Mechanism of b-catenin targeting to adhesive or transcriptional complexes

描述(申请人提供)：B-catenin是一种双功能蛋白，在Wnt介导的转录和基于钙粘附素的细胞间黏附中发挥重要作用。由于b-连环蛋白与钙粘附素结合介导肿瘤抑制，而b-连环蛋白转录功能驱动细胞转化，了解是什么控制了b-连环蛋白针对转录或黏附复合体的靶向，将有助于考虑寻求抑制致癌而不抑制b-连环蛋白的肿瘤抑制活性的策略。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)结合，但不能与钙粘附素类型的黏附受体结合。这种信号形式是单体的，受b-catenin的C末端调控，我们认为b-catenin通过分子内折叠机制选择性地与钙粘素结合竞争。相反，b-连环蛋白的主要钙粘附素结合形式是b-连环蛋白/a-连环蛋白二聚体，这表明存在一种不同的分子形式的b-连环蛋白，它可以与钙粘连蛋白和a-连环蛋白相互作用。这项建议的总体目标是确定b-catenin的细胞质调节如何决定黏附和信号功能，这与b-catenin作为肿瘤抑制因子和癌基因的双重作用有关。我们假设b-catenin的末端区域指导着b-catenin的这些不同的功能。为此，我们建议使用体外亲和力结合分析，结合构象特异性抗体和突变方法，鉴定控制其与钙粘素与TCP结合的b-连环蛋白序列(目标1)。磷酸化在调节b-连环蛋白结合选择性中的作用将使用磷酸酶和磷酸肽图技术来确定(目标2)。此外，b-catenin的C末端如何在体内调节黏附功能将通过细胞黏附试验来确定，该实验专门定量钙粘附素的黏附活性(目标3)。这些实验将帮助我们了解b-catenin是如何针对钙粘蛋白和tcp转录复合体进行调控的，这将为细胞如何在正常发育和肿瘤进展过程中协调b-catenin的黏附和信号功能提供洞察力。

DESCRIPTION (provided by applicant): 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 with 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.