c-Jun氨基末端激酶(JNK)的磷酸化是神经细胞损伤的重要原因。脑缺血再灌注后，细胞分裂周期蛋白42(Cdc42)与ASK1-MKK4-JNK信号模块结合，并促进其组装，增强JNK的磷酸化。但是，Cdc42是与该模块中的ASK1、MKK4或JNK直接作用还是与该模块的支架蛋白JIP3作用，以及具体的作用位点，目前尚不清楚。作用位点若能被探明，则可能成为新的药物靶点。.本项目拟通过哺乳动物双杂交技术，从JIP3、ASK1、MKK4和JNK中筛选出能与Cdc42直接作用的"靶蛋白"；用Pull-down、激光共聚焦、免疫沉淀、Cluspro蛋白模拟对接等技术验证与其作用的靶蛋白；再分别构建Cdc42和“靶蛋白”的位点突变体，用上述技术筛选并验证两者的作用位点；突变作用位点，探索其在ASK1-MKK4-JNK信号通路中调控神经细胞损伤的作用和机制，为缺血性脑损伤的治疗提供理论基础和实验依据。

The phosphorylation of c-Jun N-terminal kinase (JNK) is the key reason for neuronal injury. It was indicated that cerebral ischemia could facilitate the combination of cell division cycle protein 42 (Cdc42) with ASK1-MKK4-JNK signal module and induce the assembling of it, increasing the phosphorylation of JNK. However, which protein of ASK1, MKK4, JNK in this module or its scaffold protein.JIP3 can directly combine with Cdc42, and where are interacting specific sites between them, are still unclear. If the sites are found clearly, they may become new drug targets. In this project, mammalian two-hybrid system will be used to screen the ‘target protein’ which can directly combine with Cdc42 from JIP3, ASK1, MKK4 and JNK. Pull-down, confocal laser scanning , immunoprecipitetion and Cluspro protein-protein docking will be used to validate the ‘target protein’ . The sites mutagenesis of Cdc42 and ‘target protein’ will be constructed to screen and validate the interacting sites between Cdc42 and ‘target protein’ using the above technologies. These interacting sites will be mutated to explore the effects and mechanisms on the regulating ASK1-MKK4-JNK signaling pathway in neuronal injury after cerebral ischemia. It will provide theoretical and experimental basis for the treatment of cerebral ischemic injury.