Robinson J M, Heyworth P G, Badwey J A
Department of Anatomy, Ohio State University, Columbus.
Biochim Biophys Acta. 1990 Oct 15;1055(1):55-62. doi: 10.1016/0167-4889(90)90090-z.
Neutrophils exhibit an intense phosphorylation of a 47 kDa protein and release large quantities of superoxide (O2-) upon stimulation with phorbol 12-myristate 13-acetate (PMA) or fMet-Leu-Phe (fMLP). Antagonists of protein kinases (e.g., 200 microM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7); 15 nM staurosporine) inhibited these phenomena when the stimulus was PMA (Badwey, J.A. et al. (1989) J. Biol. Chem. 264, 14947-14953). In this paper, we now report that while neutrophils treated with 15 nM staurosporine and PMA release little O2-, cells in the presence of these compounds can be stimulated to release near normal quantities of O2- by the subsequent addition of fMLP. Surprisingly, staurosporine (15 nM) reduced the incorporation of 32P into the 47 kDa protein in fMLP stimulated cells at least as effectively as H-7, yet, while the staurosporine treated cells released substantial amounts of O2-, the cells treated with H-7 did not. These data suggest that a stimulatory pathway exists in neutrophils that contains a protein kinase 'distinct' from that which is activated when PMA is the stimulus and that this pathway may enable the O2- producing system to become functional with little or no phosphorylation of the 47 kDa protein. They further suggest that the steps which are sensitive to H-7 in the signal-transduction pathways utilized by PMA and fMLP may be different.
在用佛波酯12 -肉豆蔻酸酯13 -乙酸酯（PMA）或甲酰甲硫氨酸 -亮氨酸 -苯丙氨酸（fMLP）刺激时，中性粒细胞会出现一种47 kDa蛋白的强烈磷酸化，并释放大量超氧化物（O₂⁻）。当刺激物为PMA时，蛋白激酶拮抗剂（如200 μM 1 -（5 -异喹啉磺酰基）- 2 -甲基哌嗪（H - 7）；15 nM星形孢菌素）可抑制这些现象（Badwey，J.A.等人（1989年）《生物化学杂志》264，14947 - 14953）。在本文中，我们现在报告，虽然用15 nM星形孢菌素和PMA处理的中性粒细胞释放的O₂⁻很少，但在这些化合物存在的情况下，随后添加fMLP可刺激细胞释放接近正常量的O₂⁻。令人惊讶的是，星形孢菌素（15 nM）在fMLP刺激的细胞中减少³²P掺入47 kDa蛋白的效果至少与H - 7一样有效，然而，虽然用星形孢菌素处理的细胞释放大量O₂⁻，但用H - 7处理的细胞却不释放。这些数据表明，中性粒细胞中存在一条刺激途径，该途径包含一种与PMA作为刺激物时激活的蛋白激酶“不同”的蛋白激酶，并且这条途径可能使O₂⁻产生系统在47 kDa蛋白很少或没有磷酸化的情况下发挥功能。它们还进一步表明，PMA和fMLP利用的信号转导途径中对H - 7敏感的步骤可能不同。
