Alpha catenin phosphorylation in adhesion

描述（由申请人提供）：细胞-细胞凝聚力是多细胞生物体的定义特征，并且从癌症到心脏病的许多疾病状态是由将细胞彼此保持在一起的结构“维可牢尼龙搭扣”中的缺陷驱动的。广泛认为钙粘蛋白/连环蛋白粘附复合物是这种粘合剂“Velcro”的主要调节剂。该复合物包括介导Ca++依赖性嗜同性识别的跨膜“钙粘蛋白”组分，以及将钙粘蛋白连接到底层细胞骨架的许多相关“连环蛋白”。由于α-连环蛋白（？- cat）是钙粘蛋白/连环蛋白复合物的唯一肌动蛋白结合组分，而丝状（F）-肌动蛋白对于强细胞间粘附是关键的， ?- CAT长期以来被认为是钙粘蛋白粘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对于许多细胞类型的细胞间粘附是必需的，但对其发生的方式知之甚少。我们的实验室发现了一个高度保守的磷酸化结构域，cat，位于丝状肌动蛋白结合位点的N-末端。这些磷酸化也位于？- cat，它限制另一种肌动蛋白结合蛋白通过？-因此，本研究试图了解磷酸化是如何指导蛋白质的构象调节、肌动蛋白结合和动态细胞-细胞粘附功能的。cat使用体外和基于细胞的测定。为此，目的1试图表明如何在体外磷酸化？-酪蛋白激酶2（CK 2）在S641处的cat增强或“引发”了CK 1在残基S652、S655和T658处的磷酸化，导致更开放的构象，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将确定这些磷酸化的贡献，细胞粘附在体内，使用狗上皮细胞系，已取代内源性？具有GFP标记形式的猫，其含有、缺乏或组成型模拟磷酸化。这些目标共同解决了如何实现的根本问题。猫使用体外和基于细胞的方法在专门的粘附连接处与肌动蛋白相互作用，这将对不同细胞类型的细胞间粘附产生广泛影响，并且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相关。

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.