Roles of cofilin in regulation of actin cytoskeleton dynamics

丝切蛋白在肌动蛋白细胞骨架动力学调节中的作用

基本信息

批准号：
17570152
负责人：
OHASHI Kazumasa
金额：
$ 2.24万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570152/
关键词：
Actin cytoskeleton cofilin LIM-kinase Slingshot GFP Phosphorylation Inhibitor イメージングダイナミクス

项目摘要

Actin cytoskeletal dynamics and reorganization play essential roles in various intracellular events, including cell migration, cytokinesis, endocytosis, and polarity formation. Actin dynamics and reorganization are spatiotemporally regulated by a large number of actin-binding proteins and upstream signaling molecules. Cofilin, a key regulator of actin filament dynamics, stimulates depolymerization and severing of actin filaments. Cofilin is inactivated by phosphorylation of Ser-3 by LIM-kinase (LIMK). The Ser-3-phosphorylated cofilin (P-cofilin) is dephosphorylated and reactivated by the Slingshot phosphatase. The purposes of this project were to reveal the function of cofilin in actin cytoskeleton reorganization. In the present study, we have developed a new probe that detects the actin-binding activity of cofilin using a bimolecular fluorescence complementation (BiFC) method. The probe was improved higher intensity and sensitivity in this study. We expressed and purified these probe proteins in insect cells using a baculovirus expressing system, and developed methods to measure the actin-binding activity of cofilin, the kinase activity of LIMK, and the phosphatase activity of Slingshot with the fluorescence of complemented-GFP. We screened the inhibitors of cofilin, LIMK and Slingshot with over five hundred chemical compounds. As a consequence, LIMK was inhibited two compounds which was known as Src tyrosine kinase inhibitor. The compounds inhibited the hyper-polymerization of actin in LIMK overexpressing cells. It was reported that one of the compounds inhibited cell migration and neurite extension at the concentration of LIMK inhibition. We are tring to analyze the role of cofilin phosphorylation in the actin cytoskeleton reorganization using the compounds in living cells. We are going to screen more specific inhibitors for LIMK in derivative forms of the compounds. These inhibitors will be seeds of the drugs for inhibition of metastasis and inflammation.

肌动蛋白细胞骨架动力学和重组在各种细胞内事件中起着重要作用，包括细胞迁移，细胞因子，内吞作用和极性形成。肌动蛋白动力学和重组由大量肌动蛋白结合蛋白和上游信号分子进行空间调节。 Cofilin是肌动蛋白丝动力学的关键调节剂，刺激肌动蛋白丝的解聚和切断。 Cofilin通过LIM-激酶（Limk）的Ser-3磷酸化灭活。 SER-3磷酸化的Cofilin（P-Cofilin）被弹弓磷酸酶脱磷酸化并重新激活。该项目的目的是揭示Cofilin在肌动蛋白细胞骨架重组中的功能。在本研究中，我们开发了一种新的探针，该探针使用双分子荧光互补（BIFC）方法检测Cofilin的肌动蛋白结合活性。在这项研究中，探针提高了较高的强度和灵敏度。我们使用杆状病毒表达系统在昆虫细胞中表达并纯化了这些探针蛋白，并开发了测量Cofilin的肌动蛋白结合活性，Limk的激酶活性以及Slingshot伴有互补GFP的荧光的磷酸酶活性。我们筛选了Cofilin，Limk和Slingshot的抑制剂，并具有超过五百个化合物的抑制剂。因此，limk被抑制了两种化合物，这些化合物被称为SRC酪氨酸激酶抑制剂。这些化合物抑制了过表达细胞中肌动蛋白的过度聚合。据报道，其中一种化合物以limk抑制的浓度抑制细胞迁移和神经突延伸。我们正在使用活细胞中的化合物分析Cofilin磷酸化在肌动蛋白细胞骨架重组中的作用。我们将在化合物的衍生形式中筛选更特异性的抑制剂。这些抑制剂将是抑制转移和炎症的药物的种子。