A critical role of protein kinase Cδ activation loop phosphorylation in formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of p47phox and rapid activation of nicotinamide adenine dinucleotide phosphate oxidase
A critical role of protein kinase Cδ activation loop phosphorylation in formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of p47phox and rapid activation of nicotinamide adenine dinucleotide phosphate oxidase
复制标题
DOI：
10.4049/jimmunol.179.11.7720
复制
发表时间：
2007-12-01
影响因子：
4.4
通讯作者：
Ye, Richard D.
中科院分区：
文献类型：
--
作者：
Cheng, Ni;He, Rong;Ye, Richard D.
专业吞噬细胞产生超氧化物是宿主防御细菌感染的重要机制。一些蛋白激酶C （PKC）异构体被发现磷酸化p47(phox)，导致其膜易位和NADPH氧化酶的激活。然而，具体的PKC异构体调节NADPH氧化酶激活的机制仍有待阐明。在这项研究中，我们报道了fMLF在其激活环中快速诱导PKC δ磷酸化，这对于其催化p47（phox）磷酸化的能力至关重要。通过转染表达gp91（phox）、p22（phox）、p67（phox）和p47(phox) （COS-phox细胞）的COS-7细胞，我们发现p47（phox）磷酸化和NADPH氧化酶重组需要一个功能活跃的PKC delta。PKC β II不能代替PKC δ。PKC δ /PKC β II嵌合体的表征导致PKC δ的催化结构域被鉴定为fMLF调控的靶标，fMLF诱导小鼠PKC δ激活环中Thr（505）的双相（30和180 s）磷酸化。在转染的COS-phox细胞中，Thr（505）突变为丙氨酸，使PKC δ在体外催化p47（phox）磷酸化和重建NADPH氧化酶的能力丧失。在分化的PLB-985人髓母细胞中，fmlf诱导的活化环磷酸化与超氧化物产生之间也存在相关性。我们得出结论，激动剂诱导的PKC δ磷酸化是NADPH氧化酶激活的新机制。诱导PKC三角洲磷酸化的能力可以区分超氧化物产生的完全激动剂和部分激动剂。
Generation of superoxide by professional phagocytes is an important mechanism of host defense against bacterial infection. Several protein kinase C (PKC) isoforms have been found to phosphorylate p47(phox) resulting in its membrane translocation and activation of the NADPH oxidase. However, the mechanism by which specific PKC isoforms regulate NADPH oxidase activation remains to be elucidated. In this study, we report that PKC delta phosphorylation in its activation loop is rapidly induced by fMLF and is essential for its ability to catalyze p47(phox) phosphorylation. Using transfected COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS-phox cells), we found that a functionally active PKC delta is required for p47(phox) phosphorylation and reconstitution of NADPH oxidase. PKC beta II cannot replace PKC delta for this function. Characterization of PKC delta/PKC beta II chimeras has led to the identification of the catalytic domain of PKC delta as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr(505) in the activation loop of mouse PKC delta. Mutation of Thr(505) to alanine abolishes the ability of PKC delta to catalyze p47(phox) phosphorylation in vitro and to reconstitute NADPH oxidase in the transfected COS-phox cells. A correlation between fMLF-induced activation loop phosphorylation and superoxide production is also established in the differentiated PLB-985 human myelomonoblastic cells. We conclude that agonist-induced PKC delta phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKC delta phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.