Mechanism of b-catenin targeting to adhesive or transcriptional complexes

β-连环蛋白靶向粘附或转录复合物的机制

• 批准号: 7912120
• 负责人: Cara J Gottardi
• 金额: $ 11.9万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-16 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912120
• 关键词: Adherens Junction; Adhesions; Adhesives; Affect; Affinity; Antibodies; Automobile Driving; Back; Binding; Biological Assay; C-terminal; Cadherins; Cell Adhesion; Cell physiology; Cell-Matrix Junction; Cells; Complex; Cytoplasmic Tail; Data; Development; Exhibits; Gene Targeting; Genetic Transcription; In Vitro; Knock-out; Malignant Neoplasms; Maps; Measures; Mediating; Modification; Molecular; Molecular Conformation; Morphogenesis; Mutagenesis; N-terminal; Oncogenic; Pathway interactions; Peptidylprolyl Isomerase; Phosphopeptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phylogenetic Analysis; Play; Post-Translational Protein Processing; Proteins; RNA Interference; Regulation; Relative (related person); Reporter; Research Personnel; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Structure; TCF Transcription Factor; Techniques; Testing; Tissues; Transcriptional Activation; Tumor Suppression; Tumor Suppressor Genes; Tumor Suppressor Proteins; adhesion receptor; base; dimer; in vivo; insight; metaplastic cell transformation; mutant; programs; research study; transcription factor; tumor initiation; tumor progression

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.

b-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发挥重要作用。 基于细胞间粘附。由于 b-连环蛋白与钙粘蛋白结合介导肿瘤抑制，而 b- 连环蛋白转录功能驱动细胞转化，了解控制 b-连环蛋白的因素 针对转录或粘附复合物将与考虑寻求以下策略相关： 抑制β-连环蛋白的致癌作用，但不影响其抑癌活性。 我们的初步研究表明，可以生成一种形式的 β-连环蛋白，优先与 转录因子、T 细胞因子 (TCP)，但不包括钙粘蛋白型粘附受体。该信号形式是 单体并受 b-连环蛋白 C 末端调节，我们建议选择性竞争 钙粘蛋白通过分子内折回机制结合。相反，主要的钙粘蛋白结合 b-连环蛋白的形式是 b-连环蛋白/a-连环蛋白二聚体，表明 b-连环蛋白存在独特的分子形式 可以与钙粘蛋白和α-连环蛋白相互作用。该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附功能与信号传导功能，这与 b-相关 连环蛋白具有肿瘤抑制基因和癌基因的双重作用。我们假设 b- 的末端区域 连环蛋白指导 β-连环蛋白的这些独特功能。为此，我们建议识别序列 使用体外亲和力结合测定，控制其与钙粘蛋白结合的 b-连环蛋白与 TCP 的结合 使用构象特异性抗体和诱变方法（目标 1）。磷酸化的作用 将使用磷酸酶和磷酸肽作图来确定调节 b-连环蛋白结合选择性 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 可以使用专门量化钙粘蛋白粘附活性的细胞附着测定来确定（目标 3）。 这些实验将帮助我们了解 β-catenin 如何靶向钙粘蛋白和 TCP 转录 复合物受到调节，这将有助于了解细胞如何协调粘附和信号传导 β-连环蛋白在整个正常发育和肿瘤进展过程中的功能。