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

描述(由申请人提供)：单个细胞黏附和结合成不同组织的能力是多细胞生物体的主要特征。细胞间的黏附在很大程度上是由一种蛋白质复合体介导的，这种复合体从细胞表面突出，形成一种结构性的“魔术贴”，将细胞相互连接在一起。这个复合体由一个跨膜的“钙粘附素”成分和相关的“连接素”组成，前者介导钙依赖的同型亲和性识别，后者将钙粘附素连接到底层细胞骨架。上皮(E)-钙粘附素是存在于上皮细胞的典型的“经典钙粘附素”。E-钙粘附素的减少或丢失在许多人类上皮性癌症中被观察到，并被认为是肿瘤转移的关键限速步骤。经典钙粘附素的细胞质结构域与具有双重功能的黏附/转录共激活蛋白β-catenin结合，后者又与F-肌动蛋白结合蛋白β-catenin结合，有效地将黏附与肌动蛋白细胞骨架偶联。十多年来，人们已经知道E-钙粘蛋白的细胞质尾部在？-连环蛋白结合区被强烈地磷酸化，这种磷酸化在体外增加了与-连环蛋白的亲和力。然而，E-钙粘蛋白在体内的功能和调节仍不清楚。我们发现，E-钙粘蛋白的磷酸化是有效结合β-连环蛋白所必需的，这表明E-钙粘蛋白磷酸化的调节引导了细胞-细胞黏附的变化。我们试图使用dsRNA果蝇细胞筛选和质谱仪(目标1)来确定使E-钙粘素细胞质尾部磷酸化的一个或多个激酶以及被磷酸化的特定氨基酸。在目标2中，我们试图确定E-钙粘附素的磷酸化和β-连环素结合对其转运到质膜和从质膜内吞的贡献。总之，这些目标将有助于理解E-钙粘蛋白磷酸化是如何调节的，并将定义其在细胞-细胞黏附中的作用，这两个问题与正常的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.