The adaptor protein Crk in immune responses

项目摘要 自然杀伤(NK)细胞在人类对感染和恶性肿瘤的免疫反应中发挥着重要作用。 这些细胞如何有效地区分疾病组织和健康组织是尚未解决的关键问题之一 当今免疫学中的问题。拟议的工作试图确定(S)通过什么机制，小的 调控NK细胞活化和抑制的调节蛋白CT10蛋白及其磷酸化 通过使用人类NK细胞和新的NK细胞特异性条件性基因敲除小鼠。长期目标是 利用这些知识和在此开发的新的成像技术来揭示NK的分子基础 细胞的激活和抑制，以及开发治疗人类原发免疫缺陷疾病的新方法 以及癌症和病毒感染等慢性病。NK细胞通过极化释放杀伤靶细胞 裂解颗粒通过细胞-细胞接触的特殊区域，称为免疫突触(IS)。 通过以前对细胞毒性(Liu，D.等，免疫，2009，封面文章)和抑制性(Liu，D. 免疫等人，2012)，我们发现，Crk在IS中扮演着重要的上游角色，影响 激活和抑制所需的信号事件。这一双重现象背后的分子机制 然而，角色仍不清楚。我们假设受体驱动的Crk的磷酸化作为一种分子 开关，驱动构象变化，这反过来决定了CRK与关键分子相互作用的能力 下游的信号分子，并最终将肌动蛋白细胞骨架塑造成一个功能性的IS。指导原则 强大的初步数据，我们将通过三个具体目标来检验这些假设：1)定义准确的 Crk控制NK细胞激活和抑制的分子机制。拟议中的工作 利用最近单分子成像的爆炸性进展，将带来尖端的单分子 将影像技术引入NK研究领域，重点研究人类原发免疫缺陷疾病；2) 部分DiGeorge综合征患者NK细胞Crk样蛋白(CrkL)的作用 (Pdgs)。使用最常见的免疫缺陷疾病之一(每4,000名新生儿中就有一例)pDGS(主要由 ，我们将确定CrkL功能丧失如何影响NK细胞介导的细胞毒作用。 PDGS及其分子机制；3)确定体内NK功能是否需要Crk。 利用我们已经产生的新的NK细胞特异性Crk基因敲除小鼠，我们将确定 Crk分子在体内NK细胞介导的病毒感染和癌症免疫反应中的作用这个 拟议的工作涉及关键的信号参与者和新颖的监管机制，并与 治疗原发免疫缺陷疾病和癌症的直接临床意义，因为 Crk的高表达和高磷酸化在各种癌症(例如，肺癌， 乳腺癌、胶质母细胞瘤、肉瘤、卵巢癌和造血癌)。拟议的工作还包括 产生了一个新的模型系统，在其中确定Crk作为主调节分子的角色。 好了！

Project Summary Natural killer (NK) cells play an important role in the human immune response to infection and malignancy. How these cells effectively distinguish between diseased and healthy tissue is one of the key unsolved problems in immunology today. The proposed work seeks to identify the mechanism(s) by which the small adaptor protein CT10 regulator of kinase (Crk), and its phosphorylation, control NK cell activation and inhibition by using both human NK cells and novel NK cell-specific conditional knockout mice. The long-term goal is to use this knowledge and the novel imaging techniques developed herein to uncover the molecular basis of NK cell activation and inhibition, and to develop new treatments for human primary immunodeficiency diseases and chronic diseases such as cancer and viral infection. NK cells kill target cells through the polarized release of lytic granules through a specialized region of cell-cell contact known as the immunological synapse (IS). Through previous studies of the cytotoxic (Liu, D. et al., Immunity, 2009, Cover Article) and inhibitory (Liu, D. et al., Immunity, 2012) IS, we discovered that Crk plays an essential upstream role at the IS, influencing signaling events required for both activation and inhibition. The molecular mechanisms underlying this dual role, however, remain unclear. We hypothesize that receptor-driven phosphorylation of Crk acts as a molecular switch, driving a conformational change, which in turn determines Crk's ability to interact with critical downstream signaling molecules and ultimately shapes the actin cytoskeleton into a functional IS. Guided by strong preliminary data, we will test these hypotheses via three Specific Aims: 1) Define the precise molecular mechanisms by which Crk controls NK cell activation and inhibition. The proposed work capitalizes on the recent explosion in single molecule imaging and will bring cutting-edge single molecule imaging technology to the field of NK research, with a focus on human primary immunodeficiency diseases; 2) Determine the role of Crk-like protein (CrkL) in NK cells from patients with partial DiGeorge syndrome (pDGS). Using one of most common (1 in 4,000 births) immunodeficiency diseases, pDGS (mainly caused by CrkL haploinsufficiency), we will determine how loss of CrkL function affects NK cell-mediated cytotoxicity in pDGS and its molecular mechanisms; 3) Determine whether Crk is required for NK function in vivo. Leveraging novel NK cell-specific Crk knockout mice that we have already generated, we will determine the roles that Crk molecules play in NK cell-mediated immune responses to viral infection and cancer in vivo. The proposed work involves key signaling players and novel regulatory mechanisms and is broadly relevant with direct clinical implications for the treatment of primary immunodeficiency diseases and cancer because elevated expression and increased phosphorylation of Crk are common to various cancers (e.g., lung cancer, breast cancer, glioblastoma, sarcomas, ovarian cancer, and hematopoietic cancers). The proposed work also generates a novel model system in which to determine Crk's role as a master regulatory molecule. !