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）细胞溶解功能的膜近端事件。NK细胞是先天免疫的关键角色，负责细胞内细菌、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，但这些相互作用引发的信号机制尚未完全解决。NK受体没有内在的激酶活性，依赖于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，它带有一个基于酪氨酸的免疫受体激活基序（ITAM），允许与Syk/Zap70结合。另一方面，DAP10具有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DAP - 12的初步数据，靶细胞接合激活NK细胞中的Fyn和显性阴性（DN） Fyn，而DN- lck不阻断NK细胞的抒情功能。此外，在该系统中Syk而不是Zap70被激活。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。我们提出假设，NK细胞中存在特定的Src激酶（即Lck， Fyn或Lyn）和Syk或Zap70可以选择，这取决于NK受体/接头的组合。Aim 1定义了不同NK受体对DAP12激活及其调控的机制。Src激酶在DAP12或NK受体上的结合位点，以及Src激酶磷酸化的DAP12上酪氨酸的鉴定将使用免疫沉淀/western blotting系统进行分析。GST融合蛋白、缺失突变体和定点诱变将被采用。目的2分析Syk/Zap70磷酸化的分子基础。Src激酶突变体DAP12、Zap70和Syk在293细胞中的共表达将有助于确定所鉴定的Src激酶是直接磷酸化Syk/Zap70，还是需要DAP12作为接头来招募Syk/Zap70进行后续磷酸化。目的3分析DAP10的激活机制以及NKG2D-S结合DAP10和DAP12的双重能力。目的4探讨脂筏和细胞溶解突触在细胞溶解信号级联传播中的作用。目的5分析LGL白血病患者NK细胞的溶解性信号通路，以确定所发现的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，了解不同类别的NK受体如何提供调节裂解功能的信号特异性。

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.