Mechanism of b-catenin targeting to adhesive or transcriptional complexes

描述（由申请人提供）：b-catenin是一种双功能蛋白，在wnt介导的转录和基于钙粘蛋白的细胞间粘附中起重要作用。由于b-catenin与钙粘蛋白结合介导肿瘤抑制，而b-catenin的转录功能驱动细胞转化，了解是什么控制了b-catenin靶向转录或粘附复合物，将有助于考虑寻求抑制致癌的策略，但避免b-catenin的肿瘤抑制活性。我们的初步研究表明，一种b-连环蛋白可以优先结合转录因子t细胞因子（TCP），而不是钙粘蛋白型粘附受体。这种信号形式是单体的，受b-catenin的c端调控，我们提出b-catenin通过分子内折叠机制选择性地与钙粘蛋白结合竞争。相比之下，b-catenin的主要钙粘蛋白结合形式是b-catenin/a-catenin二聚体，这表明b-catenin有一种独特的分子形式，可以与钙粘蛋白和a-catenin相互作用。本提案的总体目标是确定b-catenin的细胞质调节如何决定粘附功能和信号功能，这与b-catenin作为肿瘤抑制因子和致癌基因的双重作用有关。我们假设b-连环蛋白的末端区域指导着b-连环蛋白的这些不同功能。为此，我们建议使用体外亲和结合实验，结合构象特异性抗体和诱变方法，鉴定控制其与钙粘蛋白结合的b-catenin序列（目的1）。磷酸化在调节b-连环蛋白结合选择性中的作用将通过磷酸酶和磷酸肽定位技术来确定（目的2）。此外，b-catenin的c端如何调节体内的粘附功能将通过细胞附着实验来确定，该实验可以特异性地量化钙粘蛋白的粘附活性（Aim 3）。这些实验将帮助我们了解b-catenin靶向钙粘蛋白和TCP转录复合物是如何被调节的，这将为细胞在正常发育和肿瘤进展过程中如何协调b-catenin的粘附和信号功能提供见解。

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.