Alpha catenin phosphorylation in adhesion

描述(申请人提供)：细胞-细胞凝聚力是多细胞生物体的一个决定性特征，从癌症到心脏病，许多疾病状态都是由将细胞相互连接的结构性“魔术贴”中的缺陷驱动的。人们普遍认为钙粘蛋白/连环蛋白黏附复合体是这种粘合剂的主要调节剂。这种复合体包括一个跨膜的“钙粘附素”成分，它介导了钙离子依赖的亲水性识别，以及一些相关的“连锁素”，它将钙粘附素连接到底层的细胞骨架上。由于α-连环蛋白(？-CAT)是钙粘连蛋白/连环蛋白复合体中唯一结合肌动蛋白的成分，而丝状(F)-肌动蛋白对细胞间的强烈黏附至关重要， ？-CAT一直被认为是钙粘附素黏附受体和肌动蛋白细胞骨架之间的关键连接蛋白。虽然功能丧失研究表明？-CAT在许多细胞类型的细胞间的黏附中是必不可少的，但其发生的方式却鲜为人知。我们的实验室在？-CAT中发现了一个高度保守的磷酸化结构域，它位于一个丝状肌动蛋白结合位点的N端。这些磷酸化也存在于？-CAT的机械敏感的自身抑制区域，该区域通过？-CAT的中心区限制另一种肌动蛋白结合蛋白的募集到钙粘蛋白/连环蛋白复合体。因此，这一建议试图通过体外和基于细胞的分析来理解磷酸化如何指导？-CAT的构象调节、肌动蛋白结合和动态细胞-细胞黏附功能。为此，目标1试图展示酪蛋白激酶2(CK2)如何在体外使S641位的？-CAT被酪蛋白激酶2(CK2)磷酸化，或为CK1在残基S652、S655和T658处的磷酸化提供“启动”，从而产生更开放的构象，有利于？-CAT与F-肌动蛋白和其他肌动蛋白结合蛋白的结合。目的2将利用一种狗上皮细胞系来确定这些磷酸化对细胞黏附的贡献，该细胞系已经用含有、缺乏或结构性模仿磷酸化的GFP标记形式取代了内源性？-CAT。总之，这些目标解决了一个基本的问题，即？-CAT如何使用体外和基于细胞的方法在特殊的黏附连接与肌动蛋白相互作用，这将对不同细胞类型的细胞间黏附产生广泛的影响，并可能与影响上皮屏障功能和整体组织完整性的病理生理条件有关。

DESCRIPTION (provided by applicant): Cell-cell cohesion is a defining feature of multicellular organisms, and a number of disease states, from cancer to heart disease, are driven by defects in the structural "Velcro" that holds cells to one another. It is widely viewed that the cadherin/catenin adhesion complex is a master regulator of this adhesive "Velcro". This complex comprises a transmembrane "cadherin" component that mediates Ca++-dependent homophilic recognition, and a number of associated "catenins" that link cadherins to the underlying cytoskeleton. Since alpha-catenin (?-cat) is the sole actin-binding component of the cadherin/catenin complex, and filamentous (F)- actin is critical for strong intercellular adhesion, ?-cat has long been viewed as a key linker protein between cadherin adhesion receptors and the actin cytoskeleton. While loss of function studies show that ?-cat is essential for cell-cell adhesion across many cell types, the means through which this occurs is poorly understood. Our lab has discovered a highly conserved phosphorylation domain in ?-cat that is situated just N- terminal to a filamentous actin-binding site. These phosphorylations also lie within a mechano-sensitive, auto-inhibitory region of ?-cat, which restricts recruitment of another actin-binding protein to the cadherin/catenin complex via the central region of ?-cat. This proposal, therefore, seeks to understand how phosphorylation directs the conformational regulation, actin-binding and dynamic cell-cell adhesive functions of ?-cat using both in vitro and cell-based assays. Towards this end, Aim 1 seeks to show how in vitro phosphorylation of ?-cat at S641 by casein kinase 2 (CK2) enhances or "primes" for phosphorylation by CK1 at residues S652, S655 and T658, resulting in a more open conformation that favors ?-cat binding to F-actin and other actin-binding proteins. Aim 2 will determine the contribution of these phosphorylations to cell adhesion in vivo, using a dog epithelial cell line that has replaced the endogenous ?-cat with GFP-tagged forms that contain, lack or constitutively mimic phosphorylation. Together, these aims address the fundamental question of how ?-cat interacts with actin at specialized adhesive junctions using both in vitro and cell-based approaches, which will have broad implications for intercellular adhesion across diverse cell types, and may be relevant to pathophysiological conditions that impact epithelial barrier function and overall tissue integrity.