| beta-catenin is a multifunctional protein that plays a critical role in cell-cell adhesion and functions as an essential component in Wnt-mediated cell fate decisions. Cadherins can inhibit Wnt/beta-catenin signaling, although the mechanisms responsible remain unclear. This thesis demonstrates that cadherins inhibit beta-catenin signaling by: (1) directly binding a small signaling pool of beta-catenin, and (2) catalytic inactivation of beta-catenin that depends on cadherin-based adhesion. Active beta-catenin is a monomeric, minor form of total beta-catenin that is easily sequestered by E-cadherin. N-terminally phosphorylated beta-catenin does not interact with E-cadherin, suggesting it remains in the phospho-destruction complex. In addition to the direct binding hypothesis, the data in this thesis support a second mechanism where cadherin-based adhesion, rather than cadherin protein levels, inhibits beta-catenin signaling. Axin, APC2, GSK-3beta, and Nterminally phosphorylated forms of beta-catenin localize to cell--cell contacts in a complex that is molecularly distinct from the cadherin--catenin adhesive complex. Nonetheless, cadherins can promote the N-terminal phosphorylation of beta-catenin, and cell-cell adhesion increases the turnover of cytosolic beta-catenin. These data suggest that cadherin-based cell-cell adhesion limits Wnt signals by promoting the activity of a junction-localized beta-catenin phospho-destruction complex. Together, these mechanisms imply that the mode of cadherin-based beta-catenin signaling inhibition may vary depending on the cellular context, and that regulation of the cadherin/beta-catenin interaction will affect signaling. The beta-catenin binding region of E-cadherin is phosphorylated in vivo, and phosphorylation of this domain in vitro enhances beta-catenin binding. This thesis shows that in vivo cadherin phosphorylation can be monitored by following the mobility properties of a cadherin cytoplasmic domain fragment in SDS-Polyacrylamide gels. Specifically, the slower migrating fragment is phosphorylated, and beta-catenin interacts exclusively with this form in coimmunoprecipitation analysis. Forced membrane targeting of the cadherin cytodomain results in more efficient phosphorylation, indicating that the responsible kinase(s) are localized at or near membranes. Three serine residues are required for generating the slower migrating form of the cadherin cytodomain, [32P]-incorporation, and beta-catenin binding and stabilization. Since these residues are predicted to be GSK-3beta sites, it is possible that Wnt signals may transiently inhibit GSK-3beta activity and cadherin phosphorylation, leading to enhanced beta-catenin signaling. |
