Nishiyama T, Sasaki T, Takaishi K, Kato M, Yaku H, Araki K, Matsuura Y, Takai Y
Department of Biochemistry, Kobe University School of Medicine, Japan.
Mol Cell Biol. 1994 Apr;14(4):2447-56. doi: 10.1128/mcb.14.4.2447-2456.1994.
Insulin and hepatocyte growth factor (HGF) induced morphologically different membrane rufflings in KB cells. Insulin-induced membrane ruffling was inhibited by microinjection of rho GDI, an inhibitory GDP/GTP exchange regulator for both rho p21 and rac p21 small GTP-binding proteins, but not inhibited by microinjection of botulinum exoenzyme C3, known to selectively ADP-ribosylate rho p21 and to impair its function. This rho GDI action was prevented by comicroinjection with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound rac1 p21. In contrast, HGF-induced membrane ruffling was inhibited by microinjection of rho GDI or C3. This rho GDI action was prevented by comicroinjection with GTP gamma S-bound rhoA p21, and this C3 action was prevented by comicroinjection with GTP gamma S-bound rhoAIle-41 p21, which is resistant to C3. Microinjection of either GTP gamma S-bound rac1 p21 or rhoA p21 alone induced membrane ruffling in the absence of the growth factors. The rac1 p21-induced membrane ruffling was morphologically similar to the insulin-induced kind, whereas rhoA p21-induced ruffling was apparently different from both the insulin- and HGF-induced kinds. Membrane ruffling was also induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore or microinjection of a dominant active Ki-ras p21 mutant (Ki-rasVal-12 p21). The phorbol ester-induced membrane ruffling was morphologically similar to the rhoA p21-induced kind and inhibited by microinjection of rho GDI or C3. These results indicate that rac p21 and rho GDI are involved in insulin-induced membrane ruffling and that rho p21 and rho GDI are involved in HGF- and phorbol ester-induced membrane rufflings.
胰岛素和肝细胞生长因子（HGF）在KB细胞中诱导出形态不同的膜 ruffling。胰岛素诱导的膜 ruffling 可被显微注射 rho GDI 抑制，rho GDI 是 rho p21 和 rac p21 小 GTP 结合蛋白的一种抑制性 GDP/GTP 交换调节因子，但不能被已知可选择性地对 rho p21 进行 ADP 核糖基化并损害其功能的肉毒杆菌外毒素 C3 的显微注射所抑制。这种 rho GDI 的作用可通过与结合了鸟苷 5'-(3-O-硫代)三磷酸（GTPγS）的 rac1 p21 共同显微注射来阻止。相反，HGF 诱导的膜 ruffling 可被 rho GDI 或 C3 的显微注射所抑制。这种 rho GDI 的作用可通过与结合了 GTPγS 的 rhoA p21 共同显微注射来阻止，而这种 C3 的作用可通过与对 C3 有抗性的结合了 GTPγS 的 rhoAIle-41 p21 共同显微注射来阻止。单独显微注射结合了 GTPγS 的 rac1 p21 或 rhoA p21 在没有生长因子的情况下也能诱导膜 ruffling。rac1 p21 诱导的膜 ruffling 在形态上与胰岛素诱导的相似，而 rhoA p21 诱导的 ruffling 明显不同于胰岛素和 HGF 诱导的。膜 ruffling 也可由 12-O-十四烷酰佛波醇-13-乙酸酯（TPA）诱导，TPA 是一种激活蛋白激酶 C 的佛波醇酯，但不能由 Ca2+离子载体或显性活性 Ki-ras p21 突变体（Ki-rasVal-12 p21）的显微注射诱导。佛波醇酯诱导的膜 ruffling 在形态上与 rhoA p21 诱导的相似，并可被 rho GDI 或 C3 的显微注射所抑制。这些结果表明 rac p21 和 rho GDI 参与胰岛素诱导的膜 ruffling，并且 rho p21 和 rho GDI 参与 HGF 和佛波醇酯诱导的膜 ruffling。
