Mechanism of b-catenin targeting to adhesive or transcriptional complexes

B-连环蛋白是一种双功能蛋白，在Wnt介导的转录和钙粘附素-2中发挥重要作用。 基于细胞间黏附。因为b-连环蛋白与钙粘附素结合介导了肿瘤抑制，而b-连环蛋白与钙粘附素结合则介导了肿瘤抑制。 连环蛋白转录功能驱动细胞转化，了解是什么控制了b-连环蛋白 以转录或粘附性复合体为目标将与考虑寻求 抑制致癌作用，但保留b-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，它优先与 转录因子，T细胞因子(TCP)，但不是钙粘附素型黏附受体。此信令表单为 单体，并受b-连环蛋白的C-末端调节，我们提出选择性竞争 钙粘附素通过分子内折叠机制结合。相比之下，主要的钙粘附素结合 B-连环蛋白的形式是b-连环蛋白/a-连环蛋白二聚体，这表明存在一种独特的分子形式的b-连环蛋白。 它可以与钙粘附素和α-连环蛋白相互作用。这项提案的总体目标是确定 B-连环蛋白的细胞质调节如何决定粘着功能与信号功能，这与b-连环蛋白相关。 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-的终末区域 连环蛋白指导着b-连环蛋白的这些不同功能。为此，我们建议识别序列 使用体外亲和结合试验控制其与钙粘蛋白结合的b-连环蛋白与磷酸三钙的结合 使用构象特异性抗体和诱变方法(目标1)。磷酸化在蛋白水解酶中的作用 调节b-连环蛋白的结合选择性将通过磷酸酶和磷酸肽图谱来确定。 技术(目标2)。此外，b-连环蛋白的C末端如何在体内调节黏附功能 使用细胞黏附试验，专门定量钙粘附素黏附活性(目标3)。 这些实验将帮助我们理解b-连环蛋白如何靶向钙粘蛋白和tcp转录。 复合体是受调控的，这将为细胞如何协调黏附和信号提供洞察 B-连环蛋白在正常发育和肿瘤进展过程中的作用。

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.