Pacquelet Sandrine, Johnson Jennifer L, Ellis Beverly A, Brzezinska Agnieszka A, Lane William S, Munafo Daniela B, Catz Sergio D
Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Biochem J. 2007 May 1;403(3):451-61. doi: 10.1042/BJ20061184.
Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.
嗜中性粒细胞暴露于脂多糖（LPS）会引发其氧化反应。然而，LPS刺激后Toll样受体4（TLR4）下游信号传导与氧化酶激活之间的关系仍不清楚。胞质因子p47phox的磷酸化对于NADPH氧化酶的激活至关重要。在本研究中，我们检验了以下假设：TLR4激活下游的主要调节激酶白细胞介素-1受体相关激酶4（IRAK-4）通过p47phox的磷酸化来调节NADPH氧化酶。我们发现p47phox是IRAK-4的底物。与蛋白激酶C（PKC）不同，IRAK-4不仅在丝氨酸残基上磷酸化p47phox，还在苏氨酸残基上磷酸化。通过串联质谱鉴定了靶残基，揭示了一个新的富含苏氨酸的调节结构域。我们还表明，在粒细胞中，p47phox会因LPS刺激而发生磷酸化。通过抑制p38丝裂原活化蛋白激酶（MAPK）可增强p47phox的LPS依赖性磷酸化，这证实该激酶在p38 MAPK上游起作用。在无细胞系统中，IRAK-4磷酸化的p47phox激活了NADPH氧化酶，并且IRAK-4的过表达增加了对LPS反应时的NADPH氧化酶活性。我们分别使用免疫沉淀分析和免疫荧光显微镜表明，内源性IRAK-4与p47phox相互作用，并且在LPS刺激后它们共定位于质膜。IRAK-4在嗜中性粒细胞中因LPS刺激而被激活。我们发现，体外IRAK-4磷酸化的靶标苏氨酸133、丝氨酸288和苏氨酸356在LPS刺激后的内源性p47phox中也会被磷酸化。我们得出结论，IRAK-4磷酸化p47phox并在LPS刺激后调节NADPH氧化酶的激活。
