Establishment of a novel method visualizing kinase activity in spatiotemporal mode

建立时空模式下可视化激酶活性的新方法

基本信息

批准号：
12558085
负责人：
INAGAKI Masaki
金额：
$ 8.77万
依托单位：
AICHI CANCER CENTER
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12558085/
关键词：
Protein kinase phosphorylation antibody Rho kinase Rho GEF KIAA380 Rho GEF 神経突起

项目摘要

The Rho/Rho-kinase signaling pathway plays an essential role in neurite retraction and cell rounding in response to G12/13-coupled receptor activation in neuronal cells. The Rho guanine nucleotide exchange factor (RhoGEF) involved in these processes has not been identified. To monitor the activation state of Rho-kinase, we developed a vimentin head/Rho-kinase chimera which is intramolecularly phosphorylated in a Rho-dependent manner at Ser71 of the fused vimentin head. Using this system, we identified a clone termed KIAA0380, which contains the Ga12/13-binding domain as well as a tandem of the DH/PH domain, as an activator of Rho/Rho-kinase signaling. Since KIAA0380 is composed of various functional domains that are commonly found in signaling molecules, we prepared several mutants and analyzed the physiological significance of the functional domains. Interestingly, in addition to DH/PH domain, a proline-rich motif which is C-terminally adjacent to the DH/PH domain is also essential for plasma membrane location of KIAA0380, cortical actin reorganization and cell rounding. In Neuro2a cells, the N-terminus fragment of KIAA0380 inhibited LPA-mediated neurite retraction. Immunological analysis revealed that KIAA0380 is well expressed in Neuro2a cells, but another Ga12/13 binding p115 RhoGEF, was not detected. Taken together, these findings suggest that in neuronal cells, KIAA0380 functions as a RhoGEF at the cell periphery and regulates G12/13-coupled receptor-mediated Rho activation, an event essential for neurite retraction and growth cone collapse.

Rho/Rho激酶信号通路在神经元细胞中对G12/13偶联受体激活的响应中，在神经突收缩和细胞舍入中起着至关重要的作用。尚未确定参与这些过程的Rho鸟嘌呤核苷酸交换因子（Rhogef）。为了监测Rho-kinase的激活状态，我们开发了一种波形蛋白头/Rho-kinase Chimera，该嵌合体在熔融波形蛋白头的Ser71上以Rho依赖性方式被分子内磷酸化。使用此系统，我们确定了一个称为KIAA0380的克隆，该克隆包含GA12/13结合域以及DH/pH域的串联，作为Rho/Rho-kinase信号传导的激活因子。由于KIAA0380由信号分子中通常发现的各种功能域组成，因此我们准备了几个突变体并分析了功能域的生理意义。有趣的是，除了DH/pH结构域外，与DH/pH结构域相邻的脯氨酸富基序也是KIAA0380的质膜位置，皮质肌动蛋白重组和细胞圆形的位置也是必不可少的。在Neuro2a细胞中，KIAA0380的N末端片段抑制了LPA介导的神经突收缩。免疫学分析表明，KIAA0380在Neuro2a细胞中很好地表达，但未检测到另一种GA12/13结合P115 Rhogef。综上所述，这些发现表明，在神经元细胞中，KIAA0380在细胞周围充当Rhogef，并调节G12/13偶联受体介导的Rho激活，这是神经突收回和生长锥塌陷必不可少的事件。