Regulation of CARD11 by a kinesin-like protein, GAKIN

驱动蛋白样蛋白 GAKIN 对 CARD11 的调节

• 批准号: 7406429
• 负责人: REBECCA L LAMASON
• 金额: $ 3.46万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-10-01 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7406429
• 关键词: Active Sites; Aging; Antigen-Presenting Cells; Antigens; Appearance; Binding; Biological Assay; Cell Line; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Chromosome Pairing; Complex; Defect; Development; FHA Domain; Family member; Gene Expression; Glycine; Goals; Human; Image; Immune; Immune response; Immune system; Immunoprecipitation; Kinesin; Kinetics; Lead; Life; Link; Mammalian Cell; Mediating; Molecular; Motor; NF-kappa B; Pathway interactions; Phosphotransferases; Play; Point Mutation; Predisposition; Protein Kinase C; Protein Overexpression; Proteins; RNA Interference; Reagent; Receptor Signaling; Regulation; Research; Role; Signal Pathway; Signal Transduction; Signaling Protein; Synapses; System; T Cell Receptor Signaling Pathway; T-Cell Activation; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Testing; adapter protein; aged; cellular imaging; cofactor; design; expression cloning; immunological synapse; inhibitor/antagonist; insight; membrane-associated guanylate kinase; novel; novel therapeutics; pathogen; programs; research study; response; transcription factor

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.

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做出适当反应的能力。遇到抗原后，T 细胞受体 (TCR) 会触发细胞内信号传导通路，激活一组转录因子以诱导新的基因表达程序。 TCR 信号传导对转录因子 NF-kB 的激活是正常抗原诱导的 T 细胞增殖、存活和效应功能所必需的。 CARD11 是一种多结构域衔接蛋白，是 TCR 介导的 NF-kB 激活所必需的，但其作用机制尚不完全清楚。为了分离 CARD11 信号辅助因子，进行了一种新型表达克隆筛选来分离 CARD11 活性调节剂。从该筛选中，一种类似驱动蛋白的蛋白 GAKIN 被鉴定为 CARD11 的抑制剂，初步研究表明 GAKIN 可以作为 NF-kB 的 TCR 信号传导的抑制剂。本研究的目的是研究 GAKIN 在这一重要信号通路中的功能。这些实验旨在测试以下总体假设：GAKIN 通过直接结合 CARD11 并将其运离免疫突触来抑制 TCR 向 NF-kB 发出信号。该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 T 细胞系，对 TCR 至 NF-kB 途径中的几个步骤进行分析，以确定 GAKIN 起作用的步骤。其次，将使用 RNA 干扰救援测定来确定 GAKIN 抑制活性所需的结构域。第三，结合测定和T细胞：APC（抗原呈递细胞）成像测定将用于证明GAKIN是否直接结合CARD11并影响CARD11募集至免疫突触的动力学或程度。这些研究的结果应该会扩大我们对适应性免疫反应中控制 T 细胞激活的分子机制的理解。此外，这项研究可能有助于深入了解衰老 T 细胞中观察到的信号失调，这可能导致免疫反应无效并增加对病原体的易感性。免疫细胞信号传导缺陷在老年人中很常见，这会导致对病原体的反应受损。拟议研究的结果有可能促进对免疫细胞用来发起有效反应的分子机制的理解。这些结果还可能为开发新疗法提供一个目标，这些新疗法可以增强衰老过程中失调的免疫反应。