NLK以激酶非依赖的方式抑制Smad4的转录活性

NLK inhibits the transcription activity of Smad4 independent of its kinase activity

目的验证Nemo样激酶(NLK)与Smad4之间的相互作用并探讨NLK对Smad4功能的影响。方法应用免疫共沉淀和谷胱甘肽硫基转移酶(GST)沉降技术检测NLK与Smad4之间相互作用,GST沉降技术确定Smad4与NLK相互作用的结构域,荧光素酶报告基因实验检测NLK及NLK激酶活性突变体-NLK(KM)对Smad4转录活性的影响,体内磷酸化实验检测NLK对Smad4磷酸化的影响。结果免疫共沉淀和GST沉降的结果显示,NLK与Smad4在体内和体外存在相互作用。GST沉降的结果显示Smad4通过MH2结构域与NLK结合。荧光素酶报告基因实验的结果显示,NLK和NLK(KM)均可抑制Smad4的转录活性,且抑制程度相同。体内磷酸化的结果显示,NLK不能磷酸化Smad4。结论 NLK与Smad4相互作用,以激酶非依赖的方式抑制Smad4的转录活性,NLK抑制Smad4的分子机制有待进一步探讨。

Objective To confirm the interaction betweem NLK and Smad 4 and to explore the effect of NLK on the function of Smad4.Methods Co-immunoprecipitation and GST Pull-down were used to detect the interaction between NLK and Smad4.GST Pull-down was used to map the domain through which Smad 4 interacts with NLK.Luciferase reporter gene assay was used to study the effect of NLK and NLK （KM）, the NLK mutant lacking kinase activity , on the transcrip-tion activity of Smad4.In vivo phosphorylation assay was used to detect whether NLK phosphorylated Smad 4 or not.Results The data of Co-immunoprecipitation and GST Pull-down showed that NLK interacted with Smad 4 in vivo and in vitro.The result of GST Pull-down showed that Smad4 interacted with NLK via MH2 domain.The results of luciferase reporter gene assay indicated that both NLK and NLK （KM） inhibited the transcription activity of Smad4.The result of in vivo phospho-rylation assay showed that NLK could not phosphorylate Smad 4 in vivo.Conclusion NLK interacts with Smad4 and inhibits the transcription activity of Smad 4 independent of the kinase activity of NLK .The mechanism through which NLK negatively regulates the transcription of Smad 4 requires further research .