Fontayne Alexandre, Dang Pham My-Chan, Gougerot-Pocidalo Marie-Anne, El-Benna Jamel
Institut National de la Santé et de la Recherche Médicale U-479, Centre Hospitalier Universitaire Xavier Bichat, Paris, France.
Biochemistry. 2002 Jun 18;41(24):7743-50. doi: 10.1021/bi011953s.
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 microM for PKC alpha, beta II, delta, and zeta, respectively. Phosphopeptide mapping of p47(phox) showed that, as opposed to PKC zeta, PKC alpha, beta II, and delta are able to phosphorylate all the major PKC sites. The use of p47(phox) mutants identified serines 303, 304, 315, 320, 328, 359, 370, and 379 as targets of PKC alpha, beta II, 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 p22(phox). In addition, PKC alpha, beta II, 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, beta II, 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.
人中性粒细胞NADPH氧化酶的多组分酶产生超氧阴离子的过程伴随着其胞质成分之一p47(phox)的广泛磷酸化。p47(phox)是蛋白激酶C（PKC）的优良底物，但每种PKC同工型对该过程的各自贡献尚未明确界定。在本研究中，我们发现已知存在于人中性粒细胞中的PKC同工型（PKCα、β、δ和ζ）以时间和浓度依赖性方式使p47(phox)磷酸化，PKCα、βII、δ和ζ的表观K(m)值分别为10.33、3.37、2.37和2.13μM。p47(phox)的磷酸肽图谱显示，与PKCζ不同，PKCα、βII和δ能够使所有主要的PKC位点磷酸化。使用p47(phox)突变体确定丝氨酸303、304、315、320、328、359、370和379是PKCα、βII和δ的作用靶点。磷酸肽强度的比较表明，Ser 328是磷酸化程度最高的丝氨酸。通过使用覆盖技术测试了每种PKC同工型诱导p47(phox)与p22(phox)结合的能力；结果表明，所有研究的PKC同工型均诱导p47(phox)与p22(phox)的胞质片段结合。此外，PKCα、βII、δ和ζ能够在使用重组胞质蛋白的无细胞系统中诱导超氧阴离子的产生。令人惊讶的是，使选择性p47(phox)位点的一个子集磷酸化的PKCζ诱导了更强的NADPH氧化酶激活。综上所述，这些结果表明，人中性粒细胞中表达的PKCα、βII、δ和ζ可以分别使p47(phox)磷酸化，并诱导其转位和NADPH氧化酶激活。此外，某些丝氨酸的磷酸化可能对氧化酶激活具有抑制作用。
