Mechanism of b-catenin targeting to adhesive or transcriptional complexes

β-连环蛋白是一种双功能蛋白，在Wnt介导的转录和钙粘蛋白中起重要作用。 基于细胞间粘附。由于b-连环蛋白结合钙粘蛋白介导肿瘤抑制，而B- 连环蛋白的转录功能驱动细胞转化，了解是什么控制了b-连环蛋白 靶向转录复合物或粘附复合物将与考虑寻求 抑制致癌性，但保留了β-连环蛋白的肿瘤抑制活性。 我们的初步研究表明，可以产生一种形式的b-连环蛋白，它优先结合到 转录因子，T细胞因子（TCP），但不是钙粘蛋白型粘附受体。这种信号形式是 单体，并由B-连环蛋白的C-末端调节，我们建议选择性竞争 钙粘蛋白通过分子内折叠机制结合。相反，主要的钙粘蛋白结合 β-连环蛋白的一种形式是β-连环蛋白/α-连环蛋白二聚体，这表明β-连环蛋白有一种独特的分子形式 可以与钙粘蛋白和α-连环蛋白相互作用。本提案的总体目标是确定 β-连环蛋白的细胞质调节如何决定粘附与信号传导功能，这与B- 连环蛋白作为肿瘤抑制基因和癌基因的双重作用。我们假设B- 连环蛋白指导β-连环蛋白的这些不同功能。为此，我们建议确定序列 使用体外亲和结合试验， 用构象特异性抗体和诱变方法（目的1）。磷酸化在 调节β-连环蛋白结合选择性将使用磷酸酶和磷酸肽作图来确定 技术（目标2）。此外，B-连环蛋白的C-末端如何调节体内粘附功能将 可以使用特异性定量钙粘蛋白粘附活性的细胞附着测定来确定（Aim 3）。 这些实验将有助于我们了解β-连环蛋白如何靶向钙粘蛋白和TCP转录 复合物的调控，这将提供深入了解细胞如何协调粘附和信号传导 B-连环蛋白在整个正常发育和肿瘤进展中的功能。

b-catenin is a bi-functional protein that plays essential roles in Wnt-mediated transcription and cadherin- based intercellular adhesion. Since b-catenin binding to cadherin mediates tumor suppression, while b- catenin transcriptional function drives cellular transformation, understanding what controls b-catenin targeting to transcriptional or adhesive complexes will be relevant towards considering strategies that seek to inhibit the oncogenic, but spare the tumor suppressor activities of b-catenin. Our preliminary studies show that a form of b-catenin can be generated that preferentially binds to the transcription factor, T-Cell Factor (TCP), but not cadherin-type adhesion receptors. This signaling form is monomeric and is regulated by the C-terminus of b-catenin, which we propose selectively competes cadherin binding through an intramolecular fold-back mechanism. In contrast, the main cadherin-binding form of b-catenin is a b-catenin/a-catenin dimer, indicating that there is a distinct molecular form of b-catenin that can interact with both the cadherin and a-catenin. The overall objective of this proposal is to determine how cytoplasmic regulation of b-catenin dictates adhesive versus signaling functions, which is relevant to b- catenin's dual role as a tumor suppressor and oncogene. We hypothesize that the terminal regions of b- catenin direct these distinct functions of b-catenin. Towards this end, we propose to identify the sequences of b-catenin that control its binding to cadherin versus TCP using an in vitro, affinity-binding assay, together with a conformation-specific antibody and mutagenesis approaches (Aim 1). The role for phosphorylation in regulating b-catenin binding selectivity will be determined using phosphatase and phosphopeptide mapping techniques (Aim 2). Furthermore, how the C-terminus of b-catenin modulates adhesive function in vivo will be determined using a cell attachment assay that specifically quantifies cadherin adhesive activity (Aim 3). These experiments will help us understand how b-catenin targeting to cadherins and TCP transcriptional complexes is regulated, which will provide insights into how cells coordinate adhesive and signaling functions of b-catenin throughout normal development and tumor progression.