Role for ICAT in {beta}-catenin-dependent nuclear signaling and cadherin functions

Cara J. Gottardi and Barry M. Gumbiner

Department of Cell Biology, School of Medicine, University of Virginia, Charlottesville, Virginia 22908-0732

Submitted 7 October 2003 ; accepted in final form 11 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibitor of {beta}-catenin and TCF-4 (ICAT) is a 9-kDa polypeptide that inhibits {beta}-catenin nuclear signaling by binding {beta}-catenin and competing its interaction with the transcription factor TCF (T cell factor), but basic characterization of the endogenous protein and degree to which it alters other {beta}-catenin functions is less well understood. At the subcellular level, we show that ICAT localizes to both cytoplasmic and nuclear compartments. In intestinal tissue, ICAT is upregulated in the mature, nondividing enterocyte population lining intestinal villi and is absent in the {beta}-catenin/TCF signaling-active crypt region, suggesting that its protein levels may be inversely related with {beta}-catenin signaling activity. However, ICAT protein levels are not altered by activation or inhibition of Wnt signaling in cultured cells, suggesting that ICAT expression is not a direct target of the Wnt/{beta}-catenin pathway. In cells where {beta}-catenin levels are elevated by Wnt, a fraction of this {beta}-catenin pool is associated with ICAT, suggesting that ICAT may buffer the cell from increased levels of {beta}-catenin. Distinct from TCF and cadherin, ICAT does not protect the soluble pool of {beta}-catenin from degradation by the adenomatous polyposis coli containing "destruction complex." Although ICAT inhibits {beta}-catenin binding to the cadherin as well as TCF in vitro, stable overexpression of ICAT in Madin-Darby canine kidney (MDCK) epithelial cells shows no obvious alterations in the cadherin complex, suggesting that the ability of ICAT to inhibit {beta}-catenin binding to the cadherin may be restricted in vivo. MDCK cells overexpressing ICAT do, however, exhibit enhanced cell scattering on hepatocyte growth factor treatment, suggesting a possible role in the regulation of dynamic rather than steady-state cell-cell adhesions. These findings confirm ICAT's primary role in {beta}-catenin signaling inhibition and further suggest that ICAT may have consequences for cadherin-based adhesive function in certain circumstances, implying a broader role than previously described.

cadherin-mediated cell-cell adhesion; Wnt/{beta}-catenin signaling


{beta}-CATENIN IS A MULTIFUNCTIONAL PROTEIN that plays essential roles in both cell-cell adhesion and nuclear gene expression (reviewed in Ref. 10). At the cell surface, {beta}-catenin binds the cytoplasmic domain of cadherins and the actin-binding protein {alpha}-catenin to link the basic homophilic adhesive activity of the cadherin ectodomain with the underlying actin cytoskeleton (31). This cadherin-bound pool of {beta}-catenin ultimately serves to join the cytoskeletal networks of adjacent cells, which is considered essential for normal tissue architecture and morphogenesis (reviewed in Ref. 14). In the cytoplasm and nucleus, {beta}-catenin is an essential mediator of the Wnt signal transduction pathway. Canonical Wnt signaling plays critical roles throughout vertebrate development by activating target genes that will impose particular cell fates (reviewed in Refs. 27 and 40), and excessive {beta}-catenin signaling has been strongly implicated in various human cancers (reviewed in Ref. 22). Gene activation is ultimately controlled by a transcriptional complex containing the DNA binding factor TCF and {beta}-catenin, where the COOH-terminal region of {beta}-catenin serves as a transcriptional coactivator by recruiting components of the general transcriptional machinery, including TATA binding protein, proteins involved in chromatin modification such as the histone acetyltransferase p300/cAMP binding protein, and a component of the SWI/SNF chromatin remodeling machinery, BRG-1 (1, 15, 36).

{beta}-Catenin is known to form mutually exclusive adhesive (e.g., cadherin/{beta}-catenin) and signaling complexes [e.g., lymphoid enhancer factor (LEF)/TCF-type transcription factor/{beta}-catenin, or adenomatous polyposis coli (APC) tumor suppressor gene product/{beta}-catenin] through a competitive binding mechanism that involves the central armadillo repeat region of {beta}-catenin (13, 18, 19, 25, 39). This region forms a single structural unit that features a long, positively charged groove (16), and each {beta}-catenin ligand engages this groove through a number of overlapping and, in some regions, similar molecular interfaces (reviewed in Ref. 10). In the context of such a similar binding mechanism between {beta}-catenin/cadherin and {beta}-catenin/TCF complexes, it remains unclear how the cell modulates one complex without affecting the other. Indeed, how the signaling and adhesive functions of {beta}-catenin are coordinated so that these processes are largely independent (26) yet clearly interrelated (6) remains an important area of investigation.

Inhibitor of {beta}-catenin and TCF-4 (ICAT) is an 81-amino acid protein that was previously identified in a yeast two-hybrid screen by using the armadillo repeat region of {beta}-catenin as bait and was shown to inhibit {beta}-catenin/TCF signaling in both reporter gene and Xenopus axis formation assays (35). Importantly, a dominant negative form of ICAT (ICAT {Delta}42–61) that can interact with {beta}-catenin, but not compete with TCF binding, activates {beta}-catenin signaling when injected into the ventral side of Xenopus embryos, suggesting that ICAT normally serves to antagonize {beta}-catenin signaling in certain regions of the embryo (35). Highly conserved orthologs of ICAT have been identified in humans, mice, rats, zebrafish, and frogs with no homology to other known proteins. Interestingly, ICAT transcript levels have been found to be significantly upregulated in human cancers (20). This upregulation is possibly due to a negative feedback mechanism to inhibit {beta}-catenin/TCF signaling, because forced expression of ICAT in cells where {beta}-catenin levels are elevated (through APC- or axin-inactivating- or {beta}-catenin-activating mutations) strongly inhibits the proliferation of these cells by inducing G2 arrest and cell death (33).

Recent crystallographic data reveal that ICAT inhibits the formation of TCF/{beta}-catenin complexes by occupying the {beta}-catenin groove along armadillo repeats 5–12 in a way that would be incompatible with TCF binding [which binds overlapping arm repeats 3–10 (5, 12)]. Because the armadillo repeat region of {beta}-catenin is responsible for multiple {beta}-catenin-ligand interactions (e.g., cadherin and APC), it is likely that ICAT binding might affect other {beta}-catenin-dependent functions. In this regard, ICAT has been shown to inhibit both cadherin and TCF binding to {beta}-catenin in vitro, but surprisingly, when overexpressed in Xenopus embryos, selectively inhibits formation of TCF/{beta}-catenin complexes and fails to compete the cadherin/{beta}-catenin interaction (12). To better understand the role of this polypeptide in {beta}-catenin functions, we sought to characterize endogenous ICAT protein in cultured cell systems and an in vivo model for {beta}-catenin signaling to determine its regulation, subcellular distribution, and binding partners and whether there are cellular circumstances in which ICAT may, in fact, have consequences for the cadherin/{beta}-catenin adhesive complex.


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Plasmids

The cDNA for human ICAT was generated with RNA from HCT 116 cells by using the RT-PCR method (5' primer, 5'-GTGGATCCATGAACCGCGAGGGAGCAC-3'; 3'primer, 5'-GGGAATTCCAGCTACTGCCTCCGGTCTTCCGTCTC-3', according to GenBank accession no. AB021262 [GenBank] ). The 243-base pair product was subcloned into the pFLAG-CMV-2 expression vector (so that the FLAG tag is encoded amino-terminally and in frame with the ICAT coding sequence) and the bacterial expression vector pGEX-4T-3. Both plasmids were verified by sequence analysis. Expression plasmids encoding human E-cadherin/pcDNA3 (11), Xenopus TCF-3/pCS2+ (38), C-cadherin/pcDNA3, C-cadherin {Delta}{beta}-catenin/pcDNA3 (3), cadherin-glutathione S-transferase (GST)/pGEX-4T-3 (41), and TCF-GST/pGEX-4T-3 (11) are described elsewhere.

Antibodies

Rabbit polyclonal anti-ICAT antibodies were generated against the entire ICAT coding sequence with assistance from Covance Research Products (Denver, PA). Recombinant GST/ICAT was induced and purified according to standard protocols, and ~250 µg of ICAT immunogen were prepared by thrombin cleavage (1.0 U thrombin/50 µg ICAT/GST-bound Sepharose beads incubated overnight at 22°C) and used to boost rabbits at regular intervals. The serum from this polyclonal antibody (PAb) was used at 1:1,000 for Western blotting (WB), 1:300 for immunofluorescence (IF), and 1:300 for immunoprecipitation (IP) analyses. The following antibodies were also used in this study: anti-Xenopus {beta}-catenin PAb [1:5,000 WB; 1:500 IF and IP (23), anti-Xenopus plakoglobin (1:1,000 WB)], anti-Xenopus C-cadherin [1:5,000 WB (2)], anti-dog E-cadherin [1:500 IP; 1:5,000 WB (24)], anti-human E-cadherin (HECD-1 MAb, Zymed Laboratories; 1:10,000 WB), anti-APC NH2 terminus (Ab-1, MAb, Oncogene Research Products), anti-murine axin (rabbit polyclonal antiserum, 1:100 IP, 1:2,000 WB, kindly provided by Roel Nusse, Stanford University), and anti-FLAG epitope (M2 MAb, Sigma, 1:5,000 WB, 1:500 IF and IP).

Cell Culture, Transfection, Fractionation, Metabolic Labeling, Immunofluorescence, and Immunohistochemistry

Madin-Darby canine kidney (MDCK; DMEM), HCT 116 (McCoy's 5a), DLD-1 (RPMI 1640), and Chinese hamster ovary (CHO; Ham's F-12) cells were obtained from American Type Culture Collection and grown in their respective media supplemented with 10% FBS. Rat1 fibroblasts with or without Wnt1 were kindly provided by J. Kitajewski (Columbia University). Transfections were done using Lipofectamine 2000 reagent (GIBCO BRL). Stable transfections were selected in G418 (800 µg/ml) and isolated using cloning cylinders. Cell fractionation was carried out as described in Reinacher-Schick and Gumbiner (30). Metabolic protein labeling was performed overnight using 0.5–1.0 mCi/10-cm dish [35S]methionine/cysteine (TranS-Label, Amersham). The pulse-chase biotinylation procedure is as essentially described in Ref. 9. Both immunofluorescence and immunoperoxidase experiments were performed according to standard protocols and as described in Gottardi et al. (8).

Immunopreciptiation and In Vitro Binding Assays

Immunoprecipitation. Cells were solubilized in a nonionic detergent buffer [1% Nonidet P-40 (NP-40), 50 mM Tris, pH 7.5, 150 mM NaCl, and 2 mM EDTA including protease inhibitors] and centrifuged at 14,000 g to remove insoluble material, and the resulting supernatant was incubated with various antibodies for 90 min at 4°C followed by protein A- and protein G-coupled Sepharose (Pierce) precipitation. We examined the effect of ICAT on the interaction between {beta}-catenin and cadherin in vitro essentially according to Tago et al. (35): {beta}-catenin (~3 ng, produced by the baculovirus system), cadherin cytoplasmic domain-GST (6 ng, bacterial expression), or TCF-GST (6 ng) were incubated in 150 ml of buffer A for 60 min at 4°C with shaking (buffer A: 10 mM Tris, pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.1% NP-40, and 10 µg/ml leupeptin and aprotinin) in the presence or absence of increasing amounts of thrombin-cleaved ICAT (0.001, 0.01, 0.1, 1.0, and 5.0 µg). {beta}-Catenin that was affinity precipitated by cadherin-GST and TCF-GST immobilized to glutathione-coupled Sepharose (Sigma) was washed with buffer A (four 1-ml washes) and subjected to SDS-PAGE and immunoblotting analysis using standard methods. Because of its small size, the ICAT polypeptide was best observed (in both WB and [35S]methionine/cysteine labeling techniques) with NuPAGE 10% Bis-Tris gel (Invitrogen) or tricine gel systems.

Hepatocyte Growth Factor and Scatter Factor Experiments

Essentially according to Fujita et al. (7), mock- and ICAT-transfected MDCK cells were plated at subconfluency and incubated with 6 ng/ml of recombinant human hepatocyte growth factor (HGF; no. 294-HG, R&D Systems) for 16 h. Control, mock-transfected MDCK cells would scatter at a concentration of 18 ng/ml after 12 h.


    RESULTS
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 METHODS
 RESULTS
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 REFERENCES
 
Characterization and Subcellular Localization of ICAT

Toward understanding the role of ICAT in cells, we generated a PAb against the entire 81-amino acid coding sequence. This antibody immunoprecipitated two bands from [35S]methionine/cysteine-labeled MDCK epithelial cells: 9- and 90-kDa bands, which corresponded to ICAT and {beta}-catenin, respectively (Fig. 1A). We determined that the 9-kDa band was ICAT by the following four criteria: 1) this band migrated at the predicted molecular weight for an 81-amino acid protein and could be competed with an excess of cold ICAT fusion protein (Fig. 1A, compare lanes 2–4 with lane 6); 2) this [35S]methionine/cysteine-incorporated band comigrated perfectly with a band that was detected by immunoblot analysis (Fig. 1C, lanes 1 and 2); 3) the coassociated 90-kDa band comigrated perfectly with {beta}-catenin; and 4) antibodies to {beta}-catenin specifically coimmunoprecipitated a 9-kDa band (Fig. 1B) that comigrated perfectly with ICAT by immunoblotting (not shown). It is unlikely that the 90-kDa band is a protein that cross-reacts with ICAT, because this band disappeared when the immunoprecipation was carried out under stringent SDS-denaturing conditions, arguing that it is coassociated with ICAT rather than independently recognized by the antibody (Fig. 1A, lanes 7–10). We also found that ICAT can interact with plakoglobin but not with more distantly related members of the armadillo-repeat family, such as p120ctn or the armadillo repeat region of APC (Fig. 1, D and E). Thus {beta}-catenin appears to be the major, stoichiometric binding partner of ICAT in cells, as determined within the limits of steady-state metabolic labeling studies. Biochemical fractionation of cultured cells revealed that ICAT is a cytosolic protein, largely found in a 100,000-g postnuclear supernatant fraction with little or no protein detected in nuclear or pelletable (e.g., membrane) fractions (Fig. 1C). At the IF level, ICAT is diffusely distributed, localizing to both cytoplasmic and nuclear compartments (Figs. 1F and 5B for epitope-tagged ICAT). The nuclear localization appears somewhat enhanced under subconfluent plating conditions and is consistent with ICAT's established role in inhibiting {beta}-catenin/TCF-dependent transcription (35, 36).



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Fig. 1. Characterization of endogenous inhibitor of {beta}-catenin and TCF-4 (ICAT) protein in epithelial cells. A: polyclonal antibody (PAb) characterization. Madin-Darby canine kidney (MDCK) cells were metabolically labeled overnight and solubilized, and lysates were subjected to immunoprecipitation with rabbit preimmune serum (lane 1), ICAT-immune sera (3 different bleeds; lanes 2–4), anti-cadherin antibody (MAb RR1; lane 5), and ICAT-immune sera competed with an excess of "cold" ICAT-glutathione S-transferase (GST) fusion protein (lane 6). Association of the 90-kDa band with ICAT is SDS sensitive (lanes 7–10: lane 7, non-ionic detergent wash; lane 8, 1% SDS-wash; lane 9, 2% SDS wash; lane 10, preimmune control). Note that because {beta}-catenin contains 10 times more cysteine/methionine residues than ICAT, the observation that the {beta}-catenin and ICAT band intensities are equivalent or darker for ICAT (lanes 2–4 and 7) argues that ICAT is present in excess of cytosolic {beta}-catenin in these cells. B: metabolically labeled MDCK cell lysates were subjected to immunoprecipitation with a polyclonal antibody to {beta}-catenin ({beta}-cat; lane 3, isotonic salt wash; lane 4, 500 mM salt wash), the corresponding preimmune (pre-imm) control (lanes 1 and 2), and affinity precipitation using cadherin cytoplasmic domain-GST fusion protein (lane 5). C: subcellular fractionation of ICAT. MDCK cell lysates were fractionated using the hypotonic lysis method followed by centrifugation at 100,000 g. Membrane/pelletable (lane 3), cytosolic/soluble (lane 4), and crude nuclear fractions (lane 5) were subjected to SDS-PAGE/Western analysis and immunoblotted with antibodies to ICAT. Control immunoprecipitation of ICAT from a total detergent lysate is shown in lanes 1 and 2. D and E: ICAT interacted with plakoglobin (plako) but not with p120ctn or the armadillo repeat region of adenomatous polyposis coli (APC). MDCK cell detergent lysates were subjected to immunoprecipitation with preimmune and ICAT antisera, separated by SDS-PAGE, transferred, and immunoblotted with antibodies to {beta}-catenin, plakoglobin, and p120ctn. E: recombinant myc-APC encoding only its armadillo repeat region (myc-APC arm) was incubated with GST and ICAT-GST bacterial fusion proteins and affinity precipitated with glutathione-coupled Sepharose. Samples were subjected to SDS-PAGE and Western analysis using an antibody against the myc epitope. ICAT-GST is a functional fusion protein by criterion of being capable of interacting with {beta}-catenin and plakoglobin from cell lysates (not shown). F: subcellular distribution of ICAT. Confocal immunofluorescence images from paraformaldehyde-fixed, human colon carcinoma-derived DLD-1 cells incubated with the following antibodies: anti-ICAT (Cy-3, left) and anti-{beta}-catenin (FITC) overlay (right), preimmune ICAT antiserum (top insert), and anti-ICAT preincubated with ICAT-GST fusion protein (bottom insert). Note that ICAT was diffusely localized to both cytoplasmic and nuclear compartments, and the nuclear localization appears enhanced under subconfluent plating conditions (for FLAG-tagged ICAT localization, see Figs. 4B and 5B).

 


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Fig. 5. ICAT does not stablize {beta}-catenin when expressed in CHO cells. A and B: cadherin and TCF, but not ICAT, upregulated {beta}-catenin in cadherin-negative CHO cells. A: cells were transiently transfected with either FLAG-ICAT or E-cadherin plasmid. After 36 h, cell lysates were fractionated by SDS-PAGE and total {beta}-catenin levels were determined by WB analysis. B: likewise, cells were transiently transfected with either FLAG-ICAT, E-cadherin, or TCF-3-myc plasmids and then subjected to immunofluorescence analysis. Note that E-cadherin and TCF upregulated {beta}-catenin robustly compared with ICAT. C: ICAT could be found in a complex with APC and Axin. Lanes 1–4: ICAT-GST, but not TCF-GST, affinity-precipitated endogenous APC from HCT 116 cells. HCT 116 cell lysates were incubated with the GST-fusion proteins shown, and affinity precipitates were separated on a 3% agarose gel and blotted with an antibody to APC, as described in Reinacher-Schick and Gumbiner (30). Lanes 5–8: APC could be coimmunoprecipitated with ICAT. Endogenous (lane 8) and FLAG-tagged ICAT [after stable transfection (lane 6)] were immunoprecipitated from MDCK cell lysates and blotted with an antibody to APC. Lanes 9–11: axin could be coimmunoprecipiated with ICAT. FLAG-ICAT-expressing MDCK cells were immunoprecipitated with an anti-FLAG antibody, separated by SDS-PAGE, and blotted with an antibody to axin. Nonimmune and anti-axin controls are also shown.

 



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Fig. 4. ICAT overexpression in MDCK cells potentiates hepatocyte growth factor (HGF)-induced cell scattering. A and B: stable overexpression of FLAG-tagged ICAT in MDCK cells. A: cell lysates from mock-transfected (clones 1–3) or ICAT-transfected (clones 3, 4, and 15) cells were analyzed by SDS-PAGE and Western blotted with an anti-FLAG antibody. Subcellular localization of FLAG-ICAT is shown in stably expressing MDCK cells (clone 4). B: cells were fixed and stained with anti {beta}-catenin (FITC-green) and anti-FLAG (CY-3-red) antibodies. C and D: effect of ICAT on HGF-induced cell scattering and cadherin levels. C: mock- or ICAT-transfected MDCK cells were incubated without or with 6 ng/ml HGF for 18 h. Representative phase-contrast images are shown. D: cell lysates from HGF (6 ng/ml)-treated and untreated mock and transfected clones were analyzed by SDS-PAGE and Western blotted with antibodies to E-cadherin and {beta}-catenin. Note that ICAT-expressing cells show reduced cadherin levels on HGF treatment. E: cell surface turnover of cadherin in ICAT-overexpressing MDCK cells during HGF treatment. Cells were pulse labeled overnight with [35S]methionine/cysteine (+HGF) and chased in cold media (+HGF) for 0, 5, and 10 h. After each time point, cell surface proteins were biotinylated with NHS-SS-Biotin (Pierce) according to a modification of the standard method (9). Lysates were immunoprecipitated with an anti-canine cadherin PAb and then eluted and incubated with streptavidin-agarose beads. Recovered proteins were analyzed by SDS-PAGE, fluorography, and densitometric scanning. Note that 2.5 times less cadherin arrived at the cell surface at time 0 in cells overexpressing ICAT, whereas there is no significant difference in the turnover rates of surface-labeled cadherin (39 vs. 37% cadherin remaining after 10-min chase for control and ICAT-expressing lines, respectively) or total {beta}-catenin as a consequence of ICAT overexpression (21 vs. 30% {beta}-catenin remaining for control and ICAT-expressing cells, respectively). Rabbit IgG was used in C as a control.

 

Characterization of ICAT During Wnt Signaling

To gain insight into the developmental regulation of ICAT in vivo, we chose to examine ICAT localization along the crypt villus axis of the intestinal mucosal epithelium, where cells undergo a coordinated series of events involving proliferation, differentiation, and migration from the crypt toward the intestinal lumen (29). Stem cells are located in the base of the crypt and give rise to a population of undifferentiated, proliferative, progenitor cells that will ultimately differentiate into one of the functional cell types along the villus. {beta}-Catenin/TCF signaling is thought to maintain this crypt progenitor phenotype because disruption of this signaling activity induces G1 arrest and promotes differentiation (37). In this regard, we were interested to find that endogenous ICAT protein was not detected in the epithelial cells lining intestinal crypts, where persistent {beta}-catenin/TCF nuclear signaling is known to impose a crypt progenitor phenotype, but rather was detected in the population of cells that line the intestinal villus (Fig. 2A, i–iv, arrows), where {beta}-catenin signaling is thought to be diminished.



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Fig. 2. ICAT protein levels in situ and as a consequence of {beta}-catenin signaling. A: immunohistochemistry of sections from paraffin-embedded dog intestine: preimmune ICAT antiserum staining of villus (i) and crypt regions (ii); anti-ICAT staining of villus (iii) and crypt regions (iv). Arrow in iii points to peroxidase staining that is apparent in the well-differentiated enterocytes along villus but not in the actively dividing crypt regions (iv). B: ICAT protein levels were not altered by transient overexpression of dominant negative or constitutively active forms of TCF (TCF-DN and TCF-VP16, respectively). DLD-1 cells were transiently transfected with empty vector (–) or increasing amounts of TCF plasmids (0.3, 1.0, 3.0 µg). After 24 h, cells were metabolically labeled overnight, solubilized, and subjected to immunoprecipitation with anti-ICAT, preimmune, and anti-myc antibodies. Transfection efficiency was ~33%. C and D: ICAT protein levels did not covary with {beta}-catenin protein levels. C: relative ICAT protein levels were determined after metabolic labeling and immunoprecipitation from an equivalent amount of total protein by using preimmune (p.i.) and anti-ICAT PAbs. Note that ICAT levels are not altered by cadherin expression (L-cells vs. CV-1 cells), Wnt expression (Rat1 cells with or without Wnt), or loss of APC (DLD-1/SW480 cells vs. MDCK). D: cytosolic (membrane free) fractions were prepared from the various cell lines, equalized for total protein, and divided: half of the sample was subjected to ICAT immunoprecipitation (top blot) and the other half was precipitated with TCA to determine total cytosolic {beta}-catenin amounts (middle blot; TCA precipitation ~30% efficiency). The TCA-precipiated half was reprobed with {beta}-tubulin to show that protein levels were similar between cell lines (bottom blot). E: quantification of total and ICAT-associated {beta}-catenin. Note that with the exception of SW480 cells, a significant fraction of cytosolic {beta}-catenin was associated with ICAT.

 

The observation that ICAT protein levels appear to be tightly regulated in intestinal epithelia suggested that ICAT expression might be coordinated with Wnt signaling. To look into this possibility, we examined the levels of endogenous ICAT protein as a consequence of transient overexpression of a dominant inhibitor and of constitutively activated forms of TCF [TCF-DN and TCF-VP16, respectively (11)] and across various cell lines that exhibit different stable modes of {beta}-catenin signaling activation, such as stable expression of Wnt in Rat1 cells or colon cancer cell lines that are mutant for the APC tumor suppressor gene product. We found that ICAT protein levels did not simply correlate with inhibition or activation of {beta}-catenin signaling, suggesting that ICAT is not likely to be a direct transcriptional target of the Wnt pathway (Fig. 2, B and C). When we compared the amount of {beta}-catenin that coimmunoprecipitated with ICAT vs. total cytosolic levels of {beta}-catenin [estimated by precipitating total cytosolic proteins with TCA at an efficiency of precipitation of ~30%], we found that a significant proportion of the cytosolic {beta}-catenin pool is bound by ICAT (Fig. 2, D and E). One notable exception is the SW480 colon carcinoma cell line, in which the ICAT-bound pool of {beta}-catenin is closer to 10%. Because the efficiency of precipitating total cytosolic protein with the TCA method was determined to be ~30% and the amount of total vs. ICAT-bound {beta}-catenin pools are roughly equivalent (Fig. 2E), it seems that much (~30%) of the soluble pool of {beta}-catenin in these cell lines is buffered or kept transcriptionally inactive by ICAT. The following observations further suggest that ICAT is in excess of {beta}-catenin in normal cells and likely serves to buffer a significant proportion of the {beta}-catenin that accumulates during Wnt signaling: 1) ICAT levels were the same in the absence or presence of Wnt (Fig. 2C); 2) ICAT appeared to be in excess of cytosolic {beta}-catenin in cells that were not receiving a Wnt signal (judged from [35S]methionine/cysteine band intensities in Fig. 1A); and 3) the amount of {beta}-catenin that associated with ICAT was approximately fourfold greater under Wnt-stimulated conditions (Fig. 2D, lanes 3 and 4). We cannot exclude the possibility, however, that Wnt signaling might even stimulate the formation of ICAT/{beta}-catenin complexes as part of a negative feedback mechanism.

Role of ICAT in Cadherin Regulation

The fact that ICAT binds the armadillo repeat region of {beta}-catenin, a region that mediates multiple {beta}-catenin-ligand interactions, raises the interesting possibility that ICAT might have functions beyond its role in inhibiting {beta}-catenin-dependent TCF signaling. In this regard, we wanted to determine whether ICAT could compete {beta}-catenin binding to cadherins. Indeed, ICAT could inhibit {beta}-catenin binding to the cadherin cytoplasmic domain in vitro (Fig. 3A), and similarly, endogenous ICAT/{beta}-catenin complexes were not found to be associated with cadherins in vivo (Fig. 1, A and B, lanes 5). Moreover, transient overexpression of ICAT resulted in a reduction in the amount of {beta}-catenin that could be coimmunoprecipitated with E-cadherin (Fig. 3B). This reduction, however, appeared to be largely due to a decrease in the amount of total E-cadherin that accumulates in these cells as a result of ICAT expression. Because {beta}-catenin binding to the cadherin cytoplasmic tail has been shown to play a critical role in cadherin stability (4, 17), we suggest that ICAT overexpression competes {beta}-catenin from the cadherin, resulting in its destabilization and turnover. Consistent with this model, ICAT overexpression did not alter the levels of a truncated cadherin protein lacking the {beta}-catenin binding region (Fig. 3C).



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Fig. 3. ICAT competes {beta}-catenin binding to cadherin in vitro and when overexpressed in cells. A: in vitro competition. Recombinant {beta}-catenin was incubated with cadherin-GST (or TCF-GST)-coupled Sepharose beads with increasing amounts of recombinant ICAT (prepared by cleaving GST-ICAT with thrombin). Top: Western blot incubated with antibodies to ICAT; bottom; 3 blots with antibodies to {beta}-catenin. B: in vivo competition. Cadherin-negative Chinese hamster ovary (CHO) cells were transiently transfected with plasmid encoding E-cadherin (0.5 µg) without or with increasing amounts of ICAT plasmid (0.1, 0.25, 0.5, 1.0, 3.0 µg). Total amounts of translatable plasmid were normalized with {beta}-galactosidase. After 36 h, cells were solubilized, immunoprecipitated with antibodies to E-cadherin or FLAG-tagged ICAT, and Western immunoblotted (WB) with the designated antibodies. C: CHO cells stably transfected with wild-type C-cadherin or with C-cadherin lacking the {beta}-catenin binding region (–{Delta}{beta}-cat) were transfected with increasing amounts of FLAG-tagged ICAT (0, 0.1, 0.3, 1.0, 3.0 µg) normalized with {beta}-galactosidase plasmid. Total cell lysates were fractionated by SDS-PAGE, and the resultant immunoblots are shown. Note that for ICAT overexpression to reduce cadherin levels, the {beta}-catenin binding region of cadherin was required.

 

To further explore a role for ICAT in cell-cell adhesion-dependent processes, we generated three independent, stably transfected MDCK cell clones that overexpress epitope-tagged ICAT (Fig. 4). In all of the transfected clones, exogenous ICAT was similarly expressed at ~10-fold higher levels than endogenous ICAT (Fig. 4A and not shown). Stable ICAT expression did not alter endogenous E-cadherin levels (not shown) or cell morphology (Fig. 4B). However, ICAT overexpression did enhance the effect of HGF on MDCK cell scattering (Fig. 4C). This effect correlated with a reduction in cadherin levels (Fig. 4D) and an increase in ICAT-bound {beta}-catenin (not shown), suggesting that under dynamic cell-cell rearrangement conditions associated with HGF treatment, ICAT may compete the {beta}-catenin/cadherin binding interaction and reduce the amount of cadherin that accumulates under these conditions. Pulse-chase turnover analysis of the surface-labeled cadherin pool suggests that ICAT overexpression preferentially affects the amount of cadherin that actually reaches the cell surface, implicating ICAT's effects at the level of cadherin-catenin complex biosynthesis (Fig. 4E).

ICAT Does Not Interfere With {beta}-catenin Turnover

Because ICAT can alter {beta}-catenin-dependent TCF signaling and cadherin functions, we wanted to explore the degree to which ICAT might alter the normal APC-mediated turnover of {beta}-catenin in cells. Cytosolic {beta}-catenin is normally degraded rapidly through the combined activities of the APC-axin-GSK-3{beta} phosphorylation complex and the ubiquitin-ligase machinery. In cadherin-negative CHO cells, transfection of the cadherin cytoplasmic domain is sufficient to upregulate and stabilize endogenous {beta}-catenin (34), presumably because the cadherin competes {beta}-catenin binding to both APC and axin. Using this upregulation of {beta}-catenin in CHO cells as an assay, we examined whether ICAT could stabilize endogenous {beta}-catenin, as has been shown previously for cadherin. In Fig. 5, A and B, we show that in contrast to cadherin and TCF, ICAT was unable to stabilize {beta}-catenin in CHO cells. Because ICAT has been shown to interact with armadillo repeats 5–12 of {beta}-catenin (5, 12), and APC and axin interact with more amino-terminally localized armadillo repeats (39), we postulate that ICAT fails to protect {beta}-catenin from turnover because ICAT cannot compete {beta}-catenin from interacting with APC and axin. Consistent with this interpretation, antibodies to ICAT could coimmunoprecipitate APC and axin (Fig. 5C), suggesting that {beta}-catenin can bind APC/Axin and ICAT simultaneously.


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 DISCUSSION
 REFERENCES
 
ICAT is a 9-kDa polypeptide that was shown previously to function as an inhibitor of {beta}-catenin nuclear signaling by competing the {beta}-catenin/TCF binding interface (5, 12, 35), but the manner and degree to which it inhibits {beta}-catenin signaling compared with adhesive functions is not well understood. Toward this end, we sought to characterize endogenous and overexpressed ICAT polypeptides in intestine and cultured cell models. Our data suggest that most cells contain a free pool of ICAT that is available to buffer cytosolic {beta}-catenin during Wnt signaling activation, because ICAT protein levels remain relatively constant in the presence or absence of Wnt, whereas the amount of {beta}-catenin that becomes associated with ICAT greatly increases (Fig. 2). Interestingly, although ICAT protein levels do not appear to be regulated in the cell lines tested, ICAT protein is clearly differentially expressed along the intestinal crypt-villus axis in vivo (Fig. 2). In particular, ICAT is upregulated along the well-differentiated cells that line the intestinal villus and is conspicuously absent in crypts, where {beta}-catenin/TCF signaling is known to maintain a crypt cell progenitor fate (37). Because {beta}-catenin appears to be the major biochemical target of ICAT in cells (Fig. 1), upregulation of ICAT along the villus likely serves to restrict {beta}-catenin/TCF signaling to the progenitor-crypt region. Although we found no evidence that ICAT protein levels are directly regulated by {beta}-catenin/TCF signaling (Fig. 2, B and C), what controls ICAT expression along the crypt-villus axis is presently unclear.

Because {beta}-catenin interacts with many of its key binding partners (e.g., cadherin, APC) through overlapping binding interfaces along the armadillo-repeat groove of {beta}-catenin, it was important to determine whether ICAT could alter either of these functional interactions. Cadherins have been shown to bind {beta}-catenin and protect it from constitutive turnover in CHO cells (34), in part because {beta}-catenin forms mutually exclusive complexes with cadherins and the APC-containing degradation machinery (25). In striking contrast to both cadherin and TCF, ICAT fails to protect {beta}-catenin from being turned over in CHO cells because it does not compete {beta}-catenin binding to APC and axin (Fig. 5). This inability of ICAT to stabilize cytosolic {beta}-catenin might ensure that ICAT/{beta}-catenin complexes would not persist long after the cessation of Wnt signaling. Thus ICAT appears to inhibit {beta}-catenin signaling through both active and permissive mechanisms, i.e., ICAT inhibits {beta}-catenin/TCF complex formation and permits {beta}-catenin degradation via the APC-axin destruction complex.

We have confirmed that ICAT inhibits {beta}-catenin binding to TCF and cadherin proteins similarly in vitro (Ref. 12; Fig. 3) and can be associated with a reduction in cadherin levels when transiently overexpressed in cells (Fig. 3). Interestingly, despite ICAT's ability to compete the {beta}-catenin/cadherin interaction in vitro, stable overexpression of ICAT in MDCK epithelial cells shows no obvious alterations in the cadherin complex or cell-cell adhesion. Thus the ability of ICAT to inhibit {beta}-catenin binding to cadherin can be somehow restricted in vivo. This selectivity is not simply due to compartmentalization of the polypeptide, because ICAT localizes to cytoplasmic as well as nuclear compartments. An alternative explanation is that adhesion and signaling are different processes with likely different sensitivities to ICAT. Nevertheless, MDCK cells overexpressing ICAT do exhibit enhanced cell scattering after HGF induction, accompanied with a reduction in E-cadherin protein levels, suggesting a possible role for ICAT in the regulation of dynamic as opposed to steady-state cell-cell adhesions (Fig. 4). How do we reconcile these differences in ICAT's ability to compete {beta}-catenin binding to cadherin in the presence or absence of scatter factor? It is known that the affinity of {beta}-catenin binding to cadherin can be altered both by cadherin serine phosphorylation (18, 21) and {beta}-catenin tyrosine phosphorylation at residue 654 (32). Thus ICAT's ability to modulate {beta}-catenin binding to the cadherin might depend on {beta}-catenin/cadherin binding affinities, which may in turn be dependent on cell-specific differences in the phosphorylation state of cadherins and {beta}-catenin. Because enhanced cell scattering was observed under overexpression conditions, the degree to which normal levels of ICAT protein affect {beta}-catenin/cadherin complex formation or catabolism remains to be elucidated. In this context, ICAT RNA (20) and protein levels (Gottardi CJ and Reinacher-Schick A, unpublished observations) are found to be significantly upregulated in human tumors, demonstrating that ICAT upregulation does happen in certain physiological cases. Whether ICAT overexpression could promote tumorigenesis, perhaps by inhibiting cadherin function, needs to be further examined.

Inappropriate activation of {beta}-catenin is associated with a variety of cancers (28), and inhibition of {beta}-catenin-mediated transcription is being considered as a potential therapeutic strategy. Previous studies have suggested that the ICAT binding region of {beta}-catenin may be an attractive target for inhibitors specific for transcription but not the adhesive function of {beta}-catenin. Our findings that ICAT can inhibit the {beta}-catenin/cadherin interface, and in certain circumstances (e.g., during HGF-induced cell scattering) adhesive function of cadherins, suggest that therapeutic inhibitors, which target the COOH-terminal region of {beta}-catenin, may also need to be evaluated in terms of their ability to affect intercellular adhesion.


    ACKNOWLEDGMENTS
 
We thank Jean-Jacques Fontaine (Ecole Nationale Veterinaire d'Alfort) for paraffin-embedded dog intestine sections and Ellen Wong for technical assistance.

GRANTS

This work was supported by National Institute of General Medical Sciences Grant R37 GM-374432 (to B. M. Gumbiner).


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. J. Gottardi, Dept. of Cell Biology, School of Medicine, Univ. of Virginia, PO Box 800732, Charlottesville, VA 22908-0732 (E-mail: gottardc{at}mskcc.org).

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.


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