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）对于胚胎发育过程中形态发生的成功完成和分化上皮中转移表型的抑制至关重要。-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意义。迄今为止，在脊椎动物中区分β -连环蛋白的粘附和Tcf/左依赖性转录辅激活因子功能，以及将详细的蛋白质结构信息与体内功能相关联一直具有挑战性。秀丽隐杆线虫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，建立一种新的，多学科的方法来研究β -连环蛋白，目的如下：首次在体内测试钙粘蛋白C端关键磷酸化残基的作用。我们将使用生物化学和高分辨率显微镜在活胚胎中测试一个保守丝氨酸磷酸化的体内重要性，该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2。首次在体内测试了关键磷酸酪氨酸在β -连环蛋白粘附功能中的作用。我们将测试两种高度保守的酪氨酸在体内的重要性，一种调节cadherin/ β -catenin结合，另一种调节β -catenin/ β -catenin结合。我们还将利用亲和纯化和质谱技术评估内源性HMP-2的磷酸化状态，并鉴定一组新的HMP-2等位基因，这将提供关于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缺乏一个螺旋结构域（“Helix C”），仅在第12臂重复的C端，这在所有已知具有典型转录辅激活子功能的β -连环蛋白中是保守的。我们将通过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Tcf的体外结合和体内共激活剂的功能。

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.