| Objective: Cell stress is a protective response when that is under adverse conditions. There are many kinds of stress ways, such as oxidative stress, virus infection, anoxia stress and so on. Cell will response to the stress with different kinds of pathways, the formation of stress granules is an important aspect in cell stress. Tudor-SN is a multifunctional protein, playing an important role in the progress of cell stress. For example, in plant, Tudor-SN can stabilize the level of sensitive m RNA, that is good for the growth under the condition of high salt and hyperosmosis. In recent years, several papers have reported Tudor-SN can participate in the formation of stress granules, playing important role in antiapoptosis and promoting cell survival. This project aims at the studying of the key phosphorylation sites of Tudor-SN participating in the formation of stress granules, discussing the important function of Tudor-SN in stress.Methods: The article is divided into three parts.the first part is to construction of two kinds of site mutant plasmids, p CMV-N-Tudor-SN(T103A)-Flag and pm Cherry-C1-Tudor-SN(T103A). The Threonine 103(T103)site of Tudor-SN was changed into Alanine by site-directed mutagenesis technique, and then Tudor-SN. Mutants were inserted into p CMV-N-Flag and pm Cherry-C1 vector. The recombinant plasmids were used for further Co IP and immunofluorescence. The second part is the effect of mutation of T103 site on Tudor-SN participating in the formation of stress granules. This part is divided into three aspects. The first aspect is to study the role of T103 site of Tudor-SN in the co-localization with stress granules by recombinant plasmids. The second aspect is to use Co IP to detect the combination of Tudor-SN and the protein components of stress granules under site mutation. The third aspect is to use the recombinant plasmids to study the combination of Tudor-SN and the m RNA component of stress granules. The third part is to analyze the upstream kinase of T103 site. This part is divided into five parts. The first aspect is to predict the potential kinases of T103. The potential kinases are predicted by Phosphorylation database. The second part is to detect the combination of potential kinase and Tudor-SN. Co IP is used to detect the combination of potential phosphorylated /unphosphorylated kinase and Tudor-SN. The third aspect is the identification of potential kinase of T103. The kinases which can phosphorylate T103 directly are identified by in vitro kinase experiment. The fourth aspect is to detect the effect of JNK inhibitor on phosphorylated level of Tudor-SN and participating in the formation of stress granules, including to detect the phosphorylated level of Tudor-SN by Western blot and the co-localization of Tudor-SN and stress granule by immunofluorescence after JNK inhibitor treatment.Result:(1)The p CMV-N-Tudor-SN(T103A)-Flag and pm Cherry-C1-Tudor-SN(T103A) were expressed normally in cells, suggesting the recombinant plasmids were constructed successfully, which can be used for further study.(2)Tudor-SN could not co-localization with stress granules after T103 site mutation.(3)The combination of Tudor-SN and G3BP(or TIAR)turns attenuated after T103 site mutation by Co IP.(4)T103 site mutation has no effect on the combination of Tudor-SN and m RNA component of stress granules AGTR1-3'UTR by RIP.(5)Tudor-SN can bind with kinase JNK rather than p38.(6)Activated JNK can phosphorylate T103 of Tudor-SN directly.(7)The phosphorylated level of T103 turn declining and the quantity of phosphorylated T103 located in stress granules turn declining after JNK inhibitor treatment.Conclusion:(1)The phosphorylation of T103 is necessary for Tudor-SN to enter stress granules.(2)The phosphorylation of T103 promotes the combination of Tudor-SN and the protein component(G3BP,TIAR) in stress granules, however it has no effect on the m RNA component(AGTR1-3'UTR).(3)JNK is the direct kinase to phosphorylate T103 of Tudor-SN. |
PDF Full Text Request