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

描述（由申请人提供）：钙粘蛋白-连环蛋白复合物（CCC）对于胚胎发育期间形态发生的成功完成以及抑制分化上皮细胞中的转移表型至关重要。β-连环蛋白是CCC的核心组成部分;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意义。迄今为止，区分β-连环蛋白的粘附和Tcf/Lef-dependent转录共激活因子功能，并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。 C. elegans提供了一个独特的机会来分析β-连环蛋白的粘附特异性作用，因为它具有多种专门的β-连环蛋白。 我们将利用这一独特的专业化，结合来自合作X射线晶体学项目的详细结构信息的强大基础，建立一个新的，多学科的方法来研究β-连环蛋白，目标如下：目标1。首次在体内检测钙粘蛋白C端一个关键磷酸化残基的作用。 我们将在活胚胎中使用生物化学和高分辨率显微镜检测一种保守丝氨酸磷酸化的体内重要性，这种丝氨酸在体外上调钙粘蛋白对β-连环蛋白的亲和力数百倍。 目标二。首次在体内测试关键磷酸酪氨酸在β-连环蛋白粘附功能中的作用。 我们将测试两个高度保守的酪氨酸在体内的重要性，一个调节钙粘蛋白/β-连环蛋白结合，另一个调节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，并确定一组新的HMP-2等位基因，这将提供有关β-连环蛋白粘附功能的重要氨基酸的信息。 目标3.测试β-连环蛋白中保守螺旋结构域作为转录辅激活因子的作用。 HMP-2缺少恰好在第12臂重复序列的C末端的螺旋结构域（“HMP C”），其在已知具有典型转录共激活因子功能的所有β-连环蛋白中是保守的。 BAR-1以高亲和力结合POP-1/Tcf，并保留HepG 2 C。 我们将使用HMP-2和BAR-1之间的结构域交换实验，并评估体外Tcf结合和体内共激活因子功能，来测试HVC的作用。

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.