Mitochondrial Dok-4 recruits Src kinase and regulates NF-κB activation in endothelial cells
Mitochondrial Dok-4 recruits Src kinase and regulates NF-κB activation in endothelial cells
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DOI：
10.1074/jbc.m410262200
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发表时间：
2005-07-15
影响因子：
4.8
通讯作者：
Abe, J
中科院分区：
文献类型：
--
作者：
Itoh, S;Lemay, S;Abe, J
衔接蛋白的激酶下游（Dok）家族至少包含五个成员，其结构特征为一个N端保守的普列克底物蛋白同源结构域和磷酸酪氨酸结合结构域的串联结构，该结构与一个独特的C端区域相连。为了确定新型衔接蛋白Dok - 4在内皮细胞中的作用，我们首先研究了Dok - 4的细胞定位。最令人惊讶的是，免疫荧光显微镜、细胞分级分离研究以及增强型绿色荧光蛋白嵌合体研究表明，野生型Dok - 4（Dok - 4 - WT）特异性定位于线粒体。Dok - 4的一个N端缺失突变体（Dok - 4 -（ΔN11 - 29）），由于缺失线粒体靶向序列，不能在线粒体中积累。免疫共沉淀显示内皮细胞中c - Src与Dok - 4 - WT存在相互作用。最有趣的是，Dok - 4 - WT的过表达（而非Dok - 4（ΔN11 - 29））增加了线粒体c - Src的表达，而用小干扰RNA载体敲低内源性Dok - 4则极大地抑制了c - Src的线粒体定位，这表明Dok - 4作为c - Src在线粒体中的锚定蛋白具有独特功能。Dok - 4 - WT显著降低了39 - kDa亚基复合物I的表达。PP2，一种特异性的Src激酶抑制剂，阻止了Dok - 4介导的复合物I的降低，这表明Src激酶参与调节复合物I的表达。Dok - 4 - WT增强了肿瘤坏死因子 - α（TNF - α）介导的活性氧（ROS）产生，支持了线粒体中Dok - 4 - Src - 复合物I/ROS信号通路的功能相关性。最后，Dok - 4增强了TNF - α介导的NF - κB激活，而用Dok - 4小干扰RNA转染则抑制了这种激活。此外，用显性负性形式的c - Src转染也抑制了Dok - 4诱导的NF - κB激活。这些数据表明线粒体Dok - 4作为酪氨酸激酶c - Src的锚定分子发挥作用，进而作为TNF - α介导的ROS产生和NF - κB激活的调节因子。
The downstream of kinase (Dok) family of adapter proteins consists of at least five members structurally characterized by an NH2-terminal tandem of conserved pleckstrin homology and phosphotyrosine binding domains linked to a unique COOH-terminal region. To determine the role of the novel adapter protein Dok-4 in endothelial cells, we first investigated the cell localization of Dok-4. Most surprisingly, immunofluorescence microscopy, cell fractionation studies, and studies with enhanced green fluorescent protein chimeras showed that wild type Dok-4 (Dok-4-WT) specifically localized in mitochondria. An NH2-terminal deletion mutant of Dok-4 (Dok-4-(Delta N11-29)), which lacks the mitochondrial targeting sequence, could not accumulate in mitochondria. Co-immunoprecipitation revealed an interaction of c-Src with Dok-4-WT in endothelial cells. Most interestingly, overexpression of Dok-4-WT, but not Dok-4(Delta N11-29), increased mitochondrial c-Src expression, whereas knock-down of endogenous Dok-4 with a small interfering RNA vector greatly inhibited mitochondrial localization of c-Src, suggesting a unique function for Dok-4 as an anchoring protein for c-Src in mitochondria. Dok-4-WT significantly decreased 39-kDa subunit complex I expression. PP2, a specific Src kinase inhibitor, prevented the Dok-4- mediated complex I decrease, suggesting the involvement of Src kinase in regulation of complex I expression. Dok-4-WT enhanced tumor necrosis factor-alpha (TNF-alpha)-mediated reactive oxygen species (ROS) production, supporting the functional relevance of a Dok-4- Src-complex I/ROS signaling pathway in mitochondria. Finally, Dok-4 enhanced TNF-alpha-mediated NF-kappa B activation, whereas this was inhibited by transfection with Dok-4 small interfering RNA. In addition, Dok-4-induced NF-kappa B activation was also inhibited by transfection of a dominant negative form of c-Src. These data suggest a role for mitochondrial Dok-4 as an anchoring molecule for the tyrosine kinase c-Src, and in turn as a regulator of TNF-alpha-mediated ROS production and NF-kappa B activation.