The scaffold MyD88 acts to couple protein kinase Cε to toll-like receptors
The scaffold MyD88 acts to couple protein kinase Cε to toll-like receptors
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DOI：
10.1074/jbc.m710330200
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发表时间：
2008-07-04
影响因子：
4.8
通讯作者：
Parker, Peter J.
中科院分区：
文献类型：
--
作者：
Faisal, Amir;Saurin, Adrian;Parker, Peter J.
缺乏蛋白激酶C β（PKC β）的小鼠对革兰氏阳性和革兰氏阴性细菌感染都非常敏感;然而，PKC β与负责病原体检测的Toll样受体（TLR）偶联的机制知之甚少。在这里，我们试图研究的机制，PKC β参与TLR信号转导，发现PKC β被招募到TLR 4和磷酸化的两个最近确定的网站，响应脂多糖（LPS）的刺激。这两个位点（Ser-346和Ser-368）的磷酸化导致PKC β与14-3-3 β结合。LPS诱导的PKC β磷酸化、14-3-3 β结合和TLR 4的募集都依赖于支架蛋白MyD 88的表达。在MyD 88基因敲除小鼠的小鼠胚胎成纤维细胞和活化的巨噬细胞中，与野生型细胞相比，LPS刺激的PKC β磷酸化减少。在LPS应答的293细胞中，MyD 88的急性敲除也导致Ser-346磷酸化和TLR 4/PKC β结合的完全丧失。相比之下，MyD 88在293细胞中的过表达导致PKC β的组成性磷酸化。MyD 88的一般作用由以下发现证明：PKC β的磷酸化由RAW细胞和原代人巨噬细胞中通过MyD 88发出信号的所有测试TLR（即，除了TLR 3之外的所有TLR）的激活诱导。在功能上，已经确定在这两个位点的PKC β的磷酸化是在重构的PKC β（-/-）细胞中TLR 4和TLR 2诱导的NF κ B报告子激活和I κ B降解所必需的。因此，本研究确定了支架蛋白MyD 88作为连接TLR与PKC β募集、磷酸化和下游信号传导的连接。
Mice lacking protein kinase C epsilon(PKC epsilon) are hypersensitive to both Gram-positive and Gram-negative bacterial infections; however, the mechanism of PKC epsilon coupling to the Toll-like receptors (TLRs), responsible for pathogen detection, is poorly understood. Here we sought to investigate the mechanism of PKC epsilon involvement in TLR signaling and found that PKC epsilon is recruited to TLR4 and phosphorylated on two recently identified sites in response to lipopolysaccharide (LPS) stimulation. Phosphorylation at both of these sites (Ser-346 and Ser-368) resulted in PKC epsilon binding to 14-3-3 beta. LPS-induced PKC epsilon phosphorylation, 14-3-3 beta binding, and recruitment to TLR4 were all dependent on expression of the scaffold protein MyD88. In mouse embryo fibroblasts and activated macrophages from MyD88 knock-out mice, LPS-stimulated PKC epsilon phosphorylation was reduced compared with wild type cells. Acute knockdown of MyD88 in LPS-responsive 293 cells also resulted in complete loss of Ser-346 phosphorylation and TLR4/PKC epsilon association. By contrast, MyD88 overexpression in 293 cells resulted in constitutive phosphorylation of PKC epsilon. A general role for MyD88 was evidenced by the finding that phosphorylation of PKC epsilon was induced by the activation of all TLRs tested that signal through MyD88 (i.e. all except TLR3) both in RAW cells and in primary human macrophages. Functionally, it is established that phosphorylation of PKC epsilon at these two sites is required for TLR4- and TLR2-induced NF kappa B reporter activation and I kappa B degradation in reconstituted PKC epsilon(-/-) cells. This study therefore identifies the scaffold protein MyD88 as the link coupling TLRs to PKC epsilon recruitment, phosphorylation, and downstream signaling.