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）细胞溶解功能的近膜事件。NK细胞是先天免疫的关键参与者，负责对细胞内细菌，病毒和肿瘤的早期防御。尽管在NK受体及其配体的鉴定方面取得了重大进展，但由这些相互作用引发的信号机制尚未完全解决。NK受体没有固有的激酶活性，并依赖于衔接子来启动导致裂解功能的信号过程。衔接子本身不具有激酶功能，但是在DAP 12、zeta、Fc ε Rgamma的情况下，它带有基于免疫受体酪氨酸的激活基序（ITAM），其允许结合Syk/Zap 70。另一方面，DAP 10具有PI 3-激酶的结合位点。目前尚不清楚DAP 10和DAP 12如何被激活。基于集中于DAP 12的初步数据，靶细胞接合激活NK细胞中的Fyn，并且显性阴性（DN）Fyn而非DN-Lck阻断NK细胞中的抒情功能。此外，Syk而不是Zap 70在该系统中被激活。然而，在另一个利用DAP 10的NK系统中，我们有提示性数据表明Lck也可能参与其中。我们提出的假设是，一个特定的Src激酶存在于NK细胞（即Lck，Fyn，或林恩）和Syk或Zap 70可以选择，这取决于NK受体/接头组合。目的1明确DAP 12的活化机制及其受不同NK受体的调控。将使用免疫沉淀/蛋白质印迹系统分析DAP 12或NK受体上Src激酶的结合位点，并鉴定DAP 12上发生Src激酶磷酸化的酪氨酸。将采用GST融合蛋白、缺失突变体和定点诱变。目的2分析Syk/Zap 70磷酸化的分子基础。Src激酶突变体构建体DAP 12、Zap 70和Syk在293细胞中的共表达将有助于鉴定的Src激酶是否直接磷酸化Syk/Zap 70或需要DAP 12作为衔接子以募集Syk/Zap 70用于随后的磷酸化。目的3分析DAP 10活化的机制和NKG 2D-S结合DAP 10和DAP 12的双重能力的机制。目的4阐明脂筏和溶细胞突触膜参与了细胞裂解信号级联反应的传播。目的5分析LGL白血病患者NK细胞中的裂解信号通路，以确定每个NK受体/接头对的信号通路是否普遍适用。通过这些研究，将获得重要的见解，了解不同类型的NK受体如何提供信号特异性，调节溶解功能。

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.