Avdi N J, Winston B W, Russel M, Young S K, Johnson G L, Worthen G S
Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 80206, USA.
J Biol Chem. 1996 Dec 27;271(52):33598-606. doi: 10.1074/jbc.271.52.33598.
Mechanisms of neutrophil activation in response to chemoattractants remain incompletely understood. We have recently reported a Ras-mediated c-Raf pathway leading to the activation of mitogen-activated protein (MAP) kinase in human neutrophils stimulated with the chemoattractant formyl-Met-Leu-Phe (FMLP). However, concern that Raf activation may not fully account for the early FMLP-mediated human neutrophil responses prompted us to investigate the activation of MAP kinase/ERK kinase (MEK) by MEK kinase (MEKK). In cell lysates we identified protein species at 180, 160, 110, 72, and 54 kDa with a monoclonal antibody to MEKK. Activation of MEKK was determined on immunoprecipitates from FMLP-stimulated neutrophils by in vitro kinase assay, which utilized both MEK1 and MEK2 as substrates. It was rapid, detectable at 30 s and reaching a plateau at 5 min, and it was inhibited in a dose-dependent fashion by a specific phosphatidylinositol 3-kinase inhibitor, wortmannin. Partial inhibition by pertussis toxin was observed. We were unable to show inhibition of the MEKK response by GF 109203X, a protein kinase C-specific inhibitor. These data indicate that in neutrophils activation of MEKK in addition to Raf may underlie stimulation of MAP kinase and other MAP kinase homologues by FMLP.
中性粒细胞对趋化因子作出反应的激活机制仍未完全明了。我们最近报道了一条由Ras介导的c-Raf信号通路，该通路可导致在趋化因子甲酰甲硫氨酸-亮氨酸-苯丙氨酸（FMLP）刺激下的人中性粒细胞中丝裂原活化蛋白（MAP）激酶的激活。然而，由于担心Raf的激活可能无法完全解释FMLP介导的人中性粒细胞的早期反应，促使我们研究MEK激酶（MEKK）对MAP激酶/ERK激酶（MEK）的激活作用。在细胞裂解物中，我们用抗MEKK的单克隆抗体鉴定出了分子量为180、160、110、72和54 kDa的蛋白条带。通过体外激酶分析，以MEK1和MEK2作为底物，在FMLP刺激的中性粒细胞的免疫沉淀物上测定MEKK的激活情况。其激活迅速，在30秒时即可检测到，5分钟时达到平台期，并且被特异性磷脂酰肌醇3激酶抑制剂渥曼青霉素以剂量依赖的方式抑制。观察到百日咳毒素有部分抑制作用。我们未能显示蛋白激酶C特异性抑制剂GF 109203X对MEKK反应的抑制作用。这些数据表明，在中性粒细胞中，除了Raf之外，MEKK的激活可能是FMLP刺激MAP激酶和其他MAP激酶同系物的基础。
