Proteasome Activity Is Inhibited By Casein Kinase ?

The ubiquitin-proteasome system is one of the main pathways for protein degradation in cells.Its main function is to degrade damaged or misfolded proteins.It has been reported that phosphorylation of 20 S proteasome alpha subunit C8(?7)stabilizes the 26 S proteasome by Casein kinase II(CK?).However,the effect of CK? on the activity of the proteasome is still unresolved.Thus,we performed series of molecular and cellular experiments to answer this question.Methods: series of in vitro and in vivo experiments were designed to evaluate the effect of CK? on the activity of the proteasome.Firstly,to evaluate the effect of CK? on proteasome activity in the cells,NRK or 293 T cells were treated with CK? inhibitors(DMAT,CX4945,TBB)for different time before the cells were harvested,and then to prepare nuclear extracts.The chymotrypsin-like peptidase activity and trypsin-like peptidase activity of the proteasome were evaluated with fluorogenic peptide substrates Suc-LLVY-AMC or Boc-LSTR-AMC respectively.To evaluate the accumulation of the ubiquitinated proteins,NRK or 293 T cells were treated with CK? inhibitors(0.5/1 uM DMAT,0.25/0.5/1/5 uM CX-4945,1/5/10 uM TBB)overnight respectively,the precipitates of the cell lysate were collected and dissolved with 1X SDS sample buffer,The dissolved pellets were then resolved with SDS-PAGE and blotted with anti-ubiquitin antibody.The GFP-CL1 plasmid was transfected into 293 T cells,and the cells were treated with CK? inhibitor(1 uM DMAT,5 uM CX-4945,10 uM TBB)overnight,and the fluorescence content of GFP in the cells was measured.Secondly,we observed the existence and localization of CK? in cells.The holoenzyme of CK? is a teterotetramer,which is composed of two catalytic subunits(? or ?')and two ?regulatory subunits.Immunofluorescence assay was used to detect the localization of CK? subunits in cells.The nuclear proteins and cytosolic proteins of normal NRK and293 T cells were islolated,followed by Western blot to detect the expression of CK? ?and CK? ?'.Thirdly,to determine the effect of CK? on proteasome activity in vitro,NRK cell nuclear extract,purified 26 S or 20 S proteasome were treated with 1U CK?,the peptidase activities were detected with the fluorogenic peptide substrates.Results:(1)CK? inhibitors activate proteasome activity in the cell.Treatment of NRK cells with 1 uM DMAT,5 uM CX-4945,10 uM TBB significantly activatedchymotrypsin-like and trypsin-like peptidase activities in nuclear extract at 16 h.Time course experiment showed that 5 uM TBB was able to activate the peptidase activity in NRK cells as early as 30 min,while DMAT and CX-4945 showed activation at 2h and4 h respectively.In 293 T cells,CK? inhibitors DMAT(0.5 uM),CX-4945(0.25 uM)and TBB(1 uM)ware able to significantly activate the peptidase activities at 30 min.CK? inhibitors(0.5/1 uM DMAT,0.25/0.5/1/5 uM CX-4945,10 uM TBB)also accelerated the clearance of ubiquitinated proteins in both NRK and 293 T cells.CK? inhibitors had no significant effect on the degradation of GFP-CL1.(2)CK? ?,CK? ??and CK? ? are mainly localized in the nucleus of NRK and 293 T cells.The expression of CK? ? and CK? ?' in the nuclear extract of NRK and 293 T cells was significantly higher than that in the cytoplasm.(3)In vitro experiment showed that CK? inhibited the peptidase activities of the proteasome in NRK cell nuclear extract,as well as purified26 S and 20 S proteasome.Conclusion: Casein kinase II inhibits the activity of proteasome.It directly acts on26 S and 20 S proteasome particles.