ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts.
ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts.
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DOI：
10.1083/jcb.200112107
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发表时间：
2002-05-27
影响因子：
7.8
通讯作者：
Narumiya, Shuh
中科院分区：
文献类型：
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作者：
Tsuji, Takahiro;Ishizaki, Toshimasa;Okamoto, Muneo;Higashida, Chiharu;Kimura, Kazuhiro;Furuyashiki, Tomoyuki;Arakawa, Yoshiki;Birge, Raymond B;Nakamoto, Tetsuya;Hirai, Hisamaru;Narumiya, Shuh
小GTPase Rho作用于ROCK和mDia1两种效应物，诱导应力纤维和局灶粘连。然而，ROCK和mDia1如何单独调节这些结构的信号和动力学仍然未知。我们用LPA刺激血清饥饿的瑞士3T3成纤维细胞，并比较C3外泌酶（Rho抑制剂）与Y-27632 （ROCK抑制剂）的作用。Y-27632处理抑制了lpa诱导的应力纤维的形成和局灶粘连，C3外泌酶也抑制了应激纤维的形成，但诱导了C3外泌酶处理的细胞中不存在的膜褶和局灶复合物。这种表型被N17Rac的表达所抑制。与此一致，Y-27632在lpa刺激的细胞中显著增加GTP-Rac的数量。生化上，Y-27632抑制paxillin和黏附激酶酪氨酸磷酸化，而不抑制Cas。用PP1抑制Cas磷酸化或表达显性阴性Cas突变体可抑制y -27632诱导的膜褶皱形成。此外，缺乏与磷酸化的Cas或DOCK180结合的Crk-II突变体抑制了y -27632诱导的膜褶皱形成。最后，显性mDia1负突变体的表达也抑制了Y-27632的膜褶皱形成。因此，这些结果揭示了rho依赖的Rac激活信号是由mDia1通过Cas磷酸化介导的，并被ROCK拮抗。
The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 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 focal adhesion kinase 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 mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.