Nauseef W M, Volpp B D, McCormick S, Leidal K G, Clark R A
Department of Medicine, University of Iowa, Iowa City 52242.
J Biol Chem. 1991 Mar 25;266(9):5911-7.
Activated human polymorphonuclear neutrophils (PMNs) convert molecular oxygen into superoxide anion, a process known as the respiratory burst, through the activity of a latent multicomponent NADPH-dependent oxidase. Components of this respiratory burst oxidase include the membrane-bound cytochrome b558 and the cytosolic factors p47-phox and p67-phox. We initiated these studies based on three observations: 1) that stimulation of PMN oxidase activity is associated with translocation of the cytosolic oxidase components to the plasma membrane; 2) that p47-phox is phosphorylated during PMN activation and that there is a sequential relationship between phosphorylation of p47-phox in the cytosol and appearance of the phosphoprotein in the membran; and 3) that the predicted amino acid sequences of p47-phox and of p67-phox contain regions of homology to the SH3 or A domain of the src family of tyrosine kinases, a region found in a variety of proteins which interact with the cytoskeleton or the subplasmalemmal cytoskeleton. Thus the purpose of our studies was to examine the role of protein kinase C (PKC)-dependent phosphorylation in the stimulus-induced association of p47-phox and p67-phox with the plasma membrane and the cytoskeleton. Using the PKC activator phorbol myristate acetate (PMA) as the agonist, we found that activation of the respiratory burst oxidase was associated with translocation of cytosolic p47-phox and p67-phox to the plasma membrane as well as redistribution of p47-phox to the Triton-insoluble cytoskeleton. Furthermore, the PKC inhibitor staurosporine inhibited phosphorylation of p47-phox, interrupted the redistribution of cytosolic oxidase factors, and blocked PMA-induced generation of superoxide anion. Taken together these results indicate that PKC-dependent phosphorylation of p47-phox correlates with association of p47-phox with the cytoskeleton and with translocation of p47-phox and p67-phox to the plasma membrane, with the ensuing assembly of an active superoxide-generating NADPH-dependent oxidase.
活化的人类多形核中性粒细胞（PMN）通过一种潜在的多组分NADPH依赖性氧化酶的活性，将分子氧转化为超氧阴离子，这一过程被称为呼吸爆发。这种呼吸爆发氧化酶的组分包括膜结合细胞色素b558以及胞质因子p47 - phox和p67 - phox。我们基于三项观察开展了这些研究：1）PMN氧化酶活性的刺激与胞质氧化酶组分向质膜的转位相关；2）p47 - phox在PMN活化过程中被磷酸化，并且胞质中p47 - phox的磷酸化与膜中磷蛋白的出现之间存在顺序关系；3）p47 - phox和p67 - phox的预测氨基酸序列包含与src家族酪氨酸激酶的SH3或A结构域同源的区域，该区域存在于多种与细胞骨架或质膜下细胞骨架相互作用的蛋白质中。因此，我们研究的目的是检验蛋白激酶C（PKC）依赖性磷酸化在刺激诱导的p47 - phox和p67 - phox与质膜及细胞骨架的结合中的作用。使用PKC激活剂佛波酯肉豆蔻酸酯乙酸酯（PMA）作为激动剂，我们发现呼吸爆发氧化酶的激活与胞质p47 - phox和p67 - phox向质膜转位以及p47 - phox向Triton不溶性细胞骨架重新分布有关。此外，PKC抑制剂星形孢菌素抑制p47 - phox的磷酸化、中断胞质氧化酶因子的重新分布，并阻断PMA诱导的超氧阴离子生成。综合这些结果表明p47 - phox的PKC依赖性磷酸化与p47 - phox与细胞骨架的结合以及p47 - phox和p67 - phox向质膜的转位相关，随之组装成有活性的产生超氧阴离子的NADPH依赖性氧化酶。
