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应答中起着重要作用。 这些细胞如何有效地区分病变和健康组织是尚未解决的关键问题之一 免疫学的问题。拟议的工作旨在确定小企业 衔接蛋白CT 10调节激酶（Crk）及其磷酸化，控制NK细胞的活化和抑制 通过使用人类NK细胞和新型NK细胞特异性条件性敲除小鼠。长期目标是 利用这些知识和本文开发的新成像技术来揭示NK的分子基础 细胞激活和抑制，并开发用于人类原发性免疫缺陷疾病的新疗法 以及慢性疾病如癌症和病毒感染。NK细胞通过极化释放杀伤靶细胞 通过一个称为免疫突触（IS）的细胞-细胞接触的专门区域。 通过先前的细胞毒性研究（Liu，D.例如，Immunity，2009，封面文章）和抑制性（Liu，D. 例如，Immunity，2012）IS，我们发现Crk在IS中起着重要的上游作用，影响 激活和抑制所需的信号事件。这种双重的分子机制 然而，作用仍然不明确。我们假设受体驱动的Crk磷酸化作为一种分子机制， 开关，驱动构象变化，这反过来又决定了Crk与关键分子相互作用的能力。 下游的信号分子，并最终形成肌动蛋白细胞骨架成为一个功能的IS。指导 强有力的初步数据，我们将通过三个具体目标来测试这些假设：1）定义精确的 Crk控制NK细胞活化和抑制的分子机制。拟议工作 利用最近单分子成像的爆炸性发展， 将影像技术引入NK研究领域，重点关注人类原发性免疫缺陷疾病; 2） 确定部分DiGeorge综合征患者NK细胞中Crk样蛋白（CrkL）的作用 （pDGS）。使用一种最常见的（每4,000名新生儿中有1名）免疫缺陷疾病，pDGS（主要由 CrkL单倍不足），我们将确定CrkL功能的丧失如何影响NK细胞介导的细胞毒性。 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型NK细胞特异性Crk敲除小鼠，我们将确定 Crk分子在体内NK细胞介导的对病毒感染和癌症的免疫应答中发挥的作用。的 拟议的工作涉及关键的信号球员和新的监管机制，并广泛相关， 对治疗原发性免疫缺陷疾病和癌症有直接的临床意义， Crk的升高的表达和增加的磷酸化是各种癌症所共有的（例如，肺癌， 乳腺癌、胶质母细胞瘤、肉瘤、卵巢癌和造血系统癌症）。拟议的工作还 产生了一个新的模型系统，以确定Crk的作用作为一个主调节分子。 !

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. !