Tsuji Takahiro, Ishizaki Toshimasa, Okamoto Muneo, Higashida Chiharu, Kimura Kazuhiro, Furuyashiki Tomoyuki, Arakawa Yoshiki, Birge Raymond B, Nakamoto Tetsuya, Hirai Hisamaru, Narumiya Shuh
Department of Pharmacology, Kyoto University Faculty of Medicine, 606-8501, Japan.
J Cell Biol. 2002 May 27;157(5):819-30. doi: 10.1083/jcb.200112107. Epub 2002 May 20.
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.
小GTP酶Rho作用于两种效应器ROCK和mDia1，并诱导应力纤维和粘着斑的形成。然而，ROCK和mDia1如何分别调节这些结构的信号和动力学仍不清楚。我们用溶血磷脂酸（LPA）刺激血清饥饿的瑞士3T3成纤维细胞，并比较了Rho抑制剂C3外切酶和ROCK抑制剂Y-27632的作用。Y-27632处理抑制了LPA诱导的应力纤维和粘着斑的形成，C3外切酶也有同样的效果，但Y-27632诱导了膜皱褶和粘着斑复合体的形成，而C3外切酶处理的细胞中没有这些结构。这种表型被N17Rac的表达所抑制。一致地，在LPA刺激的细胞中，Y-27632使GTP-Rac的量显著增加。在生化方面，Y-27632抑制桩蛋白和粘着斑激酶的酪氨酸磷酸化，但不抑制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诱导的膜皱褶形成。此外，缺乏与磷酸化Cas或DOCK180结合能力的Crk-II突变体抑制了Y-27632诱导的膜皱褶形成。最后，显性负性mDia1突变体的表达也抑制了Y-27632诱导的膜皱褶形成。因此，这些结果揭示了由mDia1通过Cas磷酸化介导并被ROCK的作用拮抗的Rho依赖性Rac激活信号通路。
