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Regulatory mechanisms for Ras family and Rho family GTP-binding protein networks

Ras 家族和 Rho 家族 GTP 结合蛋白网络的调控机制

基本信息

批准号：
15570117
负责人：
SATOH Takaya
金额：
$ 2.37万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570117/
关键词：
GEF Dbl family GTP-binding protein Rho family Cdc42 Sec14 インスリン骨格筋

项目摘要

Dbl family guanine nucleotide exchange factors (GEFs), which target the Rho family, direct various cellular functions, such as cell motility, cell adhesion, and cell polarity, through cytoskeletal rearrangements. Here, we investigated the role of the Sec14-like domain of the Dbl family proteins Dbl and Ost, which is localized at the N terminal portion.Splice variants containing this domain, which are localized in the perinuclear region, did not induce change of cell shape, whereas those without the Sec14-like domain were localized in the plasma membrane, and induced filopodia formation. By employing a novel method to visualize the activation of the Rho family in situ, we demonstrated that Cdc42 colocalized with Dbl in the plasma membrane is in fact activated. Therefore, the Sec14-like domain has an important role in the regulation of subcellular localization and activation of Dbl family GEFs. We also analyzed insulin-dependent activation of Rac and Cdc42 in the muscle cell line L6 to examine the involvement of the Rho family in insulin-stimulated glucose uptake in the muscle. We clarified that the activation of Rac was partially dependent on phosphoinositide 3-kinase (PI3K), and PI3K-dependent activation of Rac occurred in the ruffling membrane areas, whereas PI3K-independent activation was observed in endomembranes.

以Rho家族为靶标的DBL家族鸟嘌呤核苷酸交换因子(GEF)，通过细胞骨架重排，指导细胞运动、细胞黏附和细胞极性等多种细胞功能。在这里，我们研究了位于N末端的DBL家族蛋白DBL和Ost的Sec14样结构域的作用。含有该结构域的剪接变体定位于核周区域，不会引起细胞形状的改变，而没有Sec14样结构域的剪接变体定位于质膜，并诱导丝状足的形成。通过使用一种新的方法来原位显示Rho家族的激活，我们证明了质膜中与DBL共定位的CDC42实际上是被激活的。因此，Sec14样结构域在调节DBL家族GEF的亚细胞定位和激活过程中具有重要作用。我们还分析了肌肉细胞系L6中Rac和CDC42的胰岛素依赖激活，以检查Rho家族在肌肉中胰岛素刺激的葡萄糖摄取中的参与。我们阐明RAC的激活部分依赖于磷脂酰肌醇3-激酶(PI3K)，PI3K依赖的RAC激活发生在褶皱的膜区，而PI3K不依赖的激活发生在内膜。