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的稳定性和功能是必不可少的。 对于多层表皮尤其如此，因为它暴露在高水平的机械应力下 维持必要的身体和免疫屏障。对表皮维持能力的关键贡献者 同时调节压力的表皮屏障是角蛋白中间体之间的连接 细丝(IF)和细胞间连接称为桥粒。在桥粒中，一种细胞骨架连接蛋白 所谓的桥粒蛋白(DP)负责将IF锚定到连接处。DP无处不在地表达在 桥粒形成细胞，受其C末端尾部结构域的翻译后修饰(PTM)调控 控制DP-IF亲和力。与DP的这个磷酸调控区对桥粒的重要性一致 功能，C末端区域的基因缺失会导致与严重心脏疾病相关的多种疾病 皮肤和致命的表皮屏障缺陷。在功能上，当C-末端基序处于次要状态时- 在磷酸化状态下，DP对IF的亲和力最高。在实验模型中，增加的DP-IF 联合通过产生更强大、更稳定的桥粒在成熟的细胞膜中起到保护作用。 相反，在桥粒组装过程中，低磷酸化的DP在IF网络上积累，破坏 它运输到桥粒连接，干扰新细胞的形成和成熟 桥粒连接。考虑到与DP的低磷酸化形式相关的强烈分子表型 在体外，必须存在一个协调的过程来调节DP的磷酸化，以实现正确的桥粒功能。我们 以前发现Gsk3是负责磷酸化DP的激酶；然而，蛋白磷酸酶 负向调节DP磷酸化的作用以前是未知的。我的初步数据显示 PP2A-B55能够与DP的C末端结合并使其去磷酸化。这项提案将考验 PP2A-B55在桥粒动态过程中调节DP磷酸化的假说 组装并响应机械应力，以允许细胞对不断变化的属性做出反应，并 层状表皮的特殊功能。AIM 1将使用尖端显微镜和蛋白质组学- 基于确定PP2A在桥粒组装过程中调节DP的机制的方法。目标2 将确定PP2A对DP的磷酸化调节如何影响1)桥粒依赖的细胞黏附和2) 利用新的机械生物学技术对机械应力进行桥粒反应。拟议的工作将 对磷酸酶信号如何控制桥粒组装和功能提供了重要的见解 维持人类表皮屏障，因此，失调的磷酸酶信号转导如何起作用 与屏障相关的疾病。总之，拟议的研究和职业发展计划将提供一个 我作为一名独立调查员的发展和计划中的K99/R00申请的跳板。

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.