Establishment of a novel method visualizing kinase activity in spatiotemporal mode

Rho/Rho-Kinase信号通路在神经细胞G12/13偶联受体激活后的轴突收缩和细胞回绕中起重要作用。参与这些过程的Rho鸟嘌呤核苷酸交换因子(Rhogef)尚未确定。为了监测Rho-Kinase的激活状态，我们开发了一个Vho-Head/Rho-Kinase嵌合体，该嵌合体在融合的Vimentin Head的Ser71处以Rho依赖的方式分子内磷酸化。利用这个系统，我们鉴定了一个命名为KIAA0380的克隆，它含有GA12/13结合结构域和一个串联的DH/PH结构域，是Rho/Rho-Kinase信号的激活剂。由于KIAA0380是由信号分子中常见的各种功能结构域组成的，我们制备了几个突变体，并分析了这些功能结构域的生理意义。有趣的是，除了DH/PH结构域之外，一个C端邻近DH/PH结构域的富含Pro的基序对于KIAA0380的质膜定位、皮质肌动蛋白重组和细胞圆形也是必不可少的。在Neuro2a细胞中，KIAA0380的N末端片段可抑制LPA介导的突起回缩。免疫学分析表明，KIAA0380在Neuro2a细胞中有很好的表达，但没有检测到另一个与GA12/13结合的p115 Rhogef。综上所述，这些发现表明，在神经细胞中，KIAA0380作为细胞外围的Rhogef发挥作用，并调节G12/13偶联受体介导的Rho激活，这是轴突回缩和生长锥体崩溃所必需的事件。

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.