Motor protein regulation of T cell receptor signaling

T 细胞受体信号传导的运动蛋白调节

• 批准号: 7648345
• 负责人: Joel L Pomerantz
• 金额: $ 41万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-10 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7648345
• 关键词: Affect; Age; Antigen Receptors; Antigen-Presenting Cells; Antigens; Autoimmune Diseases; B-Lymphocytes; Biochemical; Biological Assay; Cell Line; Cell Proliferation; Cell membrane; Cell physiology; Cells; Complex; Cues; Development; Gene Expression; Growth; Human; Hyperactive behavior; Image; Immune; Immune System Diseases; Immune response; Immune system; Immunologic Deficiency Syndromes; Kinesin; Leukocytes; Lymphocyte; Mediating; Microtubule-Organizing Center; Microtubules; Molecular; Molecular Target; Motor; Mutation; NF-kappa B; Oncogenic; Pathway interactions; Pattern; Process; Proteins; RNA Interference; Reagent; Receptor Signaling; Receptors, Antigen, B-Cell; Regulation; Reporting; Resistance; Role; Set protein; Signal Pathway; Signal Transduction; Stimulus; Synapses; T Cell Receptor Signaling Pathway; T-Cell Receptor; T-Lymphocyte; Testing; Translating; adapter protein; aged; cancer type; cell type; cofactor; design; expression cloning; immunological synapse; large cell Diffuse non-Hodgkin' s lymphoma; lentiviral-mediated; mutant; novel; overexpression; pathogen; programs; public health relevance; receptor; research study; scaffold; therapy design; transcription factor

DESCRIPTION (provided by applicant): The engagement of the T cell receptor (TCR) on T lymphocytes initiates signaling pathways that regulate cell proliferation, activation, and survival through the activation of transcription factors that regulate programs of gene expression. TCR signaling is initiated when an antigen presenting cell (APC) presents antigen, in the appropriate molecular context, to a T cell. After APC:T cell conjugation, the immunological synapse forms in the region of cell:cell contact. Signaling from the synapse to transcription factors requires the recruitment of signaling intermediates to the immunological synapse in a process that is incompletely understood. CARD11 is a multi-domain adapter protein that coordinates the signal-induced association of a set of proteins that are required for TCR-mediated activation of NF-kappaB. In order to expand our understanding of how CARD11 relays signals from the TCR to NF-kappaB, we have developed a novel expression cloning strategy for the identification of modulators of CARD11 signaling activity. Using this strategy, we isolated the kinesin-like motor protein GAKIN as a negative regulator of CARD11. In our preliminary studies, we have determined that GAKIN overexpression inhibits CARD11 activity and TCR signaling while the reduction in GAKIN expression results in enhanced CARD11 and TCR signaling. GAKIN and CARD11 associate at endogenous levels in T cells in a signal-inducible manner. Imaging studies suggest that the cellular localization of GAKIN changes during TCR signaling, which may depend on GAKIN's ability as a motor protein to move cargo along microtubules. In this application, we propose to test our overall hypothesis that GAKIN is a critical negative regulator of TCR signaling that regulates the scaffolding function and cellular localization of CARD11. Using biochemical approaches, we will investigate which domains of GAKIN are required for its association with CARD11 and for its ability to negatively regulate TCR signaling. We will also determine whether GAKIN modulates the association of signaling cofactors with CARD11. In imaging experiments, we will characterize how the localization of GAKIN is determined and whether GAKIN regulates the recruitment of CARD11 and signaling factors to the immunological synapse. GAKIN associates with CARD11 in a region in which oncogenic mutations have been found in Diffuse Large B cell Lymphoma (DLBCL). We will investigate whether these mutations affect GAKIN-mediated regulation of CARD11 activity and determine whether GAKIN can inhibit the dysregulated growth in DLBCL. Our results should add to the understanding of how the molecular machinery of immune cells can recognize and interpret environmental cues, including pathogenic and nonpathogenic stimuli, and respond appropriately. Since this machinery is impaired in aged T cells, dysregulated in immunodeficiencies, and is abnormally hyperactive in autoimmune disease and in several types of cancer, our results may illuminate molecular targets for the development of new therapies designed to treat multiple diseases of the immune system. PUBLIC HEALTH RELEVANCE: The normal function of the immune system depends on the ability of white blood cells, or lymphocytes, to detect foreign pathogens and respond appropriately so that a pathogen is eliminated without damage to the host. This proposal is designed to expand understanding of the molecular machinery that is used by T lymphocytes in this process. Since this machinery is impaired in aged T cells, dysregulated in immunodeficiencies, and is abnormally hyperactive in autoimmune disease and in several types of cancer, our results may illuminate molecular targets for the development of new therapies designed to treat multiple diseases of the immune system.

描述（由申请人提供）：T细胞受体（TCR）在T淋巴细胞上的参与启动了信号传导途径，这些途径通过调节基因表达程序的转录因子的激活来调节细胞增殖，激活和存活。当抗原呈递细胞（APC）在适当的分子环境中呈现抗原时，TCR信号传导开始。 APC：T细胞结合后，在细胞区域中形成免疫突触：细胞接触。从突触到转录因子的信号传导需要在未完全理解的过程中募集信号传导中间体到免疫突触。 Card11是一种多域衔接子蛋白，它可以协调一组TCR介导的NF-kappab激活所需的一组蛋白的信号诱导的关联。为了扩展我们对Card11如何将信号从TCR转移到NF-Kappab的理解，我们开发了一种新颖的表达克隆策略来识别Card11信号活动的调节剂。使用这种策略，我们将驱动蛋白样的运动蛋白Gakin分离为Card11的负调节剂。在我们的初步研究中，我们确定Gakin的过表达抑制了Card11活性和TCR信号传导，而Gakin表达的降低会导致Card11和TCR信号的增强。 Gakin和Card11以信号诱导的方式在T细胞中的内源水平助理。成像研究表明，在TCR信号传导过程中，Gakin的细胞定位变化，这可能取决于Gakin作为运动蛋白沿微管移动货物的能力。在此应用中，我们建议测试我们的总体假设，即Gakin是TCR信号的关键负面调节剂，可调节Card11的脚手架功能和细胞定位。使用生化方法，我们将研究Gakin与Card11相关的哪些域以及其对TCR信号进行负调控的能力。我们还将确定Gakin是否调节信号辅因子与Card11的关联。在成像实验中，我们将表征Gakin的定位方式，以及Gakin是否调节了Card11的募集和免疫学突触的信号因子。 Gakin与Card11相关的区域，其中在弥漫性大B细胞淋巴瘤（DLBCL）中发现了致癌突变。我们将研究这些突变是否影响Gakin介导的Card11活性调节，并确定Gakin是否可以抑制DLBCL的失调生长。我们的结果应加深对免疫细胞的分子机制如何识别和解释环境线索，包括致病性和非致病性刺激，并做出适当反应。由于这种机械在老年T细胞中受损，在免疫缺陷中失调，并且在自身免疫性疾病和几种类型的癌症中异常活跃，因此我们的结果可能会阐明分子靶标，用于开发旨在治疗多种免疫系统疾病的新疗法。公共卫生相关性：免疫系统的正常功能取决于白细胞或淋巴细胞检测异物病原体并做出适当反应的能力，以便消除病原体而不会损害宿主。该建议旨在扩大对T淋巴细胞在此过程中使用的分子机械的理解。由于这种机械在老年T细胞中受损，在免疫缺陷中失调，并且在自身免疫性疾病和几种类型的癌症中异常活跃，因此我们的结果可能会阐明分子靶标，用于开发旨在治疗多种免疫系统疾病的新疗法。