Sequential binding of cytosolic phox complex to phagosomes through regulated adaptor proteins: Evaluation using the novel monomeric kusabira-green system and live imaging of phagocytosis
Sequential binding of cytosolic phox complex to phagosomes through regulated adaptor proteins: Evaluation using the novel monomeric kusabira-green system and live imaging of phagocytosis
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DOI：
10.4049/jimmunol.181.1.629
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发表时间：
2008-07-01
影响因子：
4.4
通讯作者：
Saito, Naoaki
中科院分区：
文献类型：
--
作者：
Ueyama, Takehiko;Kusakabe, Tomoko;Saito, Naoaki
我们设计了一种方法，用于检测分子内和分子间phox蛋白在细胞中的相互作用，通过荧光显微镜使用的珊瑚荧光报告蛋白（单体草比绿）的互补片段的融合蛋白。我们证实了单体Kusabira-Green系统的有效性，表明p40（phox）的PX和PB 1结构域在完整细胞中相互作用，我们建议保持这种蛋白质处于无活性的封闭构象。使用该系统，我们还探讨了p47（phox）内的分子内相互作用，并表明PX结构域与自抑制串联Src同源3结构域相互作用，该结构域与自抑制区保持接触，沿着残基341-360。此外，我们证明了在Fc γ R介导的吞噬作用期间，p67（phox）与涉及衔接蛋白p47（phox）和p40（phox）的吞噬体的顺序相互作用。虽然p67（phox）本身不靶向吞噬体，但p47（phox）在吞噬体关闭之前的吞噬作用的早期阶段作为三元复合物（p47（phox），-p67（phox）-p40（phox））的衔接子发挥作用，而p40（phox）在吞噬体关闭之后的后期阶段发挥作用。有趣的是，突变的p40（phox）的“开放”形式，连接p47（phox），关闭吞噬体和延长p47（phox）和p67（phox）保留在吞噬体上。这些结果表明，结合的三元复合物的吞噬体可以暂时调节之间的衔接蛋白，具有不同的脂质结合特异性PX结构域切换。
We engineered a method for detecting intramolecular and intermolecular phox protein interactions in cells by fluorescence microscopy using fusion proteins of complementary fragments of a coral fluorescent reporter protein (monomeric Kusabira-Green). We confirmed the efficacy of the monomeric Kusabira-Green system by showing that the PX and PB1 domains of p40(phox) interact in intact cells, which we suggested maintains this protein in an inactive closed conformation. Using this system, we also explored intramolecular interactions within p47(phox) and showed that the PX domain interacts with the autoinhibited tandem Src homology 3 domains maintained in contact with the autoinhibitory region, along with residues 341-360. Furthermore, we demonstrated sequential interactions of p67(phox) , With phagosomes involving adaptor proteins, p47(phox) and p40(phox), during Fc gamma R-mediated phagocytosis. Although p67(phox) is not targeted to phagosomes by itself, p47(phox) functions as an adaptor for the ternary complex (p47(phox), -p67(phox)-p40(phox)) in early stages of phagocytosis before phagosome closure, while p40(phox) functions in later stages after phagosomal closure. Interestingly, a mutated "open" form of p40(phox), linked p47(phox), to closed phagosomes and prolonged p47(phox) and p67(phox) retention on phagosomes. These results indicate that binding of the ternary complex to phagosomes can be temporally regulated by switching between adaptor proteins that have PX domains with distinct lipid-binding specificities.