Regulation of NK Cell Function by Vav-family Proteins

说明(申请人提供)：Vav蛋白是鸟嘌呤核苷酸交换因子和分子接头，在淋巴细胞抗原受体的信号转导中发挥关键作用。最近的几项研究也表明Vav1参与了自然杀伤细胞(NK细胞)介导的肿瘤细胞毒作用。然而，Vav1是否影响了所有的NK细胞激活途径尚不清楚，可能还有其他Vav家族分子参与了NK细胞的触发。NK细胞通过具有不同结构、特异性和信号转接子的多种激活受体识别肿瘤或病毒感染的细胞。激活的NK细胞受体NKG2D识别主要在病毒感染和肿瘤细胞中高水平表达的内源性主要组织相容性复合体(MHC)I类相关分子，而Ly49H介导对M157的选择性识别，M157是一种表达在感染细胞上的小鼠巨细胞病毒(MCMV)编码的I类分子。NKG2D和Ly49H都提供刺激信号，触发干扰素-β的分泌。以及释放含有穿孔素和颗粒酶的细胞毒性颗粒。然而，目前还不清楚这种刺激信号是如何在NK细胞内传递的。NKG2D和Ly49D/H缺乏胞质信号元件，只能通过结合跨膜接头蛋白来传递刺激信号。Ly49D和Ly49H通过DAP12(也称为Karap)传递信号，DAP12包含基于免疫受体酪氨酸的激活基序(ITAM)，这些基序是磷酸化的，可以作为Syk和ZAP70蛋白酪氨酸激酶的对接位点。相比之下，NKG2D通过DAP10发出信号，DAP10是一种独特的适配器，包含一个YxNM基序，它招募磷脂酰肌醇3-激酶(PI3-K)和GRB-2。 作为这一提议的初步研究的一部分，我们培育了缺乏单个或全部VavFamily蛋白的小鼠，并开始检查这些蛋白对NK细胞细胞毒的潜在贡献。有趣的是，这些数据表明，Vav对DAP10是必需的，但对DAP12介导的自然细胞毒性不是。因此，这些数据提示了一种新的范式，即利用Vav激活与非ITAM-vs.含ITAM适配器相关的NK细胞表面受体下游的自然细胞毒性。在这里，我们建议使用几种体外和体内方法来确定Vav在NK细胞功能中的机制以及对病毒感染和肿瘤细胞的天然细胞毒作用的发展。

DESCRIPTION (provided by applicant): Vav proteins are guanine nucleotide exchange factors and molecular adaptors that play a key role in signal transduction of lymphocyte antigen receptors. Several recent studies have also implicated Vav1 in natural killer (NK) cell-mediated cytotoxicity of tumor cells. However, whether Vav1 effects all NK cell activating pathways is not known, and it is possible that other Vav family molecules are involved in NK cell triggering. NK cells recognize tumor or virally-infected cells through multiple activating receptors with diverse structures, specificities and signaling adaptors. The activating NK cell receptor NKG2D recognizes endogenous major histocompatibility complex (MHC) class I-related molecules expressed at high levels primarily in virally infected and tumor cells while Ly49H mediates selective recognition of m157, a murine cytomegalovirus (MCMV)-encoded class I-like molecule that is expressed on infected cells. Both NKG2D and Ly49H deliver stimulatory signals which trigger secretion of IFN-? and release of cytotoxic granules that contain perforin and granzymes. However, it is not know exactly how such stimulatory signals are transduced inside the NK cells. NKG2D and Ly49D/H lack cytoplasmic signaling elements and can deliver stimulatory signals only by associating with transmembrane adaptor proteins. Ly49D and Ly49H signal through DAP12 (also called KARAP) which contains immunoreceptor tyrosine-based activation motifs (ITAM) that are phosphorylated and function as docking sites for Syk and ZAP70 protein tyrosine kinases. In contrast, NKG2D signals through DAP10, a unique adapter that contains a YxNM motif which recruits phosphatidyl inositol 3-kinase (PI3-K) and Grb-2. As part of our preliminary studies for this proposal we generated mice lacking the individual, or all, Vavfamily proteins and began to examine the potential contribution of these proteins to NK cell cytotoxicity. Intriguingly, these data indicate that Vav is essential for DAP10- but not for DAP12-mediated natural cytotoxicity. Thus, these data suggest a new paradigm regarding the utilization of Vav in activation of natural cytotoxicity downstream of NK cell surface receptors associated with non-ITAM- vs. ITAM-containing adaptors. Here, we propose to use several in vitro and in vivo approaches to determine Vav mechanism in NK cell function and development of natural cytotoxicity against virally-infected and tumor cells.