Wu Di, Jiao Meng, Zu Shicheng, Sollecito Christopher C, Jimenez-Cowell Kevin, Mold Alexander J, Kennedy Ryan M, Wei Qize
From the Department of Biological Sciences, Fordham University, Bronx, New York 10458.
From the Department of Biological Sciences, Fordham University, Bronx, New York 10458
J Biol Chem. 2014 Dec 5;289(49):34033-48. doi: 10.1074/jbc.M114.607267. Epub 2014 Oct 21.
We have reported previously that nonmuscle myosin II-interacting guanine nucleotide exchange factor (MyoGEF) plays an important role in the regulation of cell migration and cytokinesis. Like many other guanine nucleotide exchange factors (GEFs), MyoGEF contains a Dbl homology (DH) domain and a pleckstrin homology domain. In this study, we provide evidence demonstrating that intramolecular interactions between the DH domain (residues 162-351) and the carboxyl-terminal region (501-790) of MyoGEF can inhibit MyoGEF functions. In vitro and in vivo pulldown assays showed that the carboxyl-terminal region (residues 501-790) of MyoGEF could interact with the DH domain but not with the pleckstrin homology domain. Expression of a MyoGEF carboxyl-terminal fragment (residues 501-790) decreased RhoA activation and suppressed actin filament formation in MDA-MB-231 breast cancer cells. Additionally, Matrigel invasion assays showed that exogenous expression of the MyoGEF carboxyl-terminal region decreased the invasion activity of MDA-MB-231 cells. Moreover, coimmunoprecipitation assays showed that phosphorylation of the MyoGEF carboxyl-terminal region by aurora B kinase interfered with the intramolecular interactions of MyoGEF. Furthermore, expression of the MyoGEF carboxyl-terminal region interfered with RhoA localization during cytokinesis and led to an increase in multinucleation. Together, our findings suggest that binding of the carboxyl-terminal region of MyoGEF to its DH domain acts as an autoinhibitory mechanism for the regulation of MyoGEF activation.
我们之前报道过，非肌肉肌球蛋白II相互作用鸟嘌呤核苷酸交换因子（MyoGEF）在细胞迁移和胞质分裂的调节中起重要作用。与许多其他鸟嘌呤核苷酸交换因子（GEF）一样，MyoGEF包含一个Dbl同源结构域（DH结构域）和一个普列克底物蛋白同源结构域。在本研究中，我们提供证据表明，MyoGEF的DH结构域（第162 - 351位氨基酸残基）与羧基末端区域（第501 - 790位氨基酸残基）之间的分子内相互作用可抑制MyoGEF的功能。体外和体内下拉实验表明，MyoGEF的羧基末端区域（第501 - 790位氨基酸残基）可与DH结构域相互作用，但不与普列克底物蛋白同源结构域相互作用。MyoGEF羧基末端片段（第501 - 790位氨基酸残基）的表达降低了RhoA的激活，并抑制了MDA - MB - 231乳腺癌细胞中肌动蛋白丝的形成。此外，基质胶侵袭实验表明，MyoGEF羧基末端区域的外源表达降低了MDA - MB - 231细胞的侵袭活性。而且，免疫共沉淀实验表明，极光B激酶对MyoGEF羧基末端区域的磷酸化干扰了MyoGEF的分子内相互作用。此外，MyoGEF羧基末端区域的表达在胞质分裂过程中干扰了RhoA的定位，并导致多核化增加。总之，我们的研究结果表明，MyoGEF羧基末端区域与其DH结构域的结合作为一种自我抑制机制来调节MyoGEF的激活。
