Motor protein regulation of T cell receptor signaling

描述（由申请人提供）：T淋巴细胞上T细胞受体（TCR）的参与启动信号传导途径，通过激活调节基因表达程序的转录因子来调节细胞增殖、活化和存活。当抗原呈递细胞（APC）在适当的分子背景下将抗原呈递给T细胞时，TCR信号传导被启动。APC：T细胞结合后，免疫突触在细胞：细胞接触区域形成。从突触到转录因子的信号传导需要募集信号传导中间体到免疫突触，这一过程还不完全清楚。CARD 11是一种多结构域衔接蛋白，其协调TCR介导的NF-κ B活化所需的一组蛋白质的信号诱导的缔合。为了扩大我们对CARD 11如何将信号从TCR传递到NF-κ B的理解，我们开发了一种新的表达克隆策略来鉴定CARD 11信号活性的调节剂。使用这种策略，我们分离出驱动蛋白样运动蛋白GAKIN作为CARD 11的负调节因子。在我们的初步研究中，我们已经确定GAKIN过表达抑制CARD 11活性和TCR信号传导，而GAKIN表达减少导致CARD 11和TCR信号传导增强。GAKIN和CARD 11以信号诱导的方式在T细胞中以内源性水平缔合。影像学研究表明，GAKIN的细胞定位在TCR信号传导过程中发生变化，这可能取决于GAKIN作为马达蛋白沿着微管移动货物的能力。在本申请中，我们提出测试我们的总体假设，即GAKIN是TCR信号传导的关键负调节因子，其调节CARD 11的支架功能和细胞定位。使用生物化学方法，我们将研究GAKIN的哪些结构域是其与CARD 11的关联所必需的，以及其负调节TCR信号传导的能力。我们还将确定GAKIN是否调节信号辅因子与CARD 11的关联。在成像实验中，我们将描述GAKIN的定位是如何确定的，以及GAKIN是否调节CARD 11和信号传导因子向免疫突触的募集。GAKIN在弥漫性大B细胞淋巴瘤（DLBCL）中发现致癌突变的区域与CARD 11相关。我们将研究这些突变是否影响GAKIN介导的CARD 11活性调节，并确定GAKIN是否可以抑制DLBCL中的失调生长。我们的研究结果应该有助于理解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，并做出适当的反应。由于这种机制在老年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，我们的研究结果可能会阐明用于开发旨在治疗多种免疫系统疾病的新疗法的分子靶点。公共卫生相关性：免疫系统的正常功能取决于白色血细胞或淋巴细胞检测外来病原体并做出适当反应的能力，以便在不损害宿主的情况下消除病原体。该建议旨在扩大对T淋巴细胞在此过程中使用的分子机制的理解。由于这种机制在老年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，我们的研究结果可能会阐明用于开发旨在治疗多种免疫系统疾病的新疗法的分子靶点。

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.