Role of Src kinases in NK lytic function

描述(由申请人提供)：本提案的目标是了解导致自然杀伤(NK)细胞的裂解功能的膜近端事件。NK细胞是先天免疫中的关键角色，负责对细胞内细菌、病毒和肿瘤的早期防御。尽管在NK受体及其配体的识别方面取得了重大进展，但由这些相互作用触发的信号机制尚未完全解决。NK受体没有内源性的激酶活性，依赖接头来启动导致溶解功能的信号过程。接头本身没有激酶功能，但在DAP12、Zeta、FcepsilonRGamma的情况下，它带有一个基于免疫受体酪氨酸的激活基序(ITAM)，允许与Syk/Zap70结合。另一方面，DAP10有一个PI 3-激酶的结合位点。目前尚不清楚DAP 10和DAP12是如何被激活的。基于专注于DAP 12的初步数据，靶细胞参与激活NK细胞中的Fyn，显性阴性(DN)Fyn但不能阻断NK细胞中的抒情功能。此外，在该系统中激活的是Syk，而不是ZAP70。然而，在另一个利用DAP10的NK系统中，我们有提示数据表明LCK也可能参与其中。我们提出的假设是，NK细胞(即Lck、Fyn或Lyn)和Syk或ZAP70中存在的特定Src激酶可以被选择，这取决于NK受体/接头的组合。目的1明确DAP12的激活机制及其受不同NK受体的调控。将使用免疫沉淀/免疫印迹系统分析DAP12或NK受体上Src激酶的结合位置，以及受Src激酶磷酸化影响的DAP12上酪氨酸的鉴定。将采用GST融合蛋白、缺失突变体和定点突变。目的2分析Syk/ZAP70磷酸化的分子基础。在293细胞中共表达Src激酶突变结构DAP12、ZAP70和Syk将有助于确定所识别的Src激酶是直接磷酸化Syk/ZAP70还是需要DAP 12作为接头招募Syk/ZAP70进行后续磷酸化。目的3分析DAP10的激活机制以及NKG2D-S与DAP10和DAP12结合的双重能力。目的4探讨脂筏和细胞溶解突触在裂解信号级联反应中的作用。目的5分析LGL白血病患者NK细胞中的裂解信号通路，以确定已发现的每对NK受体/接头的信号通路是否具有普遍适用性。通过这些研究，我们将对不同类别的NK受体如何提供调节溶解功能的信号特异性有了重要的认识。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the membrane-proximal events that lead to lytic function in Natural Killer (NK) cells. NK cells are key players in innate immunity and are responsible for early defense against intracellular bacteria, viruses and tumors. Despite major developments in the identification of NK receptors and their ligands, the signal mechanisms triggered by these interactions have not been fully resolved. NK receptors have no intrinsic kinase activity and rely on adaptors to initiate the signal process leading to lytic function. The adaptor itself has no kinase function but, in the case of DAP12, zeta, FcepsilonRgamma, it bears an immunoreceptor tyrosine-based activatory motif (ITAM), which allows for binding to Syk/Zap70.On the other hand, DAP10 has a binding site for PI 3-kinase. It is unknown how DAP 10 and DAP12 become activated. Based on preliminary data focused on DAP 12, target cell engagement activates Fyn in NK cells and dominant-negative (DN) Fyn but not DN-Lck blocks lyric function in NK cells. In addition, Syk but not Zap70 is activated in this system. However, in another NK system that utilizes DAP10, we have suggestive data that Lck can also be involved. We propose the hypothesis that a specific Src kinase present in NK cells (i.e. Lck, Fyn, or Lyn) and Syk or Zap70 can be selected, depending on the NK receptor/adaptor combination. Aim 1 defines the mechanism of DAP12 activation and its control by different NK receptors. The binding site for Src kinases on DAP12 or the NK receptor, and the identification of the tyrosines on DAP12 subject to Src kinase phosphorylation will be analyzed using immunoprecipitation/western blotting systems. GST fusion proteins, deletion mutants, and site-directed mutagenesis will be employed. Aim 2 analyzes the molecular basis for Syk/Zap70 phosphorylation. Co-expression of Src kinase mutant constructs, DAP12, Zap 70, and Syk in 293 cells will help to identify if the identified Src kinase directly phosphorylates Syk/Zap70 or requires DAP 12 as an adaptor to recruit Syk/Zap70 for subsequent phosphorylation. Aim 3 analyzes the mechanisms of DAP 10 activation and of the dual ability of NKG2D-S to bind DAP10 and DAP12. Aim 4addresses the involvement of lipid rafts and cytolytic synapes in propagating the lytic signal cascade. Aim 5 analyzes lytic signal pathways in NK cells of LGL leukemic patients to determine if the signal pathways uncovered for each of the Nk receptor/adaptor pair are universally applicable. Through these studies, important insight will be gained into how the various classes of NK Receptors provide signal specificity that regulates lytic function.