Two Pairs Of The Human Protein (msk1 Of Ck2, Nlk, And Smad4) Interactions And Phosphorylation

This thesis is composed of two separate parts:The phosphorylation of MSK1 by CK2 and the function of it;NLK can reduce the protein level of SMAD4 through phosphorylate SMAD4.In partⅠ,we found the different subcellur localization of the two transcripts of MSK1.Identified MSK1 can be phosphorylated by CK2.MSK1 have two isoforms, trans1 and trans2.We found that the longer trans1 localized in the nuclear,but the shorter trans2 localized in the cytoplasm.This suggest that the sequence trans1 longer than shorter have the nuclear localization sequence.We are constructing series truncated mutants to identife the NLS sequence.Through yeast two-hybrid screening,we obtained CK2β.We identified the interaction between MSK1 and CK2 in vitro and in vivo.And also identified the interaction between MSK1 and CK2α.We found MSK1 can interact with CK2 under starverd and anisomycin stimulated condition. This interaction can be inhibited by SB203580,a p38 specific inhibitor.Through truncated mutants CoIP,we identified CK2βcan interact with both the N-terminal truncate and the C-terminal truncate,but CK2αcan only interact with the N-termianl truncate.Using theμRPLC-ESI-MS,we identified 10 Ser/Thr sites within the C-terminal of MSK1 can be phosphorylated by CK2:Ser-691,Ser-694,Thr-753,Thr755,Ser-757, Ser-758,Ser-759,Ser-760,Ser-762,Thr-793.We further identified Ser-657 as another site can be phorylated by CK2 using the site mutation method which is not identified by mass spectrum.Taken together,we found there is a nuclear localization sequence in the C-terminal sequence of MSK1.Identified the interection between MSK1 and CK2,the interaction is p38 MAPK signal dependent.And further identified 11 sites within the C-termianl of MSK1 can be phosphorylated by CK2.These phosphorylation sites may play an important role in the alteration of the three dimensional structure of MSK1 leading it to activation.In partⅡ,we first use yeast two-hybrid system identified the interaction between the SMAD4 and NLK.We further identified the interaction between SMAD4 and NLK in vitro and in vivo,and found the interaction is kinase activity independent.Series truncated mutants assay displayed that the linker sequence between the MH1 domain and MH2 domain of SMAD4 are sufficient and necessary for this specific interaction.The linker sequence is relevant to polyubiquitination -dependent degradation.So we suggest that NLK can induce the degradation of SMAD4.Colocalization assay indicate that the localization of SMAD4 can be altered when cotransfected with NLK.It altered from equal localization in cytoplasm and in nuclear to assemble localization in cytoplasm and a little can switch into the nuclear.We identified SMAD4 can be phosphorylated by NLK in vitro,and the phosphorylate site is within the MH1 domain and the linker sequence.When overexpress NLK in 293T cell,the protein level of SMAD4 is reduced.This reduction happened in transcription level or through polyubiquitination-dependent degradation is unknown.The mechanism need our further research.In partⅡ,we identified the interaction between SMAD4 and NLK.The interaction is kinase activity independent.We also found NLK can phosphorylate SMAD4 and can alter the localization of SMAD4.We further identified when overexpress NLK,the protein level of SMAD4 can be reduced.SMAD4 is the core member of SMADs family,is an important tumor-suppress gene.The interaction between SMAD4 and R-SMADs is important for the TGF-βsignaling transduction.NLK is one conserved Ser/Thr kinase,having important role in the tissue organization.Our research provide more advantaged information for further research of the function and control mechanism of these two proteins.