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: 8431361
• 负责人: Jeffrey D Hardin
• 金额: $ 21.04万
• 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 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8431361
• 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.

描述（由申请人提供）：cadherin-catenin复合物（CCC）对于在胚胎发育过程中成功完成形态发生至关重要，对于抑制分化性上皮的转移表型。 β-catenin是CCC的核心组成部分；因此，分析和操纵β-catenin的粘附功能对人类健康具有广泛的影响。迄今为止，区分β-catenin的粘附和TCF/LEF依赖性转录共激活函数，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具有挑战性。 秀丽隐杆线虫为分析β-catenin的粘附特定作用提供了独特的机会，因为它具有多种专门的β-蛋白蛋白。 我们将利用这种独特的专业化，再加上从协作X射线晶体学项目中得出的详细结构信息的强大基础，以建立一种新的多学科方法来研究β-catenins在以下目的中：AIM 1。测试cadherin of Cadherin for First The Vivo的cadherin of Cadherin在Vivo中的关键磷酸化残留物的作用。 我们将使用生物化学和高分辨率显微镜在生物胚胎中测试保守丝氨酸的磷酸化体内磷酸化的体内磷酸化，从而在体外上调了几百倍的β-catenin的亲和力。 AIM 2。在体内首次测试关键磷酸酪氨酸在β-catenin的粘附功能中的作用。 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-catenin结合，另一种调节β-catenin/beta-catenin结合。我们还将使用亲和力纯化和质谱法评估内源性HMP-2的磷酸化状态，并识别一系列新的HMP-2等位基因，该小组将提供有关氨基酸的信息，这些信息对于β-catenin的粘附功能很重要。 AIM 3。测试保守的螺旋结构域在β-catenin中的作用，其作为转录共激活剂的活性。 HMP-2缺少仅在第12臂重复的C末端的螺旋结构域（“ Helix c”），该重复均在所有已知具有规范转录共激活函数的β-catenins中保守。 BAR-1与高亲和力结合POP-1/TCF，并保留HelixC。我们将使用HMP-2和BAR-1之间的域S-s-swap实验测试螺旋C的作用，并评估体内体外和共激活器的TCF结合功能。