ICAT is a multipotent inhibitor of {beta}-catenin. Focus on "Role for ICAT in {beta}-catenin-dependent nuclear signaling and cadherin functions"

Jennifer L. Stow

Institute for Molecular Bioscience and School of Molecular and Microbial Science, The University of Queensland, Brisbane 4072, Queensland, Australia

THE PERIPATETIC PROTEIN {beta}-catenin is at the heart of a complex series of protein interactions involved in controlling cell growth and differentiation and now highlighted by Gottardi and Gumbiner in the current article in focus (Ref. 9, see p. C747 in this issue). {beta}-Catenin participates in three mutually exclusive protein complexes, at the cell membrane, in the cytoplasm, and in the nucleus, where at each point it subserves dynamic and distinct functional roles (8). At the adherens junctions, {beta}-catenin is well-known as a participant in mediating cadherin-based, cell-cell adhesion. {beta}-Catenin binds with high affinity to a COOH-terminal domain on classic cadherins, which stabilizes the newly synthesized cadherin and, at the junction, forms a link through {alpha}-catenin to the actin cytoskeleton. {beta}-Catenin is also an integral part of the Wnt signaling pathway involved in both development and tumorigenesis (3, 20). In the absence of Wnt signaling, a pool of non-adhesion-associated, soluble {beta}-catenin binds to the adenomatous polyposis coli (APC)-axin tumor suppressor complex, where it is phosphorylated by glycogen synthase kinase 3{beta} and targeted for proteasomal degradation. Finally, in response to Wnt signaling, {beta}-catenin can move to the nucleus, where it forms a complex with the TCF/LEF transcription factors to drive transcription of Wnt responsive genes.

Curiously, {beta}-catenin uses the same domain to bind to E-cadherin, to APC, and to TCF. This binding site in {beta}-catenin is now well characterized through mutational studies and by X-ray crystallography as a positively charged groove formed by the helices of its central armadillo repeat region (11, 12, 21). In this groove there is an extensive interaction surface and multiple, similar points of contact between {beta}-catenin and bound cadherin, TCF, or APC. So, given that {beta}-catenin can bind to multiple competitive partners, what does regulate the differential formation of these protein complexes and therefore the different functions of {beta}-catenin? The answer, in part, is a small, soluble inhibitory protein, "inhibitor of {beta}-catenin and TCF" (ICAT).

ICAT was first identified as a {beta}-catenin-binding protein from a yeast two-hybrid screen; it is an 81-amino acid, soluble protein that is widely expressed in tissues and is upregulated in some tumors (19, 20). Tago et al. (19) first demonstrated that ICAT could bind to {beta}-catenin as a competitive inhibitor of TCF and that in vivo it acts as an inhibitor of Wnt signaling. ICAT injected into Xenopus oocytes inhibited Wnt-mediated axis induction, and dominant negative forms of ICAT counteracted this inhibition.

More recently, the crystallographic structure of ICAT bound to the armadillo repeat region of {beta}-catenin shows how TCF binding is inhibited (6, 10). An NH2-terminal domain of ICAT binds to armadillo repeats 10–12 of {beta}-catenin, whereas a COOH-terminal domain of ICAT binds to the groove formed by armadillo repeats 5–9, this latter site being crucial for the binding of TCF and, coincidentally, of E-cadherin. Experimental evidence has confirmed that ICAT does block binding of {beta}-catenin to TCF and blocks the function of {beta}-catenin in Wnt signaling (10), but what about interactions with E-cadherin? On the basis of the structural data, having ICAT bound in the groove of {beta}-catenin should also potentially interfere with E-cadherin binding. Previously it was shown that ICAT could block the binding of C-cadherin to {beta}-catenin in vitro; however, overexpression of ICAT in Xenopus oocytes did not disrupt adhesion or show biochemical evidence for an in vivo inhibition of C-cadherin/{beta}-catenin complexes by ICAT (10).

Gottardi and Gumbiner (9) have now more closely explored the biology of ICAT in cells and its potential for involvement in regulating cadherin-based adhesion through inhibiting the interaction of E-cadherin with {beta}-catenin. An important contribution of this article is to reveal some of the basic physiology of ICAT's expression, localization, and response to Wnt signaling in cells and tissues. Immunostaining of ICAT is present in the cytoplasm and nucleus of cells (9). Other salient biochemical features of ICAT are its relative abundance in cells and the fact that a large proportion of soluble {beta}-catenin is stably bound to ICAT in cells. ICAT is thus well positioned to be a major regulator of {beta}-catenin, particularly during Wnt signaling, which appears to increase the amount of {beta}-catenin bound to ICAT. By staining sections of intestinal mucosa, the expression of endogenous ICAT was examined in an epithelium that undergoes Wnt-induced differentiation along the length of the villus. The data show that expression of ICAT correlates inversely with the known transcriptional activity of {beta}-catenin, i.e., ICAT is predomintantly expressed in the villus epithelial cells (where {beta}-catenin is not active in Wnt-induced transcription) and is diminished in crypt cells (where {beta}-catenin is transcriptionally active). This provides a physiological context for correlating ICAT's expression with {beta}-catenin function and with cell differentiation. However, ICAT expression levels in different cell lines did not correlate with Wnt signaling or with {beta}-catenin levels, suggesting that ICAT itself is probably not a Wnt responsive gene. These results preserve the growing reputation of ICAT as a competitive inhibitor of {beta}-catenin in its role as a transcriptional activator and as a regulator in Wnt signaling pathways.

What next for ICAT? As a possible alternative binding partner for the central groove and same armadillo repeats of {beta}-catenin, E-cadherin is notably absent, and apparently actively excluded, from the ICAT/{beta}-catenin complexes precipitated from cell extracts. New data (9) further confirm the ability of ICAT to block {beta}-catenin binding to the cytoplasmic tail domain of E-cadherin in vitro. There is a clear consensus that the precise binding domains on {beta}-catenin for ICAT and for E-cadherin do overlap, and there is the potential for competitive binding. Gottardi and Gumbiner (9) have gone on to show in vivo that, indeed, ICAT can affect the {beta}-catenin/E-cadherin interaction, but only as qualified by the physiology of the cells. At steady state, in Madin-Darby canine kidney cells stably overexpressing ICAT, there was no evidence for altered {beta}-catenin binding to E-cadherin and no overt effect on cadherin-based adhesion. However, in cells treated with hepatocyte growth factor (scatter factor; HGF), ICAT overexpression reduced E-cadherin levels, increased the {beta}-catenin bound to ICAT, and enhanced cell scattering. Thus ICAT can act as an inhibitor of a second function of {beta}-catenin: its role in cadherinbased adhesion. As suggested by these data, ICAT might only perform this inhibition in growth-stimulated or motile cells in which there is the requirement to downregulate adhesion. One mechanism suggested by the authors (9) for this discriminate binding of ICAT to {beta}-catenin is the reduced affinity of binding between E-cadherin and {beta}-catenin brought about by phosphorylation of both proteins after growth factor stimulation (11). Subsequent to this in the response to growth factor binding, E-cadherin is endocytosed (7). The stoichiometry of the E-cadherin/{beta}-catenin complex changes during endocytosis (14), providing another possible opportunity for ICAT binding to sequester {beta}-catenin and reduce adhesive capacity.

The mechanisms available for downregulating cadherin-based adhesion are only now beginning to be understood. During morphogenesis or in wound healing, cell-cell adhesion is downregulated, and several mechanisms have been identified for reducing adhesion based on regulating the cadherins themselves. The endocytosis and recycling of E-cadherin remove cadherin/catenin complexes from the cell surface and provide one efficient mechanism for transiently regulating adhesion (1, 13, 14, 1618). In epithelial-to-mesenchymal transition and in tumorigenesis there is a more permanent loss of adhesion through SNAIL- and SLUG-mediated transcriptional repression of E-cadherin (2, 4, 15) and through growth factor-induced ubiquitination, endocytosis, and degradation of E-cadherin (7). A fully detailed understanding of these endocytic and degradative pathways has yet to be pieced together, and it will be interesting to see whether ICAT is directly involved.

Thus, of the three main functional complexes formed by {beta}-catenin in cells, ICAT selectively participates in two of them. ICAT is not, however, a three-way inhibitor. ICAT does not directly regulate the cellular levels of {beta}-catenin by affecting the interaction of {beta}-catenin with APC for its degradation, and it does not compete with APC for binding (9). APC can bind to soluble {beta}-catenin simultaneously with ICAT, in keeping with predictions from APC's specific and slightly offset binding site (20). Through sequestering {beta}-catenin, ICAT inhibits both Wnt responsive gene expression and cadherin-based cell adhesion, each of which is an avenue to a role for ICAT in tumorigenesis. ICAT is not the singly selective inhibitor and drug target previously proposed (10) but may in fact be more powerful in both respects because of its dual specificity. The inappropriate transcriptional activity of {beta}-catenin is implicated in a number of cancers, through either mutations that block GSK3{beta} phosphorylation or mutations of APC/axin (3), and E-cadherin is typically downregulated in metastatic tumours (5). Intriguingly, ICAT is overexpressed in some tumors (12). Future studies should no doubt provide new insights into how manipulation of ICAT might be exploited through one or both of its inhibitory actions for new therapeutic approaches in cancer.


Address for reprint requests and other correspondence: J. L. Stow, Institute for Molecular Bioscience, Univ. of Queensland, Brisbane QLD 4072, Australia (E-mail: J.Stow{at}imb.uq.edu.au).

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