Regulation Of DHX33 By GSK-3? Through Phosphorylation Mediated Protein Degradation

DEAD/H box protein family is the largest protein family of RNA helicases.They are highly conserved in all eukaryotic cells as well as in many bacteria and archaea.DHX33 is a member of DEAD/H box protein family.As a RNA helicase,DHX33 is involved not only in several RNA metabolic activities but also in DNA metabolism.It plays diverse roles in multiple cellular activities.As a key regulator in cell cycle progression,dysregulation of DHX33,especially overexpression,has been confirmed to associate with the development of several human cancers.Therefore,DHX33 has become a potential therapeutic target for cancers and deserves further investigation.Current studies of DHX33 mainly focus on its cellular functions.However,little attention has been paid to its post-transcriptional regulation.The expression level of DHX33 protein has been found to oscillate in different phases of cell cycle,however,the change of its protein levels did not fully match the change in its m RNA levels.Based on previous data,it hypothesizes that DHX33 might be regulated at the post-translational level through GSK-3b mediated protein degradation.In this study,colon cancer cell line HCT116 was treated with Li Cl(inhibitor of GSK-3b)at different doses for different lengths of time.Western blot and Real-time quantitative polymerase chain reaction(RT-q PCR)were performed to detect the protein levels of DHX33.HCT116 cells were further treated with Li Cl and protease inhibitor MG132 simultaneously.Western blot was performed to detect the protein level of DHX33.Results suggest that GSK-3? correlates with DHX33 and might be responsible for DHX33 protein proteolysis.To explore whether GSK-3? protein interacts with DHX33 protein,we performed co-immunoprecipitation(IP)assay and the data suggest that GSK-3? protein physically associates with DHX33 protein in cells.To confirm the correlation between GSK-3? and DHX33,GSK-3? was either knocked down by sh RNAs targeting GSK-3b or overexpressed by transient transfection of GSK-3? expressing plasmids in HCT116 cells.GSK-3? inhibitor CT99021 was further applied in cells.Additionally,protein synthesis inhibitor cycloheximide(CHX)was used to treat HCT116 cells to confirm that GSK-3? causes DHX33 proteolysis.We performed Mass spectrometry(MS)to investigate whether GSK-3? protein phosphorylates DHX33 protein.Results demonstrated that human GSK-3? protein phosphorylated mouse DHX33 protein at T43 and T482.To figure out whether GSK-3? phosphorylation of DHX33 leads to DHX33 degradation,we performed site-directed mutagenesis(SDM)on DHX33 T482 to mimic its phosphorylation.Plasmids encoding wild type DHX33 or the phosphomimetic mutant DHX33 were transfected into HCT116 cells,respectively.Through protein stability analysis by cycloheximide treatment,we confirmed that phosphorylation of DHX33 by GSK-3?significantly decreased the half-life of DHX33 protein and expedited its degradation.This study,for the first time,reveals an important regulatory role of GSK-3? on DHX33 through post-translational modification,and warrant further investigation.