Protein redistribution in TCR-directed NF-kB activation

描述（由申请人提供）：抗原刺激的T细胞分裂和效应功能的获得依赖于由T细胞受体（TCR）指导的信号转导，以及随后影响基因表达变化的许多转录因子的激活。NF-?的转录因子B族在这个过程中起着至关重要的作用。tcr调控的NF-？B激活途径将通过结合分子、生化和高度先进的成像技术进行研究。目的是建立1)蛋白-蛋白相互作用，2)亚细胞蛋白组织和3)胞质信号转导的生化修饰之间的机制关系。B通路。这一目标将通过执行三个具体目标来实现：确定信号中间体的局部蛋白-蛋白关联在tcr定向的NF- B激活中的机制意义。我们将检验tcr导向的NF-？B信号传导涉及特定蛋白质复合物在离散亚细胞结构域中的精心组装和拆卸，特定蛋白质复合物发挥关键的机制作用。目标2。在单个细胞中，研究抗原信号强度、信号转导复合物的组装和tcr导向的NF-？B信号转导通路。实验将旨在评估抗原信号通过TCR转化为“二元”结果的假设，即效应功能没有激活或完全激活。此外，这些功能结果依赖于单细胞水平上的信号转导决策事件。目标3。定义磷酸化和MALT1相互作用域在TCR介导的BcHO降解和激活中的作用。我们将进行实验来验证BcHO的激活和蛋白水解破坏是机制耦合的假设，并且这两个过程都受BcHO与MALT1的相互作用和BcHO的pkc依赖性磷酸化的调节。这些研究将有助于建立基础研究基础，从而可能导致开发新的免疫调节药物，这些药物将通过高度特异性抑制抗原受体介导的NF-？B信号级联。

DESCRIPTION (provided by applicant): Antigen-stimulated T cell division and acquisition of effector functions are dependent upon signal transduction directed by the T cell receptor (TCR), and the consequent activation of a number of transcription factors that affect changes in gene expression. Transcription factors of the NF-?B family are of central importance in this process. The TCR-regulated NF-?B activation pathway will be investigated through a combination of molecular, biochemical, and highly advanced imaging techniques. The goal is to establish mechanistic relationships between 1) protein-protein interactions, 2) subcellular protein organization and 3) biochemical modification of cytoplasmic signal transducers in the TCR-directed NF-?B pathway. This goal will be accomplished via the execution of three specific aims: Aim1. To determine the mechanistic significance of localized protein-protein associations of signaling intermediates in TCR-directed activation of NF-?B. We will test the hypothesis that TCR-directed NF-?B signaling involves the orchestrated assembly and disassembly of defined protein complexes into discrete subcellular domains, with specific protein complexes playing a critical mechanistic role. Aim 2. To investigate, in individual cells, the mechanistic relationship between antigen signal strength, assembly of signal transduction complexes, and successful activation of the TCR-directed NF-?B signal transduction pathway. Experiments will be directed towards the evaluation of the hypothesis that antigen signals through the TCR are converted into "binary" outcomes of either no activation or full activation of effector functions. Furthermore, these functional outcomes are dependent on signal transduction decision events made at the single-cell level. Aim 3. To define the roles of phosphorylation and the MALT1 interaction domain in the TCR- mediated degradation and activation of BcHO. We will perform experiments to test the hypothesis that activation and proteolytic destruction of BcHO are mechanistically coupled, and that both processes are regulated by BcHO interaction with MALT1 and by PKC-dependent phosphorylation of BcHO. These studies will help establish the basic research foundation that could lead to the development of novel immuno-modulatory drugs, which would function via highly specific inhibition of antigen-receptor mediated activation of the NF-?B signaling cascade.