Phosphatase-dependent regulation of desmosome intercellular junctions

项目摘要 细胞间连接及其细胞骨架连接对于维持组织稳定性和功能至关重要。 这对于多层表皮尤其如此，因为其暴露于高水平的机械应力，同时 维持基本的身体和免疫屏障。表皮细胞维持 表皮屏障在同时调节应力时是角蛋白中间体之间附着物 纤维（IF）和称为桥粒的细胞间连接。在桥粒内，一种细胞骨架连接蛋白 称为桥粒斑蛋白（DP）的蛋白负责将IF锚定到连接处。DP普遍表达于所有 桥粒形成细胞，并通过其C-末端尾部结构域的翻译后修饰（PTM）进行调节 以控制DP-IF亲和力。这与DP的磷酸化调节区对桥粒的重要性一致 功能，C-末端区域的遗传缺失导致与严重心脏病相关的多种疾病， 皮肤和致命的表皮屏障缺陷。在功能上，当C-末端基序处于其低- 在磷酸化状态下，DP对IF表现出最高的亲和力。在实验模型中，增加的DP-IF 缔合通过产生更强、更稳定的桥粒在成熟细胞片层中具有保护作用。 相反，在桥粒组装过程中，低磷酸化DP在IF网络上积累，破坏了 它运输到桥粒连接处，干扰新细胞-细胞的形成和成熟， 桥粒连接鉴于DP的低磷酸化形式具有很强的分子表型， 在体外，必须存在一个协调的过程来调节DP磷酸化以实现适当的桥粒功能。我们 先前鉴定GSK 3 β是负责磷酸化DP的激酶;然而， 负责负调节DP磷酸化的基因是以前未知的。我的初步数据显示 PP 2A-B55激酶能够结合DP的C-末端并使其去磷酸化。这项提案将考验 PP 2A-B55 β在桥粒动态过程中调节DP磷酸化的假说 组装和响应机械应力，以允许细胞响应变化的性质， 复层表皮的特殊功能。Aim 1将采用尖端的显微镜和蛋白质组学- 的方法，以确定机制，PP 2A调节DP桥粒组装。目的2 将确定DP的PP 2A磷酸化调节如何影响1）桥粒依赖性细胞粘附和2） 桥粒对机械应力的反应。拟议的工作将 提供了重要的洞察磷酸酶信号如何控制桥粒组装和功能， 维持人类表皮屏障，因此，磷酸酶信号失调如何有助于 to barrier屏障related相关diseases疾病.拟议的研究和职业发展计划将共同提供一个 作为一名独立调查员和计划K99/R 00应用程序的发展跳板。

Project Summary Intercellular junctions and their cytoskeletal connections are essential for maintaining tissue stability and function. This is particularly true of the multi-layered epidermis as it is exposed to high levels of mechanical stress while maintaining an essential physical and immune barrier. Critical contributors to the epidermis' ability to maintain the epidermal barrier while simultaneously adjusting to stress are attachments between keratin intermediate filaments (IF) and intercellular junctions called desmosomes. Within desmosomes, a cytoskeletal linker protein called desmoplakin (DP) is responsible for anchoring the IF to the junctions. DP is ubiquitously expressed in all desmosome-forming cells and is regulated by post-translational modification (PTM) of its C-terminal tail domain to control DP-IF affinity. Consistent with the importance of this phospho-regulatory region of DP for desmosome function, genetic deletions of the C-terminal region cause multiple diseases associated with severe cardio- cutaneous and lethal epidermal barrier defects. Functionally, when the C-terminal motif is in its hypo- phosphorylated state, DP exhibits its highest affinity towards IF. In experimental models, the increased DP-IF association had protective effects in mature cell sheets by generating stronger, more stable desmosomes. Conversely, during desmosome assembly hypo-phosphorylated DP accumulates on the IF network, disrupting it's trafficking to desmosome junctions, and interfering with the formation and maturation of new cell-cell desmosome junctions. Given the strong molecular phenotypes associated with DP's hypo-phosphorylated form in vitro, a coordinated process must exist to regulate DP phosphorylation for proper desmosome function. We previously identified GSK3 as the kinase responsible for phosphorylating DP; however, the protein phosphatase responsible for negatively regulating DP phosphorylation was previously unknown. My preliminary data identified PP2A-B55 as capable of binding to and dephosphorylating DP's C-terminus. This proposal will test the hypothesis that PP2A-B55 regulates DP phosphorylation during the dynamic process of desmosome assembly and in response to mechanical stress to allow cells to respond to the changing properties and specialized functions of the stratified epidermis. Aim 1 will employ cutting-edge microscopy and proteomics- based approaches to identify the mechanism by which PP2A regulates DP during desmosome assembly. Aim 2 will establish how PP2A phospho-regulation of DP impacts 1) desmosome-dependent cell adhesion and 2) the desmosomal response to mechanical stress using novel mechano-biology techniques. The proposed work will provide important insight into how phosphatase signaling controls desmosome assembly and function to maintain the human epidermal barrier and, therefore, how dysregulated phosphatase signaling could contribute to barrier-related diseases. Together, the proposed research and career development plan will provide a springboard for my development as an independent investigator and planned K99/R00 application.