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

描述(由申请人提供)：钙粘素-连环蛋白复合体(CCC)对于成功完成胚胎发育过程中的形态发生和抑制分化上皮细胞的转移表型至关重要。β-连环蛋白是CCC的核心成分，分析和操纵β-连环蛋白的黏附功能对人类健康具有广泛的意义。到目前为止，在脊椎动物中区分黏附和依赖于Tcf/Lef的转录辅助激活蛋白的功能，以及将详细的蛋白质结构信息与体内功能联系起来一直是具有挑战性的。秀丽线虫提供了一个独特的机会来分析β-连环蛋白的黏附特异性作用，因为它拥有多个专门的β-连环蛋白。我们将利用这种独特的专业化，结合来自协作X射线结晶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，目标如下：目标1.首次在体内测试钙粘蛋白C末端的一个关键的磷酸化残基的作用。我们将在活体胚胎中使用生化和高分辨率显微镜，在体外测试一种保守的丝氨酸的磷酸化的重要性，这种丝氨酸可以上调钙粘附素与β-连环蛋白的亲和力数百倍。目的2.首次在体内检测关键的磷酸酪氨酸在β-连环蛋白黏附功能中的作用。我们将测试两种高度保守的酪氨酸在体内的重要性，一种是调节钙粘素/β-连环蛋白结合，另一种是调节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仪来评估内源性HMP-2的磷酸化状态，并鉴定一组新的HMP-2等位基因，这些等位基因将提供与β-连环蛋白黏附功能相关的氨基酸信息。目的3.检测β-连环蛋白中保守的螺旋结构域作为转录辅助激活因子的作用。HMP-2没有螺旋结构域(“螺旋C”)，只是C端到第12臂重复序列，它在所有已知具有规范转录辅助激活功能的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。我们将通过HMP-2和BAR-1之间的结构域互换实验来检测Helix C的作用，并评估Tcf在体外的结合和体内的共激活功能。 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人体非常重要。这项提案研究了一种关键蛋白质--β-连环素，它调节细胞的粘附性，以及这种蛋白质如何与其他蛋白质一起工作，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。

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.