MAPK AND LYTIC FUNCTION IN HUMAN NK CELLS

细胞毒性是自然杀伤细胞（NK）和T细胞杀伤肿瘤细胞的关键策略，但其分子基础尚不清楚。信号分子分析表明，丝裂原活化蛋白激酶(MAPK)/ERK2对NK的直接裂解起关键控制作用。人NK细胞的肿瘤结扎诱导MAPK酪氨酸磷酸化和酶活性。此外，通过药物抑制PD098059或ERK2显性阴性表达使MAPK失活，干扰了51-Cr标记的肿瘤细胞的NK裂解，并中断了穿孔素/颗粒酶B的极化。因此，确定控制MAPK激活的信号分子是很重要的，一旦确定了MAPK，就有可能在癌症患者中找到增强这一途径的方法。初步研究表明NK利用一个不依赖ras的MAPK级联来裂解肿瘤细胞。法尼基转移酶抑制剂FTI-277或N17ras显性阴性表达对肿瘤细胞NK溶解、穿孔素/颗粒酶B动员或MAPK激活没有有害影响。相反，肿瘤细胞在NK细胞中将ras相关蛋白rap1A激活为GTP形式。GGTI-298阻断了rap1A用于膜定位的geranyation过程，有效抑制了NK裂解能力和rap1A的激活。本研究旨在验证rap1A是MAPK上游效应因子的假设，目的1是确定rap1A/B-raf/MEK/MAPK通路是否对裂解过程起关键控制作用。另外，其他途径也可能输入MAPK以表示重叠或单独的功能。目的2是分析PI 3-激酶和Syk70在MAPK激活中的作用，目的3是解决抑制NK受体（如KIR）通过MAPK灭活功能的假设。目的4是确定MAPK是否通过调控微管重组来控制穿孔蛋白的运动。实验计划遵循深入的生化和遗传学方法，使用药理学试剂和突变基因构建，以确定MAPK激活的特定作用。鉴定一种独特的ras独立NK溶解过程可以提供一种新的治疗策略，专门针对患者的ras转化肿瘤而不是NK功能。

Cytotoxicity is a key strategy utilized by natural killer (NK) and T cells to kill tumor cells, but its molecular basis is unclear. Analysis of signal molecules indicated that mitogen-activated protein kinases (MAPK)/ERK2 critically controlled direct NK lysis. Tumor ligation in human NK cells induced MAPK tyrosine phosphorylation and enzymatic activity. Moreover, MAPK inactivation by pharmacological inhibition with PD098059 or dominant-negative ERK2 expression interfered with NK lysis of 51-Cr labeled tumor cells, and interrupted perforin/granzyme B polarization. It is thus important to define what signal molecules control MAPK activation, with the goal of possibly finding means to enhance this pathway in cancer patients, once it is identified. Preliminary studies indicated that NK utilized a ras-independent MAPK cascade to lyse tumor cells. Farnesyl transferase inhibitor, FTI-277, or dominant-negative N17ras expression, had no deleterious effect on NK lysis of tumor cells, perforin/granzyme B mobilization, or MAPK activation. Instead, tumor cells activate a ras related protein, rap1A, to its GTP form, in NK cells. GGTI-298, which blocks geranylation, a process used by rap1A for membrane localization, effectively inhibited NK lytic capacity and rap1A activation. This proposal is to test the hypothesis that rap1A is an upstream effector of MAPK, and Aim 1 is to identify if the rap1A/B-raf/MEK/MAPK pathway critically controls the lytic process. Alternatively, other pathways may feed into MAPK to represent overlapping or separate functions. Aim 2 is analyze the roles of PI 3-kinase and Syk70 in MAPK activation and Aim 3 addresses the hypothesis that inhibitory NK receptors, such as KIR, inactivate function via MAPK. Aim 4 is to identify if MAPK control of perforin movement is via regulation of microtubule reorganization. The experimental plan follows an in-depth biochemical and genetic approach, using pharmacological agents and mutant gene constructs, to identify specific plays in MAPK activation. Identification of a distinct ras- independent NK lytic process could provide a new strategy for therapeutics that specifically targets ras-transformed tumors and not NK function in patients.