Signal Crosstalk between Rho and Tyrosine phosphorylation

Rho 和酪氨酸磷酸化之间的信号串扰

基本信息

批准号：
12670112
负责人：
ISHIZAKI Toshimasa
金额：
$ 2.18万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670112/
关键词：
Rho actin cytoskeleton cas focal adhesion Rock mDia Cell migration cell polarity チロシンリン酸化微小管細胞運動 Rac

项目摘要

The small GTPase Rho acts on two effectors, ROCK and mDial, and induces stress fibers and focal adhesions. However, how ROCK and mDial individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA, and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme, but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine-phosphorylation of paxillin and FAK and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDial mutant inhibited also the membrane ruffle formation by Y-27632. These results have thus revealed the Rho-dependent Rac activation signaling that is mediated by mDial through Cas phosphorylation, and is antagonized by the action of ROCK.

小的GTPase RHO作用于两个效应子，即岩石和MDIAL，并诱导应力纤维和局灶性粘连。但是，岩石和MDial如何单独调节这些结构的信号和动力学仍然未知。我们刺激了血清饥饿的瑞士3T3成纤维细胞与LPA，并将RHO抑制剂C3外酶与岩石抑制剂Y-27632的C3外酶的作用进行了比较。 Y-27632治疗抑制了LPA诱导的应激纤维和局灶性粘附的形成，就像C3外酶一样，但是诱导的膜褶皱和局灶性复合物在C3外酶处理的细胞中不存在。 N17RAC的表达抑制了这种表型。一致地，LPA刺激的细胞中Y-27632的GTP-RAC量显着增加。从生化上，Y-27632抑制了帕克西林和FAK的酪氨酸磷酸化，而不是CAS的磷酸化。用PP1抑制CAS磷酸化或显性阴性CAS突变体的表达抑制Y-27632诱导的膜褶皱的形成。此外，缺乏与磷酸化的CAS或DOCK180结合的CRK-II突变体抑制了Y-27632诱导的膜褶皱的形成。最后，Y-27632的显性阴性MDIAL突变体的表达也抑制了膜褶皱的形成。因此，这些结果揭示了由MDIAL通过CAS磷酸化介导的Rho依赖性RAC激活信号传导，并由岩石的作用拮抗。