Regulation of NK Cell Function by Vav-family Proteins

描述（由申请人提供）：Vav蛋白是鸟嘌呤核苷酸交换因子和分子衔接子，在淋巴细胞抗原受体的信号转导中起关键作用。最近的几项研究也表明Vav 1参与了自然杀伤（NK）细胞介导的肿瘤细胞的细胞毒性。然而，Vav 1是否影响所有NK细胞活化途径尚不清楚，其他Vav家族分子可能参与NK细胞触发。NK细胞通过具有不同结构、特异性和信号传导衔接子的多种活化受体识别肿瘤或病毒感染的细胞。活化NK细胞受体NKG 2D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Ly 49 H介导对m157（一种在感染细胞上表达的鼠巨细胞病毒（MCMV）编码的I类样分子）的选择性识别。NKG 2D和Ly 49 H都传递刺激信号，触发IFN-？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细胞内转导的。NKG 2D和Ly 49 D/H缺乏胞质信号传导元件，只能通过与跨膜衔接蛋白结合来传递刺激信号。Ly 49 D和Ly 49 H通过DAP 12（也称为KARAP）进行信号传导，DAP 12含有基于免疫受体酪氨酸的激活基序（ITAM），ITAM被磷酸化并作为Syk和ZAP 70蛋白酪氨酸激酶的对接位点发挥作用。相反，NKG 2D通过DAP 10发出信号，DAP 10是一种独特的衔接子，含有YxNM基序，可募集磷脂酰肌醇3-激酶（PI 3-K）和Grb-2。 作为我们对该提议的初步研究的一部分，我们产生了缺乏单个或全部Vav家族蛋白的小鼠，并开始检查这些蛋白对NK细胞细胞毒性的潜在贡献。有趣的是，这些数据表明，Vav是必不可少的DAP 10-但不是DAP 12-介导的天然细胞毒性。因此，这些数据表明了关于利用Vav激活与不含ITAM与含ITAM衔接子相关的NK细胞表面受体下游的天然细胞毒性的新范例。在这里，我们建议使用几种体外和体内方法来确定Vav机制在NK细胞功能和发展的自然细胞毒性对病毒感染和肿瘤细胞。

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.