Study On The Expression And Localization Of SGK3 In The Mouse One-cell Fertilized Eggs

Objective Serum and glucocorticoid-induced protein kinase（SGK）is a serine/threonine bi-specific protein kinase,which belongs to one of the AGC subfamily members.Its catalytic domain is very similar to that of protein kinase B（PKB）,SGK and PKB have similar structure,specific substrate and function.SGK is activated downstream of the phosphatidylinositol3-kinase（PI3K）signaling pathway,This process is mediated by mammalian target of rapamycin complex 2（m TORC2）and 3-phosphoinositol-dependent protein kinase 1（PDK1）.So far,SGK in mammals has been found to include three subtypes:SGK1,SGK2 and SGK3,which are highly homologous but have different functions.Although the three subtypes are highly homologous,they differ in tissue specificity:SGK3 and SGK1 are commonly expressed in all tissues,while SGK2 is only expressed in liver,kidney,pancreas,and brain.In addition,the SGK family phosphorylated substrates contain the common structural characteristics of Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr（X is any amino acid）.The SGK family has three domains,one N-terminal variable domain,one catalytic domain and one C-terminal hydrophobic domain.There are two key regulatory sites in the SGK family:Thr residues in the catalytic domain activation ring（Thr 320 in SGK3）and Ser residues in the C-terminal hydrophobic motif（Ser 486 in SGK3）.PDK1 is responsible for phosphorylation of Thr residues,while m TORC2 is responsible for phosphorylation of Ser residues.Both sites require phosphorylation to fully activate SGK.The three SGK isomers are highly similar in structure,the sequence identity in the catalytic domain is close to 80%,and the sequence identity in the C-terminal region is close to 50%.The main structural difference between isomers is in the N-terminal.SGK3 is a unique member of this family:it has a Phox（PX）domain in the N-terminal region,which is important for its protein kinase activity and is responsible for targeting SGK3 to endosomal comparttions and vesicle-like structures.SGK3,as an important serine/threonine kinase,plays a stable and conserved role in the eukaryotic G2/M transition from starfish to humans.Then,whether the three SGK isomerases exist in the mouse one-cell fertilized eggs,and how are the m RNA and protein expressions of the three subtypes?The localization of SGK3 in the G1,S,G2 and M phases in mouse one-cell stage fertilized eggs,which subtypes are mainly expressed in mouse one-cell stage fertilized eggs,and whether SGK3 regulates the activity of Cdc2 kinase to achieve G2/M transition through phosphorylation of Cdc25B have not been studied yet.At present,the study of SGK3 in G2/M transition of mouse one-cell fertilized eggs has not been reported at home and abroad.In order to verify the role of SGK3 in G2/M transition,this study took mouse one-cell fertilized eggs as research objects to explore the expression of three kinds of SGK isomerase m RNA and protein in the four phases,namely,the phases of G1,S,G2 and M.In addition,the localization of SGK3 in the four stages of mouse one-cellfertilized egg cells was observed by indirect immunofluorescence technology,and the effect of SGK3 on the G2/M transition of mouse one-cell fertilized egg cells was preliminary discussed,laying a foundation for the study of the mechanism of early development of fertilized egg.Methods:1.The similarity and consistency of amino acid sequences of mouse and human SGK3 were compared by BLAST.2.Mice were intraperitoneally injectedwith PMSG and HCG for superovulation.According to the time of HCG injection and the morphology of fertilized eggs,the fertilized eggs of G₁、S、G₂and M phases were collected.3.q RT-PCR was used to detect the relative expression of SGK1-3 m RNA in G1,S,G2 and M stages of mouse one-cell fertilized eggs.4.The expression levels of SGK1-3 protein in G1,S,G2 and M phases of the mouse one-cell fertilized eggs were detected by Western blotting.5.Indirect immunofluorescence technique was used to observe the migration and localization of SGK3 in the four stages of mouse one-cell fertilized eggs.Results:1.The amino acid sequences of human SGK3 and mouse SGK3 were very similar in primary structure,and the consistency and similarity of mouse SGK3 and human SGK3 were97%and 98%,respectively.2.q RT-PCR results showed that m RNA of SGK subtypes were expressed in four stages of mouse one-cell fertilized egg.The expression of SGK1 and SGK3 showed no significant difference in G1 and S phases,but reached the peak in G2 phase and decreased in M phase.The expression of SGK2 increased gradually from G1 phase to M phase and peaked at M phase.The m RNA expression level of SGK3 in G2 phase was significantly higher than that of G1,Sand M phases.3.Western-blotting results showed that the expression levels of three subtypes of SGK proteins in the four stages of mouse one-cell fertilized eggs were consistent with their m RNA expression levels,and the expression levels of SGK1 and SGK3 showed no significant difference in G1 and S phases,but increased in G2 phase and decreased in M phase.The expression of SGK2 increased gradually from G1 phase to M phase,with the highest expression in M phase.The protein expression level of SGK3 in G2 phase was significantly higher than that of G1,Sand M phases.4.Indirect immunofluorescence results showed that in the mouse one-cell fertilized egg,SGK3 protein conjugated red fluorescence signal was located in the cytoplasm at G1 and S phases,and part of SGK3 entered the nucleus at G2 phase,and red fluorescence signal at M phase was distributed in the whole cell.Conclusion:1.The amino acid sequence（primary structure）of human and mouse SGK3 is very similar,we speculate that their spatial structure and function are also similar or approximate.2.SGK1,SGK2 and SGK3 proteins are expressed in G1,S,G2 and M stages of mouse one-cell fertilized eggs,SGK1 and SGK3 are mainly expressed in G2 phase,and SGK2 is mainly expressed in M phase and the expression level is relatively low in the four stages.3.There was no significant difference in the expression of SGK3 in G1 and S phases,and the protein expression level of SGK3 in G2 phase was significantly higher than that of G1,Sand M phases.4.SGK3 locates in the cytoplasm in G1 and S phases,and part of SGK3 enters the nucleus in G2 phase and distributes in the whole cell in M phase.SGK3 may be involved in the activation of Cyclin B-Cdc2,as the G2/M transition of the mouse one-cell fertilized egg cells was realized with this nucleoplasmic shuttling.