Phosphorylation of p47phox sites by PKC α, βII, δ, and ζ:: Effect on binding to p22phox and on NADPH oxidase activation
Phosphorylation of p47phox sites by PKC α, βII, δ, and ζ:: Effect on binding to p22phox and on NADPH oxidase activation
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DOI：
10.1021/bi011953s
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发表时间：
2002-06-18
期刊：
影响因子：
2.9
通讯作者：
El Benna, J
中科院分区：
文献类型：
--
作者：
Fontayne, A;Dang, PMC;El Benna, J
人中性粒细胞NADPH氧化酶的多组分酶产生超氧阴离子伴随着其胞质组分之一p47（phox）的广泛磷酸化。p47（phox）是蛋白激酶C（PKC）的优良底物，但各PKC亚型对该过程的各自贡献尚不清楚。在这项研究中，我们发现，已知存在于人类中性粒细胞（PKC α，β，δ和zeta）磷酸化p47（phox）在时间和浓度依赖性的方式，与明显的K-m值为10.33，3.37，2.37，和2.13 μ M的PKC α，β II，δ和zeta，分别。p47（phox）的磷酸肽图谱显示，与PKC zeta相反，PKC α、β II和δ能够磷酸化所有主要的PKC位点。使用p47 phox突变体鉴定了丝氨酸303、304、315、320、328、359、370和379作为PKC α、β II和δ的靶标。比较磷酸化肽的强度表明Ser 328是磷酸化程度最高的丝氨酸。每种PKC亚型诱导p47（phox）与p22（phox）相关联的能力进行了测试，通过使用覆盖技术，结果表明，所有的PKC亚型的研究诱导p47（phox）结合到p22 phox的胞质片段。此外，PKC α，β II，δ和ζ能够诱导生产超氧阴离子在无细胞系统中使用重组胞质蛋白。令人惊讶的是，PKC ζ，磷酸化的一个子集的选择性p47（phox）网站，诱导更强的激活NADPH氧化酶。两者合计，这些结果表明，PKC α，β II，δ和zeta在人类中性粒细胞中表达可以单独磷酸化p47（phox），并诱导其易位和NADPH氧化酶激活。此外，某些丝氨酸的磷酸化可能对氧化酶的活化具有抑制作用。
Production of superoxide anions by the multicomponent enzyme of human neutrophil NADPH oxidase is accompanied by extensive phosphorylation of p47(phox), one of its cytosolic components. p47(phox) is an excellent substrate for protein kinase C (PKC), but the respective contribution of each PKC isoform to this process is not clearly defined. In this study, we found that PKC isoforms known to be present in human neutrophils (PKC alpha, beta, delta, and zeta) phosphorylate p47(phox) in a time- and concentration-dependent manner, with apparent K-m values of 10.33, 3.37, 2.37, and 2.13 muM for PKC alpha, betaII, delta, and zeta, respectively. Phosphopeptide mapping of p47(phox) showed that, as opposed to PKC zeta, PKC alpha, betaII, and delta are able to phosphorylate all the major PKC sites. The use of p47phox mutants identified serines 303, 304, 315, 320, 328, 359, 370, and 379 as targets of PKC alpha, betaII, and delta. Comparison of the intensity of phosphopeptides suggests that Ser 328 is the most phosphorylated serine. The ability of each PKC isoform to induce p47(phox) to associate with p22(phox) was tested by using an overlay technique; the results showed that all the PKC isoforms that were studied induce p47(phox) binding to the cytosolic fragment of p22phox. In addition, PKC alpha, betaII, delta, and zeta were able to induce production of superoxide anions in a cell-free system using recombinant cytosolic proteins. Surprisingly, PKC zeta, which phosphorylates a subset of selective p47(phox) sites, induced stronger activation of the NADPH oxidase. Taken together, these results suggest that PKC alpha, betaII, delta, and zeta expressed in human neutrophils can individually phosphorylate p47(phox) and induce both its translocation and NADPH oxidase activation. In addition, phosphorylation of some serines could have an inhibitory effect on oxidase activation.