Motor protein regulation of T cell receptor signaling

描述(由申请人提供)：T淋巴细胞上的T细胞受体(TCR)启动信号通路，通过激活调节基因表达程序的转录因子来调节细胞的增殖、激活和存活。当抗原提呈细胞(APC)在适当的分子环境中将抗原递呈给T细胞时，TCR信号被启动。APC：T细胞结合后，在细胞-细胞接触区域形成免疫突触。从突触到转录因子的信号传递需要在一个尚不完全了解的过程中向免疫突触招募信号中间体。CARD11是一种多结构域适配蛋白，它协调TCR介导的NF-kappaB激活所需的一系列蛋白的信号诱导关联。为了扩大我们对CARD11如何将信号从TCR传递到NF-kappaB的理解，我们开发了一种新的表达克隆策略来鉴定CARD11信号活性的调节子。利用这一策略，我们分离出了作为CARD11负调控因子的激动素样马达蛋白Gakin。在我们的初步研究中，我们已经确定Gakin过表达抑制CARD11活性和TCR信号，而Gakin表达降低导致CARD11和TCR信号增强。Gakin和CARD11在T细胞的内源性水平上以信号诱导的方式联系在一起。影像研究表明，Gakin的细胞定位在TCR信号传递过程中发生了变化，这可能取决于Gakin作为一种运动蛋白沿微管移动货物的能力。在这个应用中，我们建议检验我们的总体假设，即Gakin是TCR信号的关键负调节因子，调节CARD11的支架功能和细胞定位。使用生化方法，我们将调查Gakin的哪些结构域是它与CARD11关联所必需的，以及它对TCR信号的负调控能力所必需的。我们还将确定Gakin是否调节信号辅助因子与CARD11的关联。在成像实验中，我们将表征Gakin的定位是如何确定的，以及Gakin是否调节CARD11的招募和免疫突触的信号因子。在弥漫性大B细胞淋巴瘤(DLBCL)中发现致癌突变的区域，Gakin与CARD11相关。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。我们的结果应该有助于理解免疫细胞的分子机制如何识别和解释环境提示，包括致病和非致病刺激，并做出适当的反应。由于这种机制在衰老的T细胞中受损，在免疫缺陷中调节失调，在自身免疫性疾病和几种类型的癌症中异常过度活跃，我们的结果可能为设计用于治疗多种免疫系统疾病的新疗法的开发提供分子靶点。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外来病原体并做出适当反应的能力，从而在不损害宿主的情况下消除病原体。这项建议旨在扩大对T淋巴细胞在这一过程中使用的分子机制的理解。由于这种机制在衰老的T细胞中受损，在免疫缺陷中调节失调，在自身免疫性疾病和几种类型的癌症中异常过度活跃，我们的结果可能为设计用于治疗多种免疫系统疾病的新疗法的开发提供分子靶点。

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.