The Mechanism Of Interaction Between Sedlin And RB1

Objective To construct the plasmids of Sedlin mutants and investigate their expression and co-localization with RB1 in mammalian cells.On this basis,the interaction between Sedlin mutant proteins and RB1 mutant proteins were verified by GST pulldown and co-immunoprecipitation(co-IP),respectively.The function of Sedlin in mammalian cells and its effect on the regulation of RB1 in cell cycle were explored by RNA interference(si RNA and sh RNA).Methods Sedlin mutants were amplified by PCR with the template including the full length c DNA fragment of Sedlin.Eukaryotic plasmids p CDGFP-Sedlin N/Y115A/ Y115 F and prokaryotic plasmids p GEX-3X-Sedlin N/Y115A/Y115 F were constructed,respectively.Immunofluorescence experiments in COS7 cells were performed to detect the co-localization of Sedlin mutants with RB1.The GST-Sedlin N fusion protein was purified,and the FLAG-tagged RB1-N plasmid was transfected into HEK 293 T cells for GST pulldown experiments to investigate the interaction between the Sedlin N and RB1-N in vitro.p CDGFP-Sedlin N/Y115A/Y115 F were transfected into HEK 293 T cells,the interaction between Sedlin N and RB1-N in vivo was studied by co-immunoprecipitation.Sedlin si RNA was transfected into osteosarcoma cell U2 OS and Lentiviral-packaged Sedlin sh RNA was infected into U2 OS cells to detect the expression of cell cycle-associated proteins RB1,P21,P27 and P53.Results The recombinant plasmids p CDGFP-Sedlin N/Y115A/Y115 F and p GEX-3XSedlin N/Y115A/Y115 F were constructed successfully,and the expression of sedlin mutants were found both in nucleus and in cytoplasms,and they colocalize with RB1 in nucleus.Sedlin-Y115A/Y115 F proteins co-localized with RB1-N protein and Sedlin N/Y115A/Y115 F proteins co-localized with RB1-C protein,while Sedlin N protein and RB1-N protein have not co-localization.Coomase brilliant blue staining showed that the fusion protein GST-Sedlin N /Y115A/Y115 F were efficiently expressed in BL21 cells,and Western blot results showed that GFP-Sedlin N/Y115A/Y115 F were highly expressed in HEK 293 T cells.GST pulldown and co-immunoprecipitation showed that there was no interaction between Sedlin N protein and RB1-N protein in vitro and in vivo.The results of RNA interference experiments showed that the expression of cell cycle-associated proteins RB1,P21,P27 and P53 were affected by the expression level of Sedlin protein.Conclusion In COS7 cells,Sedlin was mainly distributed in the nucleus,however,its mutants were expressed in both nucleus and cytoplasm,with a considerable amount in cytoplasm.The mutants were co-localized with RB1 protein,indicating that the carboxyl terminus of Sedlin and the phosphorylation of Y in NPFY motif did not affect its co-localization with RB1 protein.Sedlin N and RB1-N do not co-localize in COS7 cells and have no interaction,indicating that RB1 may bind to the N-terminus of Sedlin through its C-terminus,and Sedlin binds to the N-terminus of RB1 through its C-terminus.Inhibition of Sedlin expression by RNA interference,affects the expression levels of RB1,P21,P27 and P53 proteins,it is speculated that Sedlin may be located upstream of these genes,maybe as a transcription factor,and regulate the expression of these genes,for example,participate in the regulation of RB1 involved in cell cycle regulation.