FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.
FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.
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FMLP 激活人类中性粒细胞中的 Ras 和 Raf。
DOI：
10.1172/jci117401
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Johnson,GL
中科院分区：
文献类型：
--
作者：
Worthen,GS;Avdi,N;Buhl,AM;Suzuki,N;Johnson,GL
化学引诱剂与多形核白细胞（中性粒细胞）上的七种跨膜G蛋白连接受体结合，并诱导多种功能反应，包括激活微管相关蛋白（MAP）激酶。虽然MAP激酶在中性粒细胞中被激活的途径是未知的，但我们假设Ras/Raf途径的激活导致MAP/ERK激酶（MEK）的激活将由化学引诱物f-met-leu-phe诱导。暴露于10 nM FMLP 30 s的人中性粒细胞显示出与MEK-1共洗脱的MAP激酶激酶活性。免疫沉淀的Raf-1激酶的刺激后，FMLP显示的活性，磷酸化MEK，可检测到在30秒，并在2-3分钟达到峰值。免疫沉淀的Ras从两个完整的中性粒细胞标记的[32 P]正磷酸盐和电透性中性粒细胞孵育[32 P]GTP被用来确定FMLP治疗与Ras的激活。Ras和Raf的激活被百日咳毒素治疗的中性粒细胞抑制，表明主要的联系Gi 2蛋白。虽然佛波醇酯激活Raf，但FMLP诱导的激活似乎不依赖于蛋白激酶C，进一步表明Gi 2与Ras和Raf连接，不依赖于磷脂酶C和蛋白激酶C。双丁酰cAMP，抑制许多中性粒细胞的功能反应，阻止Raf的FMLP激活，这表明Raf/MAP激酶通路的中断影响中性粒细胞对趋化剂的反应。这些数据表明，Gi 2介导的Ras/Raf/MAP激酶途径的受体调节是对化学引诱物的主要反应。图片
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.Images