Interaction And Function Of 14-3-3 Proteins With OGT

Background and objectiveSince the 14-3-3 proteins were found,more and more functions of 14-3-3 proteins have been reported.Seven isoforms of 14-3-3 proteins were encoded by different genes form homologous dimer or heterodimer in mammals.It is a highly-conserved family of proteins which interacts with hundreds of proteins and plays a very important role in neuronal development,signal transduction,immune response,protein transportation,cell cycle progression and apoptosis.The 14-3-3 proteins family is also involved in many neurological diseases and closely related to the occurrence of a variety of tumors.O-linked N-acetylglucosamine(O-Glc NAc,O-linked ?-N-acetylglucosamine)is a dynamic and inducible post-translational modification of proteins,which occurs at serine or threonine sites of proteins in both nucleus and cytoplasm.This dynamic modification is mainly regulated by the O-Linked N-acetylglucosamine Transferase(O-Glc NAc transferase,OGT)and O-linked N-acetylglucosamine hydrolase(O-Glc NAcase,OGA).OGT can modify serine or threonine sites of proteins by O-Glc NAc modification,while OGA can catalyze cleavage of O-Glc NAc from proteins,which plays an important role in regulating gene transcription,cell signaling and stress response.The purpose of this study is to explore whether there is an interaction between 14-3-3 proteins and OGT and to reveal the molecular mechanism of the interaction,so as to provide a theoretical basis for further research on the biological function of 14-3-3 proteins on protein O-Glc NAc modification.MethodThe plasmid with target gene was constructed by molecular cloning technology.The expression level of the target protein was detected by Western blot and Coomassie brilliant blue staining.And the interaction between 14-3-3 proteins and OGT was detected by co-immunoprecipitation assay.ResultWe identified that 14-3-3 proteins were OGT-associated proteins.The interaction between 14-3-3 protiens and OGT was not regulated by phosphorylation modification,nor by DNA damage,but by O-Glc NAc modification.When cells were treated by serum starvation or EBSS culture medium to induce autophagy,the interaction between 14-3-3 proteins and OGT was enhanced.We proposed that OGT activity might be regulated by 14-3-3 proteins which served as the adaptor protein mediated the interaction between OGT and its substrates.ConclusionUsing unbiased protein purification,14-3-3 proteins were found as the novel binding partners of OGT.We uncovered that the interaction between 14-3-3 proteins and OGT may depend on the O-Glc NAc modification on OGT and was enhanced when cells were treated by serum starvation or EBSS culture medium.