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Regulation of cytoskeletons by heterotrimeric GTP-binding proteins

异源三聚体 GTP 结合蛋白对细胞骨架的调节

基本信息

批准号：
14580663
负责人：
ASANO Tomiko
金额：
$ 2.3万
依托单位：
Institute for Developmental Research, Aichi Human Service Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580663/
关键词：
G protein Rho Apoptosis Akt Protein tyrosine phosphatase Endothelin Fibronectin Neural progenitor cells カスパーゼ HeLa細胞神経細胞新生 Rac

项目摘要

In this study, we investigate the molecular mechanisms of Gαq/11-induced apoptosis in m1 muscarinic acetylcholine receptor-expressing HeLa cells. Overexpression of the constitutively active mutant of Akt inhibited carbachol-induced ROCK-I cleavage. Insulin, a major survival factor in many cells, strongly increased phosphorylation of Akt, which was completely blocked by carbachol. This latter effect was partially inhibited by treatment with the tyrosine phosphatase inhibitor, orthovanadate. In parallel with these observations, carbachol attenuated insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1, an effect eliminated by orthovanadate. On the other hand, carbachol induced rapid stimulation of endogenous RhoA, and expression of a constitutively active mutant of RhoA increased ROCK-I cleavage. Orthovanadate and the dominant negative mutant of RhoA partially, and their combination completely, inhibited carbachol-induced ROCK-I cleavage and apoptosis. These results … More demonstrate that Ga/11 signaling induces apoptosis by reducing insulin-stimulated Akt phosphorylation through tyrosine dephosphorylation and activating RhoA in HeLa cells.In order to clarify the role of Gi2 in the ventricular zone of embryonic brains, we administered pertussis toxin (PTX) into the lateral ventricle of mouse brains at E14.5. Examination at E18.5 revealed that PTX decreased total cells and bromodeoxyuridine-positive cells in the cerebral cortices, suggesting impaired proliferation of neuroepithelial cells. Then we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors, which are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [^3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of ERK, and PTX partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of ERK. These findings indicated that Gi2 mediates signaling from receptors such as endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain. Less

在这项研究中，我们研究了Gαq/11诱导表达m1毒蕈碱乙酰胆碱受体的HeLa细胞凋亡的分子机制。Akt组成型活性突变体的过表达抑制卡巴胆碱诱导的ROCK-I裂解。胰岛素是许多细胞中的主要存活因子，强烈增加Akt的磷酸化，这被卡巴胆碱完全阻断。后一种作用被部分抑制治疗与酪氨酸磷酸酶抑制剂，orthovanadate。与这些观察结果平行，卡巴胆碱减弱胰岛素刺激的胰岛素受体底物-1的酪氨酸磷酸化，原钒酸盐消除了这种作用。另一方面，卡巴胆碱诱导内源性RhoA的快速刺激，和组成型活性突变体的RhoA的表达增加ROCK-I裂解。原钒酸盐和RhoA的显性负突变体部分抑制卡巴胆碱诱导的ROCK-I裂解和细胞凋亡，而它们的组合完全抑制卡巴胆碱诱导的ROCK-I裂解和细胞凋亡。这些结果 ...更多信息研究表明Ga/11信号通过酪氨酸去磷酸化减少胰岛素刺激的Akt磷酸化和激活HeLa细胞中的RhoA来诱导细胞凋亡。为了阐明Gi 2在胚胎脑心室区的作用，我们在E14.5将百日咳毒素（PTX）注入小鼠侧脑室。在E18.5的检查显示，PTX减少了大脑皮质中的总细胞和溴脱氧尿苷阳性细胞，表明神经上皮细胞的增殖受损。然后，我们培养大鼠胚胎脑神经祖细胞，并评估了几种Gi偶联受体激动剂的促有丝分裂作用，这些受体已知在心室区表达。在所测试的激动剂中，内皮素通过内皮素-B受体在纤连蛋白存在的情况下最有效地刺激[^3H]胸苷的掺入。这与ERK的磷酸化有关，PTX部分抑制内皮素刺激的DNA合成和ERK的磷酸化。这些发现表明，Gi 2介导来自受体如内皮素-B受体的信号传导，以维持发育中脑的神经祖细胞中的促有丝分裂活性。少