MAPK AND LYTIC FUNCTION IN HUMAN NK CELLS

细胞毒性是自然杀伤（NK）细胞和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）/ERK 2关键控制直接NK细胞裂解。人NK细胞中的肿瘤连接诱导MAPK酪氨酸磷酸化和酶活性。此外，通过PD 098059或显性阴性ERK 2表达的药理学抑制来灭活MAPK干扰51-Cr标记的肿瘤细胞的NK溶解，并中断穿孔素/颗粒酶B极化。因此，重要的是要确定什么信号分子控制MAPK激活，目标是一旦确定，可能找到增强癌症患者中这一途径的方法。初步研究表明，NK利用ras非依赖性MAPK级联来裂解肿瘤细胞。法尼基转移酶抑制剂FTI-277或显性负性N17 ras表达对肿瘤细胞的NK溶解、穿孔素/颗粒酶B动员或MAPK活化没有有害作用。相反，肿瘤细胞在NK细胞中激活ras相关蛋白rap 1A，使其成为GTP形式。GGTI-298，阻断香叶基化，rap 1A用于膜定位的过程，有效地抑制NK细胞溶解能力和rap 1A激活。目的1是确定rap 1A/B-raf/MEK/MAPK通路是否对细胞裂解过程起关键控制作用。或者，其他途径可以进入MAPK以代表重叠或独立的功能。目的2分析PI 3-激酶和Syk 70在MAPK激活中的作用。目的3阐明抑制性NK受体（如KIR）通过MAPK发挥作用的假说。目的4是确定MAPK是否通过调控微管重组来调控穿孔素的运动。 实验计划遵循深入的生化和遗传方法，使用药理学试剂和突变基因构建体，以确定MAPK激活中的特定作用。鉴定不同的ras非依赖性NK裂解过程可以为特异性靶向ras转化的肿瘤而不是患者中的NK功能的治疗提供新的策略。

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.