Takaishi K, Sasaki T, Kameyama T, Tsukita S, Tsukita S, Takai Y
Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Japan.
Oncogene. 1995 Jul 6;11(1):39-48.
Rho small GTP-binding protein regulates various cell functions, such as formation of stress fibers and focal adhesions, cell motility, membrane ruffling, cytokinesis and smooth muscle contraction in mammalian cells and bud formation in the yeast Saccharomyces cerevisiae. As to the functioning sites of Rho in Saccharomyces cerevisiae, we have recently shown that RHO1 protein, a homologue of mammalian RhoA, is concentrated to the growth region of the cells where cortical actin patches are clustered. However, in mammalian cells, the functioning sites of Rho have not yet been studied. In the present study, MDCK cell lines stably expressing myc-tagged RhoA (myc-RhoA) were prepared and localization of myc-RhoA was first immunohistochemically examined using an anti-myc antibody. In the resting cells, almost all of myc-RhoA was observed in the cytosol. When the cells were stimulated with phorbol ester or hepatocyte growth factor, membrane rufflings were induced and myc-RhoA was translocated to the membrane ruffling area. Moreover, myc-RhoA was translocated from the cytosol to the cell-cell adhesion sites when the cells were transferred from a low to normal Ca2+ medium. RhoA was also concentrated to the cleavage furrows during cytokinesis in Swiss 3T3 cells. Translocation of myc-RhoA to the membrane ruffling area was inhibited by prior microinjection into the cells of Rho GDI, a negative regulator of Rho which inhibits activation of Rho, or of C3, an exoenzyme of Clostridium botulinum which ADP-ribosylates Rho and inhibits its functions, indicating that both activation and functioning of Rho are essential for the translocation of Rho. The ERM (Ezrin, Radixin, Moesin) family members were colocalized with RhoA at all of these sites. However, RhoA was not apparently observed at the focal adhesion plaque where vinculin was localized. These results suggest that at least one of the functioning sites of Rho is the ERM family-controlled actin filament/plasma membrane association sites.
Rho小GTP结合蛋白调节多种细胞功能，如应激纤维和粘着斑的形成、细胞运动、膜皱褶、胞质分裂以及哺乳动物细胞中的平滑肌收缩和酿酒酵母中的芽形成。关于Rho在酿酒酵母中的作用位点，我们最近发现，哺乳动物RhoA的同源物RHO1蛋白集中在细胞的生长区域，此处皮质肌动蛋白斑块聚集。然而，在哺乳动物细胞中，Rho的作用位点尚未得到研究。在本研究中，制备了稳定表达myc标记的RhoA（myc-RhoA）的MDCK细胞系，并首先使用抗myc抗体通过免疫组织化学方法检测myc-RhoA的定位。在静止细胞中，几乎所有的myc-RhoA都存在于细胞质中。当细胞用佛波酯或肝细胞生长因子刺激时，会诱导膜皱褶，并且myc-RhoA会转位到膜皱褶区域。此外，当细胞从低钙培养基转移到正常钙培养基时，myc-RhoA会从细胞质转位到细胞-细胞粘附位点。在瑞士3T3细胞胞质分裂期间，RhoA也集中在分裂沟处。将Rho的负调节剂Rho GDI或肉毒杆菌外切酶C3预先显微注射到细胞中可抑制myc-RhoA转位到膜皱褶区域，Rho GDI抑制Rho的激活，C3使Rho ADP核糖基化并抑制其功能，这表明Rho的激活和功能对于Rho的转位都是必不可少的。ERM（埃兹蛋白、根蛋白、膜突蛋白）家族成员在所有这些位点与RhoA共定位。然而，在纽蛋白定位的粘着斑处未明显观察到RhoA。这些结果表明，Rho的作用位点至少有一个是ERM家族控制下的肌动蛋白丝/质膜结合位点。
