Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
FASEB J. 2013 May;27(5):2066-73. doi: 10.1096/fj.12-222133. Epub 2013 Feb 11.
The phospholipase A2 (PLA2)activity of phosphorylated peroxiredoxin 6 (Prdx6) is required for activation of NADPH oxidase (NOX2). We investigated the interaction of Prdx6 with p67(phox) and its effect on NOX2 activity. With the use of specific antibodies, coimmunoprecipitation of p67(phox) and phosphorylated Prdx6 was demonstrated with lysates of mouse pulmonary microvascular endothelial cells (MPMVECs) that were stimulated with angiotensin II; the interaction of p67(phox) with nonphosphorylated Prdx6 was relatively weak. Association of p67(phox) and phosphoPrdx6 in intact MPMVECs after angiotensin II stimulation was demonstrated by proximity ligation assay and was abolished by U0126, a MAP kinase inhibitor. By isothermal titration calorimetry, p67(phox) bound strongly to phosphoPrdx6 but bound poorly to Prdx6; phosphorylated p67(phox) did not bind to either Prdx6 or phosphoPrdx6. PLA2 activity of recombinant phosphoPrdx6 was decreased by >98% in the presence of p67(phox); the calculated dissociation constant (Kd) of the p67(phox): phosphoPrdx6 complex was 65 nM. PLA2 activity (MJ33 sensitive) in cell lysates following angiotensin II treatment of MPMVECs was increased by 85% following knockdown of p67(phox) with siRNA. These data indicate that p67(phox) binds to phosphoPrdx6 and inhibits its PLA2 activity, an interaction that could function to terminate the PLA2-mediated NOX2 activation signal.-Krishnaiah, S. Y., Dodia, C., Feinstein, S. I., and Fisher, A. B. p67(phox) terminates the phospholipase A2-derived signal for activation of NADPH oxidase (NOX2).
磷酸化过氧化物还原酶 6（Prdx6）的磷脂酶 A2（PLA2）活性对于 NADPH 氧化酶（NOX2）的激活是必需的。我们研究了 Prdx6 与 p67（phox）的相互作用及其对 NOX2 活性的影响。使用特异性抗体，我们在血管紧张素 II 刺激的小鼠肺微血管内皮细胞（MPMVEC）的裂解物中证实了 p67（phox）和磷酸化 Prdx6 的共免疫沉淀；p67（phox）与非磷酸化 Prdx6 的相互作用相对较弱。在血管紧张素 II 刺激后，通过接近连接测定法证明了 p67（phox）和磷酸化 Prdx6 在完整的 MPMVEC 中的结合，并且该结合被 MAP 激酶抑制剂 U0126 所消除。通过等温滴定量热法，p67（phox）与磷酸化 Prdx6 强烈结合，但与 Prdx6 结合不良；磷酸化的 p67（phox）与 Prdx6 或磷酸化 Prdx6 均不结合。在存在 p67（phox）的情况下，重组磷酸化 Prdx6 的 PLA2 活性降低了>98％；p67（phox）：磷酸化 Prdx6 复合物的计算解离常数（Kd）为 65 nM。用 siRNA 敲低 p67（phox）后，血管紧张素 II 处理 MPMVEC 后细胞裂解物中的 PLA2 活性（MJ33 敏感）增加了 85％。这些数据表明，p67（phox）与磷酸化 Prdx6 结合并抑制其 PLA2 活性，这种相互作用可以终止 PLA2 介导的 NOX2 激活信号。-克里希纳亚，S.Y.，多迪亚，C.，费因斯坦，S.I.和费舍尔，A.B. p67（phox）终止磷脂酶 A2 衍生的 NADPH 氧化酶（NOX2）激活信号。
