丝裂原活化蛋白激酶激酶激酶3与细胞核分离基因C相互作用的研究

The study of the Interaction between MEKK3 and NudC

目的验证丝裂原活化蛋白激酶激酶激酶3(mitogen-activated protein kinase kinase ki-nase3,MAPK/ERK kinase kinase3,MEKK3)与细胞核分离基因C(Nuclear distribution gene C,NudC)的表达产物NudC相互作用的真实性,以及探讨MEKK3是否为NudC的一个上游调控激酶。方法构建了一系列的载体,通过GST-Pull down技术,免疫共沉淀技术,从体外和体内两个方面来验证MEKK3和NudC之间能否相互作用,又利用体外模拟磷酸化实验来证明MEKK3是否为NudC的一个上游调控激酶。结果GST-Pull down以及免疫共沉淀技术显示MEKK3和NudC在体内和体外均能够相互结合,体外模拟磷酸化实验表明在体外野生型MEKK3可以磷酸化NudC,而激酶失活型MEKK3不能。结论MEKK3和NudC之间能够相互作用,而且MEKK3可能为NudC的一个上游调控激酶。

Objective To confirm the interaction between MEKK3 and NudC and explore phosphorylate effct of MEKK3 on NudC. Methods We used GST-Pull down assay and Co-immunoprecipitation to verify the interaction between MEKK3 and NudC and explored in vitro kinase assay to examine if NudC can be phosphorylated by MEKK3. Results Co-immunoprecipitation assay demonstrated the interaction between MEKK3 and NudC in vivo. In vitro kinase assay showed that wild type MEKK3 （MEKK3 WT） phosphorylated NudC,whereas the kinase-dead MEKK3 failed to phosphorylate NudC. Conclusions The interaction between MEKK3 and NudC is true and NudC can be phosphorylated by MEKK3.