Although diffuse cytoplasmic and nuclear localizations of the proteins are also evident, as previously described (Henderson, 2000; Cong and Varmus, 2004), these data agree with earlier studies demonstrating APC2 and axin localization to cell contacts in and (Fig

Although diffuse cytoplasmic and nuclear localizations of the proteins are also evident, as previously described (Henderson, 2000; Cong and Varmus, 2004), these data agree with earlier studies demonstrating APC2 and axin localization to cell contacts in and (Fig. tissue structure and function. As a central component of intercellular adherens junctions, -cat links cadherin adhesion receptors to the actin cytoskeleton through the actin-binding protein -catenin. As a key component of the Wnt signaling cascade, -cat partners with lymphocyte enhancer factor/T-cell factor (TCF) family DNA-binding proteins to recruit chromatin-modifying factors required for gene expression (for review see Willert and Jones, 2006). The fact that cells use -cat in both cellCcell adhesion and transcriptional activation has long suggested that these two processes may be coordinated. Indeed, gain- and loss-of-function studies support a Rigosertib model in which cadherins directly sequester -cat from the nucleus and serve as a sink for the Wnt-generated cytosolic pool of -cat (Heasman et al., 1994; Cox et al., 1996; Fagotto et al., 1996). However, whether changes in cellCcell adhesion, rather than changes in cadherin abundance, can affect Wnt signaling has remained unanswered. In the absence of Wnt, -cat activity is largely regulated by a multiprotein complex that mediates the phosphorylation-dependent destruction of cytosolic -cat. Specifically, the scaffold protein axin recruits two Ser/Thr kinases, CK1- and GSK-3, directing an ordered phosphorylation of -cat at Ser45 (by CK1-) and subsequently at Thr41, Ser37, and Ser33 (by GSK-3; Liu et al., 2002). Phosphorylated Ser33 and Ser37 are recognized by the E3 ligase -TrCP (transducin repeatCcontaining protein), which leads to the ubiquitylation Rigosertib and degradation of -cat (Hart et al., 1999). The tumor suppressor adenomatous polyposis coli (APC) appears to facilitate the flux of -cat through the complex by displacing axin (Ha et al., 2004). APC2 is a second APC gene expressed in most epithelia (van Es et al., 1999). Unlike the APC mutated in colon cancers and found at the ends of microtubules, APC2 localizes to actin-rich structures (McCartney et al., 1999; Yu Rigosertib and Bienz, 1999; Yu et al., 1999). Although gain- and loss-of-function studies show that both APCs can inhibit -cat signaling (McCartney et al., 1999; van Es et al., 1999; Yu et al., 1999; Polakis, 2000), the different subcellular distributions of APC and APC2, along with evidence that APC2 protein expression is maintained in APC mutant tumors (Jarrett et al., 2001), suggest that they participate in different phosphodestruction complexes that are subject to distinct regulation. Wnts are the best-known inhibitors of the -cat phosphodestruction complex. These ligands engage a receptor complex and induce the local inactivation of GSK-3 within and/or disruption of the axin scaffold complex (Klaus and Birchmeier, 2008). As a result, an N-terminally hypophosphorylated form of -cat accumulates, translocates to the nucleus, and activates TCF/lymphocyte enhancer factor family transcription factors. In this study, we show that N-terminal phospho forms of -cat and components of the phosphodestruction complex are localized to sites of cell contact, where the activity of this complex can be enhanced by cadherin-based cell adhesion. Results and discussion Epithelial cadherin (E-cad) reduces levels of the signaling form of -cat by promoting its N-terminal phosphorylation The SW480 colon carcinoma cell line exhibits robust -cat/TCF signaling because of a loss-of-function mutation in APC and low levels of E-cad. Restoration of E-cad expression reduced the oncogenic signaling activity of -cat without markedly sequestering cytosolic -cat (Gottardi et al., Sirt7 2001). This finding raised the possibility that Wnts might generate a minor, transcriptionally active form of -cat used for both signaling and adhesion. Alternatively, the cadherin might inhibit -cat signaling through a mechanism that catalytically inactivates the bulk pool of -cat. It is now appreciated Rigosertib that -cat unphosphorylated at Ser33, Ser37, and Thr41 is more transcriptionally active than forms of -cat phosphorylated at these sites (Guger and Gumbiner, 2000; Staal et al., 2002). Several recently developed reagents allow for detection of these specific phosphorylated and unphosphorylated epitopes in the N terminus of -cat (Fig. 1 A). Because SW480 cells are mutant for APC, the regulation and localization of these phospho forms of -cat, which are normally coupled with ubiquitylation and degradation, can be characterized. Open in a separate window Figure 1. Restoring E-cad expression in SW480 cells alters the phosphorylation status of the -cat N terminus. (A) -cat N-terminal epitopes recognized by antibodies to ABC (unphosphorylated at Ser37 and Thr41), P33/37/41, and P41/45. The CK1- site is gray; GSK-3 sites are black. (B) Immunoblots.