| The protein kinases,which are important to cellular signal transduction and critical for regμlating a large variety of cellular processes,can catalyze transfer of phosphates to serine,threonine and tyrosine residues.RIOK3,which contains a RIO domain,is a member of the human RIO family,although it is structurally different with other members.No report is concerned with the RIO family and in this thesis,we describe the function of the RIO proteins for the first time.Subcellμlar localization assay displayed that RIOK3 accumulated in cytoplasm. The results of FACS showed that both wild type and kinase dead mutant of RIOK3 have no effect on cell cycle.To elucidate the function of RIOK3 and related molec lar mechanisms,through yeast two hybrid screening we identified four proteins, CASP10,BIM,PAK2 and CDC42,which interact with RIOK3 in yeast,and then we made a further research on two pairs of interaction of them.Caspase-10 is a kind of initiator caspase in apoptosis and the N ter minal two DEDs can activate NFκB.We validate the interaction between RIOK3 and CASP10 using GST pull down assay,co-immunoprecipitation assay and fluorescent subcellular co-localization assay.The series deletion mutants experiments suggested that the RIO domain of RIOK3 mediated the interaction between RIOK3 and CASP10:RIOK3 simultaneous interacted with both N terminal and C terminal DED of CASP10;single RIO domain had no interaction with CASP 10 and the N ter minal sequence of RIOK3 facilitated RIO domains interaction with single DED of CASP10.RIOK3 did not co-immunoprecipitated with CASPS,which is CASP10 homolog and also has two DEDs,and RIOK1,which is RIOK3 homolog and has a RIO domain,did not co-immunoprecipitated with CASP10.The results demonstrated the specificity of the interaction between RIOK3 and CASP10.RIOK3,firstly identified as a new member of NFκB pathway inhibited the NFκB activation induced by TNFα,which was dependant on its interaction with CASP10:because when co-transfected with truncated mutant of RIOK3,RIOK3N1,which had no interaction with CASP10,the effect disappeared and when co-transfected with RIOK3N2 that had interaction with CASP10,the effect restored.RIOK3 binded CASP10 competitively with RIP and NIK and then restrained the interaction between CASP10 and RIP,CASP10 and NIK by blocking the binding site,which abrogated NFκB activation.And also RIOK3 inhibited the NFκB activation induced by RIP and NIK perhaps for its competitive binding of CASP10. BIM is a kind of BH3-only protein and the activation of BIM promotes apoptosis. Co-immunoprecipitation assay and fluorescent subcellular co-localization assay validated the interaction between BIM and RIOK3.Independent of ubiquitination -proteasome pathway,BIM specifically promotes the degradation of RIOK3 and has no effect on PAK2.BIM has limited effect on the degradation of RIOK3 and co-transfected with RIOK3 and BIM,the concentration of RIOK3 increased gradually with the gradual increase of RIOK3;the concentration of RIOK3 dropped gradually with the gradual increase of BIM.Through the functional study of RIOK3,we found what the role of RIOK3 is in NFκB pathway and the research on the interaction between RIOK3 and CASP10 illu minated the mechanism;we also found BIM was a negative regulator of RIOK3 and how BIM regulated RIOK3.Combined these results we made a further discussion on the function of RIOK3 and speculated its relationship with tumor cells' genesis and metastasis.All these findings provided us clues on the regulation of NFκB pathway and tumor biogenesis.
|
PDF Full Text Request