Signal Crosstalk between Rho and Tyrosine phosphorylation

小的GTP酶Rho作用于ROCK和MDial两个效应器，并诱导应力性纤维和局部粘连。然而，ROCK和mDial如何分别调节这些结构的信号和动力学仍不清楚。我们用LPA刺激血清缺乏的瑞士3T3成纤维细胞，并比较了Rho抑制剂C3外酶和ROCK抑制剂Y-27632的作用。与C3胞外酶一样，Y-27632处理抑制了LPA诱导的应力纤维和灶性粘连的形成，但诱导了C3胞外酶处理的细胞中不存在的膜褶皱和灶性复合体。这种表型被N17Rac的表达抑制。在脂蛋白A刺激的细胞中，Y-27632能显著增加GTP-Rac的数量。生化方面，Y-27632抑制Paxlin和FAK的酪氨酸磷酸化，而不抑制CaS的酪氨酸磷酸化。用Pp1抑制CaS磷酸化或表达显性负性CaS突变体可抑制Y-27632诱导的膜褶皱形成。此外，缺乏与磷酸化CA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，显性负性mDial突变体的表达也抑制了Y-27632的膜褶皱的形成。这些结果揭示了Rho依赖的Rac激活信号，该信号是由mDial通过Cas磷酸化介导的，并被ROCK的作用所拮抗。

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.