Worthen G S, Avdi N, Buhl A M, Suzuki N, Johnson G L
Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
J Clin Invest. 1994 Aug;94(2):815-23. doi: 10.1172/JCI117401.
Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants.
趋化因子与多形核白细胞（中性粒细胞）上的七次跨膜、G蛋白偶联受体结合，并诱导多种功能反应，包括微管相关蛋白（MAP）激酶的激活。虽然中性粒细胞中MAP激酶被激活的途径尚不清楚，但我们推测趋化因子f-甲硫氨酰-亮氨酰-苯丙氨酸（FMLP）会诱导导致MAP/细胞外信号调节激酶（ERK）激酶（MEK）激活的Ras/Raf途径的激活。暴露于10 nM FMLP 30秒的人中性粒细胞表现出与MEK-1共洗脱的MAP激酶激酶活性。用FMLP刺激后对Raf-1激酶进行免疫沉淀，发现一种可使MEK磷酸化的活性，在30秒时可检测到，并在2 - 3分钟时达到峰值。从用[32P]正磷酸盐标记的完整中性粒细胞和与[32P]GTP一起孵育的电通透中性粒细胞中对Ras进行免疫沉淀，以确定FMLP处理与Ras的激活有关。用百日咳毒素处理中性粒细胞可抑制Ras和Raf的激活，表明主要与Gi2蛋白相关联。虽然佛波酯激活了Raf，但FMLP诱导的激活似乎独立于蛋白激酶C，这进一步表明Gi2与Ras和Raf的联系独立于磷脂酶C和蛋白激酶C。抑制许多中性粒细胞功能反应的二丁酰环磷腺苷（dibutyryl cAMP）阻断了FMLP对Raf的激活，表明Raf/MAP激酶途径的中断会影响中性粒细胞对趋化因子的反应。这些数据表明，Gi2介导的Ras/Raf/MAP激酶途径的受体调节是对趋化因子的主要反应。
