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

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的主要特征。细胞之间的粘附主要是由一种蛋白质复合物介导的，这种蛋白质复合物从细胞表面伸出，形成一种结构上的“尼龙搭扣”，将细胞彼此固定在一起。该复合物由一种跨膜“钙粘蛋白”成分组成，该成分介导ca2 +依赖的亲同性识别，以及将钙粘蛋白与潜在的细胞骨架连接起来的相关“连环蛋白”。上皮(E)-钙粘蛋白是存在于上皮上的典型“经典钙粘蛋白”。e-钙粘蛋白的减少或缺失已经在许多人类上皮癌中被观察到，并且被认为是肿瘤转移的关键限速步骤。经典钙粘蛋白的细胞质结构域结合具有双重功能的粘附/转录共激活蛋白。-连环蛋白，它又与f -肌动蛋白结合，-catenin，有效偶联粘连肌动蛋白细胞骨架。十多年来，人们已经知道e -钙粘蛋白的细胞质尾部在？-连环蛋白结合区域磷酸化增加了？-catenin。然而，e -钙粘蛋白磷酸化在体内的功能和调控仍不明确。我们发现e -钙粘蛋白磷酸化是有效结合？这表明E-cadherin磷酸化的调节指导了细胞-细胞粘附的变化。我们试图用dsRNA果蝇细胞筛选和质谱法确定磷酸化e -钙粘蛋白细胞质尾部的激酶（或激酶）和被磷酸化的特定氨基酸（Aim 1）。在Aim 2中，我们试图确定e -钙粘蛋白磷酸化和？-连环蛋白结合在细胞膜上的运输和胞吞作用。总之，这些目标将导致理解e -钙粘蛋白磷酸化是如何调节的，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。

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, ?-catenin, which in turn binds the F-actin binding protein, ?-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 ?-catenin binding region and that this phosphorylation increases the affinity for ?-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 ?-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.