A regulated adaptor function of p40phox:: Distinct p67phox membrane targeting by p40phox and by p47phox
A regulated adaptor function of p40phox:: Distinct p67phox membrane targeting by p40phox and by p47phox
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DOI：
10.1091/mbc.e06-08-0731
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发表时间：
2007-02-01
影响因子：
3.3
通讯作者：
Saito, Naoaki
中科院分区：
文献类型：
--
作者：
Ueyama, Takehiko;Tatsuno, Toshihiko;Saito, Naoaki
在吞噬细胞中，NADPH 氧化酶 (Nox2) 系统、细胞质调节因子（p47(phox)、p67(phox)、p40(phox) 和 Rac）易位并与跨膜黄细胞色素 b(558) 结合，从而激活超氧化物的产生。我们检查了 p40(phox) 蛋白的膜靶向性，并探索了 p40(phox) 的构象变化，这些变化在刺激时调节其向膜的易位。 GFP-p40(phox) 易位至早期内体，而 GFP-p47(phox) 响应花生四烯酸易位至质膜。相比之下，GFP-p67(phox)单独表达时不会易位至膜，但它分别依赖于p40(phox)和p47(phox)易位至早期内体或质膜。 GFP-p40(phox) 或 GFP-p47(phox) 易位至其各自的膜靶向位点被其 phox (PX) 结构域突变所消除，这些突变破坏了它们与其同源磷脂配体的相互作用。此外，GFP-p67(phox) 向任一膜的易位被破坏其与 p40(phox) 或 p47(phox) 相互作用的突变所消除。最后，我们通过缺失诱变、细胞定位和结合实验检测到静息状态下 p40(phox) 内头尾相连（PX-Phox 和 Bem1 [PB1] 结构域）的分子内相互作用，表明在没有细胞刺激的情况下，其 PX 结构域无法与 3-磷酸磷脂酰肌醇相互作用。因此，p40(phox) 和 p47(phox) 都作为不同的 p67(phox)“载体蛋白”发挥作用，通过不同机制中膜靶向结构域的暴露来调节。
In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47(phox), p67(phox), p40(phox), and Rac) translocate and associate with the membrane-spanning flavocytochrome b(558), leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40(phox) that regulate its translocation to membranes upon stimulation. GFP-p40(phox) translocates to early endosomes, whereas GFP-p47(phox) translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67(phox) does not translocate to membranes when expressed alone, but it is dependent on p40(phox) and p47(phox) for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40(phox) or GFP-p47(phox) to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67(phox) translocation to either membrane is abolished by mutations that disrupt its interaction with p40(phox) or p47(phox). Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40(phox) in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40(phox) and p47(phox) function as diverse p67(phox) "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms.