Kim Ja-Eun, Billadeau Daniel D, Chen Junjie
Department of Oncology Research, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905, USA.
J Biol Chem. 2005 Feb 18;280(7):5733-9. doi: 10.1074/jbc.M409298200. Epub 2004 Nov 15.
Epithelial cell transforming protein 2 (Ect2) is a guanine nucleotide exchange factor (GEF) for Rho GTPases, molecular switches essential for the control of cytokinesis in mammalian cells. Aside from the canonical Dbl homology/pleckstrin homology cassette found in virtually all Dbl family members, Ect2 contains N-terminal tandem BRCT domains. In this study, we address the role of the Ect2 BRCT domains in the regulation of Ect2 activity and cytokinesis. First, we show that the depletion of endogenous Ect2 by small interfering RNA induces multinucleation, suggesting that Ect2 is required for cytokinesis. In addition, we provide evidence that Ect2 normally exists in an inactive conformation, which is at least partially due to an intramolecular interaction between the BRCT domains and the C-terminal domain of Ect2. This intramolecular interaction masks the catalytic domain responsible for guanine nucleotide exchange toward RhoA. Consistent with a role in regulating Ect2 GEF activity, overexpression of an N-terminal Ect2 containing the tandem BRCT domains, but not single BRCT domain or BRCT domain mutant, leads to a failure in cytokinesis. Surprisingly, although ectopically expressed wild-type Ect2 rescues the multinucleation resulting from the depletion of endogenous Ect2, expression of a BRCT mutant of Ect2 failed to restore proper cytokinesis in these cells. Taken together, the results of our study indicate that the tandem BRCT domains of Ect2 play dual roles in the regulation of Ect2. Whereas these domains negatively regulate Ect2 GEF activity in interphase cells, they are also required for the proper function of Ect2 during cytokinesis.
上皮细胞转化蛋白2（Ect2）是一种针对Rho GTP酶的鸟嘌呤核苷酸交换因子（GEF），Rho GTP酶是哺乳动物细胞中控制胞质分裂所必需的分子开关。除了几乎所有Dbl家族成员中都存在的典型Dbl同源/普列克底物蛋白同源结构域外，Ect2还包含N端串联BRCT结构域。在本研究中，我们探讨了Ect2的BRCT结构域在调节Ect2活性和胞质分裂中的作用。首先，我们发现小干扰RNA对内源Ect2的敲低会诱导多核化，这表明Ect2是胞质分裂所必需的。此外，我们提供的证据表明，Ect2通常以无活性构象存在，这至少部分归因于BRCT结构域与Ect2 C端结构域之间的分子内相互作用。这种分子内相互作用掩盖了负责向RhoA进行鸟嘌呤核苷酸交换的催化结构域。与调节Ect2 GEF活性的作用一致，过表达含有串联BRCT结构域的N端Ect2（而非单个BRCT结构域或BRCT结构域突变体）会导致胞质分裂失败。令人惊讶的是，尽管异位表达的野生型Ect2可挽救因内源Ect2缺失而导致的多核化，但Ect2的BRCT突变体的表达未能在这些细胞中恢复正常的胞质分裂。综上所述，我们的研究结果表明，Ect2的串联BRCT结构域在调节Ect2方面发挥双重作用。这些结构域在间期细胞中负调节Ect2 GEF活性，而在胞质分裂期间它们也是Ect2正常功能所必需的。
