Structure and regulation of beta-catenin during cell-cell adhesion

细胞-细胞粘附过程中β-连环蛋白的结构和调控

• 批准号: 8320628
• 负责人: Jeffrey D Hardin
• 金额: $ 18.41万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8320628
• 关键词: Activator Appliances; Adhesions; Adhesiveness; Adhesives; Affinity; Affinity Chromatography; Alleles; Amino Acids; Binding; Biochemical; Biological Models; C-terminal; Cadherins; Caenorhabditis elegans; Cell division; Cell-Cell Adhesion; Cells; Complex; Congenital Abnormality; Cytoplasmic Tail; Data; Defect; Dependence; Diagnosis; E-Cadherin; Embryo; Embryonic Development; Ensure; Epithelium; Foundations; Health; Human; In Vitro; Invaded; Laboratories; Life; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Microscopy; Morphogenesis; Neoplasm Metastasis; Organism; Phenotype; Phosphorylation; Phosphotyrosine; Play; Protein Family; Proteins; Regulation; Resolution; Role; Serine; Signal Transduction; Structural Protein; Structure; Testing; Time; Tissue Engineering; Transcription Coactivator; Tyrosine; Vertebrates; Work; X-Ray Crystallography; arm; beta catenin; cancer cell; cell behavior; human disease; in vivo; mutant; novel; research study

DESCRIPTION (provided by applicant): The cadherin-catenin complex (CCC) is crucial for successful completion of morphogenesis during embryonic development, and for suppression of metastatic phenotypes in differentiated epithelia. Beta-catenin is a core component of the CCC; analyzing and manipulating the adhesive functions of beta-catenin thus has widespread implications for human health. To date, distinguishing between adhesion and Tcf/Lef-dependent transcriptional coactivator functions of beta-catenin, and correlating detailed protein structural information with in vivo function has been challenging in vertebrates. C. elegans presents a unique opportunity to analyze the adhesion- specific roles of a beta-catenin, because it possesses multiple, specialized beta -catenins. We will leverage this unique specialization, combined with a powerful foundation of detailed structural information derived from a collaborative X-ray crystallography project, to establish a new, multi- disciplinary approach to the study of beta-catenins in the following aims: Aim 1. Test the role of a key phosphorylated residue in the C terminus of cadherin for the first time in vivo. We will test the in vivo importace of phosphorylation of a conserved serine that upregulates the affinity of cadherin for beta-catenin several hundred fold in vitro using biochemical and high-resolution microscopy in living embryos. Aim 2. Test the role of key phosphotyrosines in the adhesive functions of beta-catenin for the first time in vivo. We will test the in vivo importance of two highly conserved tyrosines, one that regulates cadherin/beta-catenin binding, and another that regulates beta-catenin/beta-catenin binding. We will also assess the phosphorylation state of endogenous HMP-2 using affinity purification and mass spectrometry and identify a panel of new hmp-2 alleles, which will provide information about amino acids important specifically for the adhesive functions of beta-catenin. Aim 3. Test the role of a conserved helical domain in beta-catenin for its activity as a transcriptional coactivator. HMP-2 lacks a helical domain ("Helix C") just C-terminal to the 12th Arm repeat that is conserved in all beta-catenins known to have canonical transcriptional co- activator functions. BAR-1 binds POP-1/Tcf with high affinity, and retains Helix C. We will test the role of Helix C using domain-swap experiments between HMP-2 and BAR-1, and assessing Tcf binding in vitro and coactivator functions in vivo. PUBLIC HEALTH RELEVANCE: Understanding how cells attach to one another is important for understanding many common birth defects, and how cancer cells lose their connections to one another and invade the body. This proposal examines a key protein, beta-catenin that regulates cell adhesiveness, and how this protein works together with other proteins to ensure that cells make proper connections in the body. By studying how this protein works in living embryos, we will gain important information that can be used to understand and treat human disease.

描述（由申请人提供）：钙粘蛋白-连环蛋白复合物（CCC）对于胚胎发育过程中形态发生的成功完成以及分化上皮细胞中转移表型的抑制至关重要。 β-连环蛋白是 CCC 的核心成分；因此，分析和操纵 β-连环蛋白的粘附功能对人类健康具有广泛的影响。迄今为止，区分 β-连环蛋白的粘附和 Tcf/Lef 依赖性转录共激活子功能，以及将详细的蛋白质结构信息与体内功能相关联，在脊椎动物中一直具有挑战性。 线虫为分析 β-连环蛋白的粘附特异性作用提供了独特的机会，因为它拥有多种专门的 β-连环蛋白。 我们将利用这种独特的专业知识，结合来自协作 X 射线晶体学项目的详细结构信息的强大基础，建立一种新的、多学科的方法来研究 β-连环蛋白，以实现以下目标： 目标 1. 首次在体内测试钙粘蛋白 C 末端关键磷酸化残基的作用。 我们将使用生化和高分辨率显微镜在活体胚胎中测试保守丝氨酸磷酸化的体内重要性，该磷酸化可在体外将钙粘蛋白与β-连环蛋白的亲和力上调数百倍。 目标 2. 首次在体内测试关键磷酸酪氨酸在 β-catenin 粘附功能中的作用。 我们将测试两种高度保守的酪氨酸的体内重要性，一种调节钙粘蛋白/β-连环蛋白结合，另一种调节β-连环蛋白/β-连环蛋白结合。我们还将使用亲和纯化和质谱法评估内源性 HMP-2 的磷酸化状态，并鉴定一组新的 hmp-2 等位基因，这将提供有关对 β-连环蛋白的粘附功能特别重要的氨基酸的信息。 目标 3. 测试 β-连环蛋白中保守螺旋结构域作为转录共激活因子的作用。 HMP-2 在第 12 臂重复的 C 端缺少一个螺旋结构域（“螺旋 C”），该螺旋结构域在所有已知具有规范转录共激活子功能的 β-连环蛋白中都是保守的。 BAR-1 以高亲和力结合 POP-1/Tcf，并保留 Helix C。我们将使用 HMP-2 和 BAR-1 之间的结构域交换实验来测试 Helix C 的作用，并评估体外 Tcf 结合和体内共激活剂功能。 公共卫生相关性：了解细胞如何相互附着对于了解许多常见的出生缺陷以及癌细胞如何失去彼此的联系并侵入身体非常重要。该提案研究了调节细胞粘附性的关键蛋白质β-连环蛋白，以及该蛋白质如何与其他蛋白质一起发挥作用，以确保细胞在体内建立适当的连接。通过研究这种蛋白质如何在活胚胎中发挥作用，我们将获得可用于理解和治疗人类疾病的重要信息。