The role of E-cadherin phosphorylation in regulating cell-cell adhesion

E-钙粘蛋白磷酸化在调节细胞-细胞粘附中的作用

• 批准号: 8712102
• 负责人: Abbye Elizabeth McEwen
• 金额: $ 4.77万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8712102
• 关键词: Actins; Adhesions; Adhesives; Affinity; Amino Acids; Binding; CSNK1A1 gene; Cadherins; Cell Adhesion Molecules; Cell membrane; Cell surface; Cell-Cell Adhesion; Cells; Complex; Coupling; Cytoplasmic Tail; Cytoskeleton; Development; Diagnostic; Dissociation; Double-Stranded RNA; Drosophila genus; E-Cadherin; Electron Transport; Electrophoretic Mobility Shift Assay; Endocytosis; Epithelial; Epithelial Cells; Epithelium; Exhibits; F-actin-binding proteins; Felis catus; Goals; Health; Human; In Vitro; Individual; Kinetics; Knowledge; Lead; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Molecular; Mutagenesis; Neoplasm Metastasis; Organism; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Phosphotransferases; Proteins; Regulation; Role; Testing; Tissues; in vivo; mutant; protein E; protein complex; research study; tandem mass spectrometry; tool; trafficking; tumor

DESCRIPTION (provided by applicant): The ability of individual cells to adhere and coalesce into distinct tissues is a major feature of multicellular organisms. Cell-cell adhesion is largely mediated by a protein complex that projects from the cell surface to form a structural "Velcro" that holds cells to one another. This complex is comprised of a transmembrane "cadherin" component that mediates Ca++-dependent homophilic recognition, and associated "catenins" that link cadherins to the underlying cytoskeleton. Epithelial (E)-cadherin is the prototypic "classical cadherin" present on epithelia. Reduction or loss of E-cadherin has been observed in numerous human epithelial cancers and is considered a key rate-limiting step in tumor metastasis. The cytoplasmic domain of classical cadherins binds the dual function adhesion/transcriptional co-activator protein, β-catenin, which in turn binds the F-actin binding protein, α-catenin, effectively coupling adhesion to the actin cytoskeleton. It has been known for over a decade that cytoplasmic tail of E-cadherin is robustly phosphorylated in the β-catenin binding region and that this phosphorylation increases the affinity for α-catenin in vitro. However, the function and regulation of E-cadherin phosphorylation in vivo remain poorly defined. We find that E-cadherin phosphorylation is required for effective binding to β-catenin binding in vivo, suggesting the hypothesis that modulation of E-cadherin phosphorylation directs changes in cell-cell adhesion. We seek to determine the kinase (or kinases) that phosphorylates the cytoplasmic tail of E-cadherin and the specific amino acids that are phosphorylated using a dsRNA Drosophila cell screen and mass spectrometry (Aim 1). In Aim 2 we seek to determine the contribution of E-cadherin phosphorylation and ?-catenin binding on its trafficking to and endocytosis from the plasma membrane. Altogether, these aims will lead to an understanding of how E-cadherin phosphorylation is regulated and will define its role in cell-cell adhesion, two questions that are broadly relevant to both normal epithelial integrity and tumor metastasis.

描述（由申请人提供）：单个细胞粘附和合并成不同组织的能力是多细胞生物体的主要特征。细胞-细胞粘附主要由蛋白质复合物介导，该蛋白质复合物从细胞表面突出以形成将细胞彼此保持的结构“维可牢尼龙搭扣”。该复合物由介导Ca++依赖性嗜同性识别的跨膜“钙粘蛋白”组分和将钙粘蛋白连接到底层细胞骨架的相关“连环蛋白”组成。上皮（E）-钙粘蛋白是存在于上皮上的原型“经典钙粘蛋白”。在许多人上皮癌中观察到E-钙粘蛋白的减少或丢失，并认为其是肿瘤转移的关键限速步骤。经典钙粘蛋白的胞质结构域结合双重功能粘附/转录共激活蛋白β-连环蛋白，其进而结合F-肌动蛋白结合蛋白α-连环蛋白，有效地将粘附偶联至肌动蛋白细胞骨架。十多年来，人们已经知道E-钙粘蛋白的胞质尾区在β-连环蛋白结合区被强烈磷酸化，并且这种磷酸化增加了体外对α-连环蛋白的亲和力。然而，体内E-钙粘蛋白磷酸化的功能和调节仍然不清楚。我们发现E-钙粘蛋白磷酸化是体内有效结合β-连环蛋白所必需的，这表明了调节E-钙粘蛋白磷酸化指导细胞-细胞粘附变化的假设。我们试图确定激酶（或激酶），磷酸化的细胞质尾的E-钙粘蛋白和特定的氨基酸磷酸化使用dsRNA果蝇细胞筛选和质谱（目的1）。在目的2中，我们试图确定E-钙粘蛋白磷酸化和？连环蛋白结合其运输到质膜和从质膜内吞。总而言之，这些目标将导致理解如何调节E-钙粘蛋白磷酸化，并将定义其在细胞-细胞粘附中的作用，这两个问题与正常上皮完整性和肿瘤转移广泛相关。