Establishment of a novel method visualizing kinase activity in spatiotemporal mode

Rho/Rho激酶信号通路在神经元细胞中响应于G12/13偶联受体激活的神经突收缩和细胞变圆中起重要作用。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-激酶的活化状态，我们开发了波形蛋白头/Rho-激酶嵌合体，其在融合的波形蛋白头的Ser 71以Rho依赖的方式被分子内磷酸化。使用这个系统，我们确定了一个克隆称为KIAA 0380，它包含的Ga 12/13结合结构域以及DH/PH结构域的串联，作为Rho/Rho激酶信号的激活剂。由于KIAA 0380由信号分子中常见的各种功能结构域组成，因此我们制备了几种突变体并分析了功能结构域的生理意义。有趣的是，除了DH/PH结构域之外，与DH/PH结构域C端相邻的富含脯氨酸的基序对于KIAA 0380的质膜定位、皮层肌动蛋白重组和细胞变圆也是必需的。在Neuro 2a细胞中，KIAA 0380的N-末端片段抑制LPA介导的神经突收缩。免疫学分析显示KIAA 0380在Neuro 2a细胞中表达良好，但未检测到另一个Ga 12/13结合p115 RhoGEF。综上所述，这些发现表明，在神经元细胞中，KIAA 0380作为RhoGEF在细胞外周发挥作用，并调节G12/13偶联受体介导的Rho激活，这是神经突收缩和生长锥塌陷所必需的事件。

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.