NLK negatively regulate TGFβsignaling pathway via Smad4 / 重庆医学

Objective To study the effect of NLK on TGFβsignaling pathway and explore the molecular mechanism .Methods Protein stability assay was used to determine the influence of NLK on the degradation of Smad 4 .In vivo ubiquitination assay was applied to detect the effect of NLK on the ubiquitination of Smad4 .Luciferase reporter gene assay was used to detect the effect of NLK on CAGA‐luc and 3TP‐luc ,the responsive reporter of TGFβ signaling pathway .Real time PCR was applied to examine the effect of NLK on the expression of p21 and PAI‐1 ,the target genes of TGFβsignaling pathway .Results In HEK293T cell ,over ex‐pression of NLK promotes the degradation and ubiquitination of Smad4 .In HEK293T cells ,Ectopic expression of NLK inhibits the activity of CAGA luc and 3TP luc stimulated by TGFβ.In HepG2 cells ,over expression of NLK inhibits the expression of p21 and PAI 1 ,the target genes of TGFβsignaling pathway .Conclusion NLK promotes the ubiquitination and degradation of Smad4 ,conse‐quently inhibits TGFβsignaling pathway .

NLK inhibits the transcription activity of Smad4 independent of its kinase activity / 军事医学

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 .