We show that Wnt signaling generates a monomeric form of β-catenin that binds TCF selectively compared with the cadherin. In contrast, the cadherin preferentially binds a β-catenin–α-catenin dimer. This selective targeting of distinct molecular forms of β-catenin provides a mechanism by which cells could potentially separate the adhesion and signaling functions of β-catenin. We propose that segregation of these functions of β-catenin may be necessary for two reasons. First, selective targeting of β-catenin to transcriptional complexes would prevent the cadherin from competing with Wnt signaling activity, which may be important during low or transient Wnt activation where signaling may need to be especially efficient. Second, such a mechanism would ensure that cell–cell adhesion is maintained during Wnt inductions throughout development. Loading the cadherin with β-catenin monomers generated by strong Wnt signals might have undesired consequences for cell adhesion, because cadherins bound to β-catenin without α-catenin would be unable to contribute to adhesion. Thus, generation and targeting of distinct molecular forms of β-catenin could ensure that adhesion and signaling are not always coupled, and when necessary, can be regulated independently of one another.
We provide evidence that the Wnt-stimulated, TCF-binding selectivity of β-catenin is mediated by the COOH-terminal region of β-catenin. First, COOH-terminal epitopes of β-catenin are masked in the fraction of β-catenin that is unable bind the cadherin. Second, a COOH-terminal peptide of β-catenin can compete β-catenin binding to cadherin, but not to TCF. Third, deletion of the COOH terminus of β-catenin results in a loss of binding selectivity. Together with previously published data showing that the COOH-terminal region of β-catenin can bind directly to the armadillo repeat region of β-catenin (Cox et al., 1999
; Piedra et al., 2001
) and restrict cadherin binding in vitro (Castano et al., 2002
), we propose that in vivo, the COOH terminus of β-catenin adopts a folded-over conformation which controls β-catenin binding selectivity by restricting cadherin but not TCF binding. Thus, Wnts may activate β-catenin signaling not only by increasing its cytosolic levels, but by regulating the conformation of its COOH terminus.
The existence of a form of β-catenin that distinguishes between cadherins and TCF was not anticipated, given the overall structural similarity between the β-catenin–cadherin and β-catenin–TCF binding interfaces revealed by X-ray crystallography (Graham et al., 2000
; Huber and Weis, 2001
). Upon closer examination, however, the β-catenin–TCF binding interface is less extensive than the β-catenin–cadherin binding interface, spanning arm repeats 3–10 compared with all 12 armadillo repeats for the cadherin. Thus, it is possible that the COOH-terminal region of β-catenin may fold-back over the last two armadillo repeats of β-catenin, which could have consequences for cadherin but not TCF binding. Indeed, alteration of a single residue in the 12th arm repeat of β-catenin decreases β-catenin binding to the cadherin by a factor of four (Roura et al., 1999
), further arguing that small perturbations in the β-catenin–cadherin interface can have significant consequences for binding.
It has also been shown that phosphorylation of E-cadherin increases cadherin–β-catenin complex formation (Lickert et al., 2000
). In crystal structures, this phosphorylation results in interactions with β-catenin that appear to mimic TCF binding (Huber and Weis, 2001
). Indeed, we find that cadherin phosphorylation allows the cadherin to bind the monomeric, closed form of β-catenin that otherwise would be TCF selective. The fact that cadherin phosphorylation can reverse Wnt-mediated β-catenin binding selectivity suggests a mechanism by which cadherins compete for the Wnt-activated form of β-catenin. It will be important, therefore, to determine when and where cadherin modification occurs to better understand the relationship between adhesion and signaling.
Our observation that the cadherin binds preferentially to β-catenin–α-catenin dimers compared with β-catenin monomers raises the possibility that α-catenin plays a positive role in β-catenin binding to cadherin. Indeed, one study showed that preassociation of recombinant α-catenin with β-catenin increases β-catenin binding to cadherin, suggesting that α-catenin induces an open conformation of β-catenin (Castano et al., 2002
). Other evidence, however, argues that α-catenin is not required for β-catenin binding to cadherin. For example, recombinant cadherin–β-catenin complexes are readily formed in vitro (Huber et al., 2001
), and cells lacking α-catenin still form cadherin–β-catenin complexes (Bullions et al., 1997
; Vasioukhin et al., 2001
). We suggest that the form of β-catenin that binds preferentially to cadherin, also binds α-catenin.
Based on our findings from this and previous reports, we propose that cells contain a number of distinct molecular forms of β-catenin (). Thus, although an organism like C. elegans
controls the adhesive and signaling functions of β-catenin through expression of a multi-gene family, vertebrates regulate β-catenin functions by generating distinct molecular forms at the protein level. First, there is the well-known form of β-catenin that is phosphorylated at the NH2
terminus and is targeted for degradation (, phosphorylated; for review see Polakis, 1999
). We reported previously a large pool of β-catenin in the SW480 tumor cell line that cannot bind to either TCF or cadherin, and provided evidence that this was an “inactive” form for both adhesion and signaling (; Gottardi et al., 2001
). This form may be due, at least in part, to ICAT, a small 9-kD polypeptide that inhibits β-catenin binding to both TCF and cadherin (Gottardi and Gumbiner, 2004
; Tago et al., 2000
). Here, we provide evidence for a TCF-selective form of β-catenin that is targeted to transcription complexes (closed conformation), and a form that can target to adhesive complexes (β-catenin–α-catenin dimer). Although the latter form can interact with both the cadherin and TCF, there is evidence that α-catenin inhibits the transcriptional activity of β-catenin in the nucleus (Giannini et al., 2000
), suggesting that this form is specific for adhesion functions. Finally, we postulate that cells can contain a form of β-catenin that is competent for both signaling and cadherin binding (open conformation) which is observed, for example, under long-term LiCl treatment ( A, lanes 14 and 15), and would explain the many cases in which cadherin expression inhibits the transcriptional activity of β-catenin (Heasman et al., 1994
; Fagotto et al., 1996
; Sanson et al., 1996
; Orsulic et al., 1999
; Shtutman et al., 1999
; Gottardi et al., 2001
Figure 9. Multiple forms of β-catenin exist in cells. NH2-terminal phospho-β-catenin is well characterized and generated by the APC-Axin-GSK3β-CK1 complex (dashed line). Closed form of β-catenin is generated by Wnt signaling, perhaps (more ...)
Other than the targeting of β-catenin for degradation, the modifications and machinery that regulate these various forms of β-catenin are presently unknown. It is not clear, for example, whether the formation of the inactive ICAT complex is simply controlled by levels of ICAT expression, or is regulated in another way (Gottardi and Gumbiner, 2004
). Also, it is not understood what controls the formation of the β-catenin–α-catenin dimer, although it is clear that β-catenin monomers and α-catenin can coexist in the cytosol without forming complexes, even though they readily bind with high affinity in vitro (Koslov et al., 1997
). Nor is the time and place of cadherin phosphorylation known. We show that the TCF-selective, closed form of β-catenin is regulated by the Wnt pathway, and that the binding selectivity observed after short-term LiCl treatment suggests that GSK3β may be involved. However, the mechanism must be distinct from the pathway that regulates β-catenin levels in the cytosol, because the absence of GSK3β-dependent NH2
-terminal phosphorylation does not account for its binding properties, and long-term LiCl treatments and APC mutations lead to β-catenin accumulation without generating the TCF-selective form. It is tempting to speculate that the APC-axin-GSK3β–containing complex regulates the generation of these various forms of β-catenin by post-translational modifications, in addition to the targeting of β-catenin for degradation. Indeed APC mutations have been found to affect adhesive functions of β-catenin as well as Wnt signaling in Drosophila
(Hamada and Bienz, 2002
If we propose that Wnt signaling generates a TCF-selective form of β-catenin that is resistant to cadherin binding, how do we explain the fact that cadherin expression has been found to antagonize Wnt signaling in numerous model systems (Heasman et al., 1994
; Fagotto et al., 1996
; Gottardi et al., 2001
)? One possibility is that cadherin overexpression of cadherin drives the formation of complexes that do not occur under normal physiological conditions. Although the various molecular forms of β-catenin seem fairly stable in our experiments, it is possible that they are more interconvertible in the cell, or that one form is an intermediate for the other, and can be depleted during its generation. A more interesting possibility is that the relationship between cadherins and Wnt signaling may depend on the specific situation faced by each cell responding to a Wnt signal. For example, our finding that cadherin phosphorylation increases β-catenin binding to cadherin, reversing the differential binding activity observed during Wnt signaling, suggests that variations in cadherin phosphorylation may alter the extent to which adhesion and signaling are coupled. We also hypothesize that the cell can potentially generate either the open form of β-catenin, which binds to both cadherin and TCF, or the closed, TCF-selective form, and the relative proportion of these two forms may differ between different cells responding to Wnt signaling, or different strengths of Wnt signaling.
Indeed, consideration of the various findings suggests a model in which the extent of coupling between the adhesive and nuclear signaling functions of β-catenin is regulated differentially in different cell types, depending on the biological needs of the cells and tissues responding to a Wnt signal. Elucidating the cellular and biochemical mechanisms regulating the generation of the different forms of β-catenin and determining when and where they occur should provide insight into the relationship between the adhesive and signaling functions of β-catenin.