity in an oncogene co-operation assay, a function that is critically dependent upon β-catenin’s PDZ domain binding activity. Taken together, these studies demonstrate that one mechanism by which high levels of β-catenin expression can contribute to tumour progression is through downregulation of the DLG tumour supressor.

We show that β-catenin enhances DLG turnover in a proteasome-dependent manner and thereby inhibits DLG tumour suppressor activity. This involves DLG PDZ recognition by β-catenin and is regulated by sequences within the DLG N terminus. This data shows that overexpressed β-catenin can contribute to tumourigenesis through degradation of DLG.

ACKNOWLEDGMENTS

Authors are grateful to Prof. Claudio Brancolini for kindly providing the wt-βcatenin expression construct and to Dr. Miranda Thomas for critical comments on the manuscript. They also acknowledge Prof. Eric Fearon, Prof. Bert Vogelstein and Prof. Randall Moon for the various beta-catenin construct obtained through Addgene.

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