Regulation of CARD11 by a kinesin-like protein, GAKIN

描述（由申请人提供）：免疫系统防御病原体的能力主要取决于T细胞识别外来抗原并作出适当反应的能力。当遇到抗原时，T细胞受体（TCR）触发细胞内信号通路，激活一组转录因子，诱导新的基因表达程序。通过TCR信号激活转录因子NF-kB是正常抗原诱导的T细胞增殖、存活和效应功能所必需的。CARD11是一种多结构域适配蛋白，是tcr介导NF-kB活化所必需的，但其作用机制尚不完全清楚。为了分离CARD11信号辅助因子，我们采用了一种新的表达克隆筛选方法来分离CARD11活性的调节因子。从这个筛选中，一种类似于激酶的蛋白GAKIN被鉴定为CARD11的抑制剂，初步研究表明GAKIN可以作为TCR信号传导到NF-kB的抑制剂。本研究的目的是研究GAKIN在这一重要信号通路中的作用。这些实验旨在验证GAKIN通过直接结合CARD11并将其从免疫突触转运到NF-kB抑制TCR信号传导的整体假设。这一假设将通过三个具体目标进行检验。首先，使用GAKIN缺陷和GAKIN过表达的T细胞系，将分析tcr - nf - kb通路中的几个步骤，以确定GAKIN起作用的步骤。其次，GAKIN抑制活性所需的结构域将使用RNA干扰拯救试验确定。第三，结合试验和T细胞：APC（抗原呈递细胞）成像试验将用于证明GAKIN是否直接结合CARD11并影响CARD11向免疫突触募集的动力学或程度。这些研究的结果应该扩大我们对适应性免疫反应中控制T细胞活化的分子机制的理解。此外，这项研究可能会深入了解在衰老的T细胞中观察到的信号失调，这可能会导致无效的免疫反应和对病原体的易感性增加。免疫细胞信号缺陷常见于老年人，这导致对病原体的反应受损。拟议的研究结果有可能促进对免疫细胞用来进行有效反应的分子机制的理解。该结果也可能为开发新的治疗方法提供一个靶点，这些治疗方法可以增强在衰老过程中失调的免疫反应。

DESCRIPTION (provided by applicant): The ability of the immune system to defend against pathogens critically depends on the ability of T cells to recognize foreign antigens and respond appropriately. Upon encountering antigen, the T cell receptor (TCR) triggers intracellular signaling pathways that activate of a set of transcription factors to induce new programs of gene expression. The activation of the transcription factor NF-kB by TCR signaling is required for normal antigen-induced T cell proliferation, survival and effector function. CARD11 is a multi-domain adapter protein that is required for TCR-mediated activation of NF-kB, but its mechanisms of action are incompletely understood. To isolate CARD11 signaling cofactors, a novel expression-cloning screen was conducted to isolate modulators of CARD11 activity. From this screen, a kinesin-like protein, GAKIN, was identified as an inhibitor of CARD11, and preliminary studies suggest that GAKIN functions as an inhibitor of TCR signaling to NF-kB. The goal of the proposed research is to investigate how GAKIN functions in this important signaling pathway. The experiments are designed to test the overall hypothesis that GAKIN inhibits TCR signaling to NF-kB by binding CARD11 directly and transporting it away from the immunological synapse. The hypothesis will be tested with three specific aims. First, using GAKIN-deficient and GAKIN-overexpressing T cell lines, several steps in the TCR-to-NF-kB pathway will be assayed to determine the step at which GAKIN acts. Second, the domains of GAKIN that are required for its inhibitory activity will be determined using an RNA interference-rescue assay. Third, binding assays and T cell:APC (antigen presenting cell) imaging assays will be used to demonstrate whether or not GAKIN binds CARD11directly and influences the kinetics or extent of CARD11 recruitment to the immunological synapse. The results of these studies should expand our understanding of the molecular machinery that controls T cell activation in the adaptive immune response. In addition, this research may offer insight into the dysregulated signaling that is observed in aging T cells, which can contribute to ineffective immune responses and increased susceptibility to pathogens. Immune cell signaling defects are commonly seen in aged humans and this results in impaired responses to pathogens. The results of the proposed research have the potential to advance the understanding of the molecular machinery that immune cells use to mount an effective response. The results may also provide a target for the development of novel therapeutics that could enhance immune responses that are dysregulated in aging.