Analysis of subcellular region-specific activation of Ras and Rho family GTPases and its application

Ras和Rho家族GTP酶亚细胞区域特异性激活分析及其应用

• 批准号: 17370050
• 负责人: SATOH Takaya
• 金额: $ 7.74万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17370050/
• 关键词: Rac1; GLUT4; skeletal muscle; insulin; endocytosis; Ost; Rap1; M-ras; GABARAP; 筋肉

1. Analysis of the role for Rac1 in insulin-dependent glucose uptake in skeletal muscle cells: By using the L6 myoblast cell line, translocation of GLUT4 to the plasma membrane upon ectopic expression of constitutively activated Rac1 and lowered GLUT4 translocation in response to insulin upon downregulation of the expression of endogenous Rac1 by siRNA were observed. These effects were tried to be evaluated in mice by adenovirus-mediated gene transfer and targeted gene disruption. 2. Analysis of the negative regulation of clathrin-dependent receptor endocytosis by Rac1: Ectopic expression of constitutively activated Rac1 potently inhibited transferrin receptor endocytosis. The guanine nucleotide exchange factor Ost-III, but not its splice variants Ost-I and Ost-II, also abolished transferrin receptor endocytosis, suggesting that Ost-III may act as a specific activator for Rac1 that is involved in the negative regulation of receptor endocytosis. Furthermore, we isolated γ-aminobutylic acid A receptor-binding protein (GABARAP) as a specific binding partner for Ost-III, which inhibits the activity of Ost-III. 3. Analysis of physiological roles of phospholipase Cε in the mouse. 4. Analysis of physiological roles of the guanine nucleotide exchange factos for Rap1, RA-GEF-1 and RA-GEF-2, in the mouse: We generated RA-GEF-1 KO and RA-GEF-2 KO mice. RA-GEF-1 KO mice died by E9.5 due to deficiencies in vascular formation. RA-GEF-2 mice appeared healthy, but tumor necrosis factor-a-dependent integrin activation and cell adhesion was severely impaired in their splenocytes, suggesting a role for RA-GEF-2 in Rap1-dependent integrin activation.

1. Rac 1在骨骼肌细胞胰岛素依赖性葡萄糖摄取中的作用分析：通过使用L 6成肌细胞系，观察到在组成性激活的Rac 1异位表达时GLUT 4易位至质膜，以及在通过siRNA下调内源性Rac 1表达时响应于胰岛素的降低的GLUT 4易位。通过腺病毒介导的基因转移和靶向基因破坏在小鼠中评价这些作用。2. Rac 1对网格蛋白依赖性受体内吞作用负调控的分析：组成性激活Rac 1的异位表达有效抑制转铁蛋白受体内吞作用。鸟嘌呤核苷酸交换因子Ost-III，但不是它的剪接变体Ost-I和Ost-II，也废除转铁蛋白受体内吞作用，这表明Ost-III可能作为Rac 1的特异性激活剂，参与受体内吞作用的负调控。此外，我们分离出γ-氨基丁酸A受体结合蛋白（GABARAP）作为Ost-III的特异性结合伴侣，其抑制Ost-III的活性。3.磷脂酶Cε在小鼠体内的生理作用分析4.小鼠中Rap 1的鸟嘌呤核苷酸交换因子RA-GEF-1和RA-GEF-2的生理作用分析：我们产生了RA-GEF-1 KO和RA-GEF-2 KO小鼠。RA-GEF-1 KO小鼠在E9.5时由于血管形成缺陷而死亡。RA-GEF-2小鼠看起来很健康，但其脾细胞中肿瘤坏死因子-a依赖性整合素活化和细胞粘附严重受损，表明RA-GEF-2在Rap 1依赖性整合素活化中的作用。

项目成果

Ras and Rap1 activation of phospholipase Cε lipase activity

Ras 和 Rap1 激活磷脂酶 Cε 脂肪酶活性

• 发表时间: 2006
• 期刊: Methods Enzymol. 第407巻
• 作者: 岩根敦子;上田昌宏;日比野佳代;佐甲靖志;柳田敏雄;Hironori Edamatsu
• 通讯作者: Hironori Edamatsu

Phospholipase C_ε guanine nucleotide exchange factor activity and activation of Rap1

磷脂酶C_ε鸟嘌呤核苷酸交换因子活性和Rap1的激活

• 发表时间: 2006
• 期刊: Methods in Enzymology 407巻
• 作者: Takaya Satoh;et al.
• 通讯作者: et al.

Requirement of activated Cdc42-associated kinase (ACK) for survival of v-Ras-transformed mammalian cells

v-Ras 转化的哺乳动物细胞存活需要激活的 Cdc42 相关激酶 (ACK)

• 发表时间: 2005
• 期刊: Mol.Cancer Res. 未定(in press)
• 作者: Hironori Edamatsu;Takaya Satoh;Alam Nur-E-Kamal
• 通讯作者: Alam Nur-E-Kamal

Phospholipase Cε guanine nucleotide exchange factor activity and activation of Rap1

磷脂酶 Cε 鸟嘌呤核苷酸交换因子活性和 Rap1 的激活

• 发表时间: 2006
• 期刊: Methods in Enzymology 407巻(印刷中)
• 作者: Takaya Satoh;et al.
• 通讯作者: et al.

Ras and Rap1 activation of phcspholipase Cε lipase activity

Ras 和 Rap1 激活 phcsholipase Cε 脂肪酶活性

• 发表时间: 2006
• 期刊: Methods Enzymol. 第407巻
• 作者: M.Nakamura;E.Mizuno;A.Yamasaki;M.Nakamura et al.;E.Mizuno et al.;A.Yamasaki et al.;M.Nakamura;E.Mizuno;M.Maemura;a.Yamasaki;M.Nakamura;M.Komada;J.Han;E.Mizuno;M.Komada et al.;J.Han et al.;E.Mizuno et al.;M.Komada;H.Kakinuma;H.Tanaka;J.Han;E.Mizuno;Yoko Yoshikawa;Yoko Yoshikawa;Takaya Satoh;Hironori Edamatsu;Takaya Satoh;Takaya Satoh;Hironori Edamatsu;Takaya Satoh;Hironori Edamatsu
• 通讯作者: Hironori Edamatsu