Study Of The Regulation Of DNA-PKcs To SIK2

Research DNA-PKcs regulate the Stability of SIK2 with cell knockdown DNA-PKcs or treated with inhibitor Nu7026. Purify SIK2 protein in vitro, detect the phosphorylation regulation of DNA-PKcs to SIK2 and to futher define the phosphorylation site and phosphorylation mechanism.Observe the changes of SIK2 with cell knockdown DNA-PKcs or treated with inhibitor Nu7026; and then detect DNA-PKcs regulate the halftime of SIK2 with cell treated with CHX. Immune coprecipitation technology detect the Ub-mediated degradation of DNA-PKcs to SIK2 with cell knockdown DNA-PKcs or treated with inhibitor Nu7026. Construct his-tag SIK2 plasmid and then transfer to E. coil BL21 induce and purify SIK2 protein by prokaryotic expression system. Establish phosphorylation reaction system in vitro to research the phosphorylation regulation of DNA-PKcs to SIK2, and detect the(auto)phosphorylation site of SIK2 by mass spectrometric analysis. Labelled DNA with 32 P, detect SIK2 binding DNA and whether DNA-PKcs impact the bingding ability of SIK2 by Electrophoretic Mobility Shift Assay.The results shows : Knockdown or inhibited DNA-PKcs with Nu7026, SIK2’s expression decrease; and the cell treated with CHX, the harftime of SIK2 was shorten. Immune coprecipitation shows SIK2 ubiquitylation obviously enhangced while knockdown or inhibited DNA-PKcs with Nu7026. Prokaryotic expression shows, successfully construct his-tag SIK2 recombinant plasmid, induce and purify SIK2 protein in E. coil BL21. Phosphorylation reaction system in vitro shows that SIK2 can autophosphorylate and DNA-PKcs can also phosphorylate SIK2; and through mass spectrometric define the autophosphorylation site of SIK2 at site S358、S515、S534 and phosphorylation at site S512、S515、S534. SIK2 can bind to DNA, and DNA-PKcs can stable the ability of SIK2 binding DNA; while DNA-PKcs can bind to DNA,but on the contrary, SIK2 inhibits DNA-PKcs bind to DNA.DNA-PKcs can regulate the stability of SIK2, SIK2’s expression decrease and the harftime of SIK2 was shorten and ubiquitylation obviously enhangced in DNA-PKcs knockdown or inhibited with Nu7026 cell. SIK2 can autophosphorylate at site S358、S515、S534 and DNA-PKcs can also phosphorylate SIK2 at site S512、S515、S534. DNA-PKcs can autophosphorylate while SIK2 inhibit its phosphorylation. SIK2 can bind to DNA, and DNA-PKcs can stable the ability of SIK2 binding DNA. DNA-PKcs can bind to DNA,but on the contrary, SIK2 inhibits DNA-PKcs bind to DNA.