Protein redistribution in TCR-directed NF-kB activation

描述（由申请人提供）：抗原刺激的T细胞分裂和效应子功能的获得依赖于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。转录因子NF-？B家庭在这一过程中至关重要。TCR调节的NF-？B活化途径将通过分子、生物化学和高度先进的成像技术的组合来研究。我们的目标是建立1）蛋白质-蛋白质相互作用，2）亚细胞蛋白质组织和3）细胞质信号转导的生化修饰之间的机械关系，在TCR导向的NF-？B途径。这一目标将通过执行三个具体目标来实现：目标1。为了确定局部蛋白质-蛋白质协会的信号中间体在TCR介导的NF-？B。我们将测试的假设，TCR导向NF-？B信号传导涉及到特定蛋白质复合物到离散的亚细胞结构域中的协调组装和拆卸，其中特定蛋白质复合物起关键的机械作用。目标2.调查，在单个细胞中，抗原信号强度，组装的信号转导复合物，和成功激活的TCR导向的NF-？B信号转导通路。实验将针对以下假设的评估：通过TCR的抗原信号被转化为效应子功能的无激活或完全激活的“二元”结果。此外，这些功能结果依赖于在单细胞水平上进行的信号转导决策事件。目标3：明确磷酸化和MALT 1相互作用结构域在TCR介导的BcHO降解和活化中的作用。我们将进行实验，以测试的假设，激活和蛋白水解破坏BcHO的机械耦合，这两个过程是由BcHO与MALT 1的相互作用和PKC依赖的磷酸化BcHO调节。这些研究将有助于建立基础研究基础，可能导致新的免疫调节药物的发展，这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.