Peroxisomal targeting as a tool for assaying potein-protein interactions in the living cell: cytokine-independent survival kinase (CISK) binds PDK-1 in vivo in a phosphorylation-dependent manner.
Peroxisomal targeting as a tool for assaying potein-protein interactions in the living cell: cytokine-independent survival kinase (CISK) binds PDK-1 in vivo in a phosphorylation-dependent manner.
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过氧化物酶体靶向作为检测活细胞中蛋白-蛋白质相互作用的工具：细胞因子非依赖性生存激酶 (CISK) 在体内以磷酸化依赖性方式结合 PDK-1。
DOI：
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发表时间：
2004
影响因子：
4.8
通讯作者：
S. Olsnes
中科院分区：
文献类型：
--
作者：
T. Nilsen;Thomas Slagsvold;C. S. Skjerpen;A. Brech;H. Stenmark;S. Olsnes
目前检测蛋白质-蛋白质相互作用的方法要么难以实施，要么不适用于哺乳动物系统或体外方法。通过共聚焦和电子显微镜观察，将过氧化物酶体靶向信号（PTS）添加到一个蛋白质上，缺乏靶向信号的结合伙伴以“背带”方式共同运输到过氧化物酶体中。大肠杆菌素E2片段及其紧密相互作用的免疫蛋白ImmE2均在细胞质中表达。当其中任何一种含有PTS标记时，两种蛋白在过氧化物酶体中共定位。含有PTS标签的细胞因子独立生存激酶（CISK）不能有效地靶向过氧化物酶体，除非将连接蛋白质到内体膜的Phox同源性（PX）结构域去除。然而，带有删除PX结构域的pts标记的CISK能够引导3-磷酸肌醇依赖性蛋白激酶-1 （PDK-1）进入过氧化物酶体。这表明这两种蛋白质在体内相互作用。在活化的CISK中磷酸化的Ser486突变为Ala阻止了相互作用，表明CISK和PDK-1以磷酸化依赖的方式相互作用。因此，该方法允许评估依赖于细胞特异性或依赖于细胞生理状态的翻译后修饰的蛋白质-蛋白质相互作用。
Current methods to detect protein-protein interactions are either laborious to implement or not adaptable for mammalian systems or in vitro methods. By adding a peroxisomal targeting signal (PTS) onto one protein, binding partners lacking a targeting signal were co-transported into the peroxisomes in a "piggy-back" fashion, as visualized by confocal and electron microscopy. A fragment of colicin E2 and its tightly interacting immunity protein, ImmE2, were both expressed in the cytosol. When either one contained a PTS tag, both proteins were co-localized in the peroxisomes. The cytokine-independent survival kinase (CISK) containing a PTS tag was not efficiently targeted to the peroxisomes unless the Phox homology (PX) domain, attaching the protein to endosomal membranes, was removed. However, PTS-tagged CISK with deleted PX domain was able to direct 3-phosphoinositide-dependent protein kinase-1 (PDK-1) into the peroxisomes. This demonstrates that the two proteins interact in vivo. Mutating Ser486, which is phosphorylated in activated CISK, to Ala prevented the interaction, indicating that CISK and PDK-1 interact in a phosphorylation-dependent manner. The method therefore allows assessment of protein-protein interactions that depend on post-translational modifications that are cell-specific or dependent on the physiological state of the cell.
影响因子：
4.8
作者：
Kenworthy, AK
通讯作者：
Kenworthy, AK
DOI：
10.1073/pnas.96.1.151
发表时间：
1999-01-05
影响因子：
11.1
作者：
Xu, Y;Piston, DW;Johnson, CH
通讯作者：
Johnson, CH