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
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ABSTRACT |
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cadherin-mediated cell-cell adhesion; Wnt/-catenin signaling
-Catenin is known to form mutually exclusive adhesive (e.g., cadherin/
-catenin) and signaling complexes [e.g., lymphoid enhancer factor (LEF)/TCF-type transcription factor/
-catenin, or adenomatous polyposis coli (APC) tumor suppressor gene product/
-catenin] through a competitive binding mechanism that involves the central armadillo repeat region of
-catenin (13, 18, 19, 25, 39). This region forms a single structural unit that features a long, positively charged groove (16), and each
-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
-catenin/cadherin and
-catenin/TCF complexes, it remains unclear how the cell modulates one complex without affecting the other. Indeed, how the signaling and adhesive functions of
-catenin are coordinated so that these processes are largely independent (26) yet clearly interrelated (6) remains an important area of investigation.
Inhibitor of -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
-catenin as bait and was shown to inhibit
-catenin/TCF signaling in both reporter gene and Xenopus axis formation assays (35). Importantly, a dominant negative form of ICAT (ICAT
4261) that can interact with
-catenin, but not compete with TCF binding, activates
-catenin signaling when injected into the ventral side of Xenopus embryos, suggesting that ICAT normally serves to antagonize
-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
-catenin/TCF signaling, because forced expression of ICAT in cells where
-catenin levels are elevated (through APC- or axin-inactivating- or
-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/-catenin complexes by occupying the
-catenin groove along armadillo repeats 512 in a way that would be incompatible with TCF binding [which binds overlapping arm repeats 310 (5, 12)]. Because the armadillo repeat region of
-catenin is responsible for multiple
-catenin-ligand interactions (e.g., cadherin and APC), it is likely that ICAT binding might affect other
-catenin-dependent functions. In this regard, ICAT has been shown to inhibit both cadherin and TCF binding to
-catenin in vitro, but surprisingly, when overexpressed in Xenopus embryos, selectively inhibits formation of TCF/
-catenin complexes and fails to compete the cadherin/
-catenin interaction (12). To better understand the role of this polypeptide in
-catenin functions, we sought to characterize endogenous ICAT protein in cultured cell systems and an in vivo model for
-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/
-catenin adhesive complex.
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METHODS |
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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 -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
-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 0.51.0 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 -catenin and cadherin in vitro essentially according to Tago et al. (35):
-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).
-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.
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RESULTS |
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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 -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 24 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
-catenin; and 4) antibodies to
-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 710). 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
-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
-catenin/TCF-dependent transcription (35, 36).
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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. -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
-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, iiv, arrows), where
-catenin signaling is thought to be diminished.
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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 -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
-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
-catenin that coimmunoprecipitated with ICAT vs. total cytosolic levels of
-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
-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
-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
-catenin pools are roughly equivalent (Fig. 2E), it seems that much (
30%) of the soluble pool of
-catenin in these cell lines is buffered or kept transcriptionally inactive by ICAT. The following observations further suggest that ICAT is in excess of
-catenin in normal cells and likely serves to buffer a significant proportion of the
-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
-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
-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/
-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 -catenin, a region that mediates multiple
-catenin-ligand interactions, raises the interesting possibility that ICAT might have functions beyond its role in inhibiting
-catenin-dependent TCF signaling. In this regard, we wanted to determine whether ICAT could compete
-catenin binding to cadherins. Indeed, ICAT could inhibit
-catenin binding to the cadherin cytoplasmic domain in vitro (Fig. 3A), and similarly, endogenous ICAT/
-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
-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
-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
-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
-catenin binding region (Fig. 3C).
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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
-catenin (not shown), suggesting that under dynamic cell-cell rearrangement conditions associated with HGF treatment, ICAT may compete the
-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 -catenin Turnover
Because ICAT can alter -catenin-dependent TCF signaling and cadherin functions, we wanted to explore the degree to which ICAT might alter the normal APC-mediated turnover of
-catenin in cells. Cytosolic
-catenin is normally degraded rapidly through the combined activities of the APC-axin-GSK-3
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
-catenin (34), presumably because the cadherin competes
-catenin binding to both APC and axin. Using this upregulation of
-catenin in CHO cells as an assay, we examined whether ICAT could stabilize endogenous
-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
-catenin in CHO cells. Because ICAT has been shown to interact with armadillo repeats 512 of
-catenin (5, 12), and APC and axin interact with more amino-terminally localized armadillo repeats (39), we postulate that ICAT fails to protect
-catenin from turnover because ICAT cannot compete
-catenin from interacting with APC and axin. Consistent with this interpretation, antibodies to ICAT could coimmunoprecipitate APC and axin (Fig. 5C), suggesting that
-catenin can bind APC/Axin and ICAT simultaneously.
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DISCUSSION |
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Because -catenin interacts with many of its key binding partners (e.g., cadherin, APC) through overlapping binding interfaces along the armadillo-repeat groove of
-catenin, it was important to determine whether ICAT could alter either of these functional interactions. Cadherins have been shown to bind
-catenin and protect it from constitutive turnover in CHO cells (34), in part because
-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
-catenin from being turned over in CHO cells because it does not compete
-catenin binding to APC and axin (Fig. 5). This inability of ICAT to stabilize cytosolic
-catenin might ensure that ICAT/
-catenin complexes would not persist long after the cessation of Wnt signaling. Thus ICAT appears to inhibit
-catenin signaling through both active and permissive mechanisms, i.e., ICAT inhibits
-catenin/TCF complex formation and permits
-catenin degradation via the APC-axin destruction complex.
We have confirmed that ICAT inhibits -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
-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
-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
-catenin binding to cadherin in the presence or absence of scatter factor? It is known that the affinity of
-catenin binding to cadherin can be altered both by cadherin serine phosphorylation (18, 21) and
-catenin tyrosine phosphorylation at residue 654 (32). Thus ICAT's ability to modulate
-catenin binding to the cadherin might depend on
-catenin/cadherin binding affinities, which may in turn be dependent on cell-specific differences in the phosphorylation state of cadherins and
-catenin. Because enhanced cell scattering was observed under overexpression conditions, the degree to which normal levels of ICAT protein affect
-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 -catenin is associated with a variety of cancers (28), and inhibition of
-catenin-mediated transcription is being considered as a potential therapeutic strategy. Previous studies have suggested that the ICAT binding region of
-catenin may be an attractive target for inhibitors specific for transcription but not the adhesive function of
-catenin. Our findings that ICAT can inhibit the
-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
-catenin, may also need to be evaluated in terms of their ability to affect intercellular adhesion.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Institute of General Medical Sciences Grant R37 GM-374432 (to B. M. Gumbiner).
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FOOTNOTES |
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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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