| Objective Cell division cycle protein phosphatase14(Cdc14)belongs to a highly conserved serine and threonine bispecific phosphatase family,capable of down-regulating the activity of CDK(Cyclin dependant kinase),and is an important cell cycle regulator protein widely expressed in eukaryotic cells.Cdc25B(cell cycle homologous 25B)is a bispecific phosphatase that activates the CyclinBl-Cdc2 complex(MPF)by dephosphorylating Thr14 and Tyr15 of Cdc2(Cdk1),leading to the G2/M transition.Studies on Saccharomyces pombe showed that Cdc14 could bind to Cdc25 and dephosphorylate Cdc25,resulting in instability and subsequent degradation.Therefore,overexpressed Cdc14 dephosphorylated Cdc25 and blocked cells in G2 phase.Mammalian genes encode two Cdc14 subtypes:Cdc14A and Cdc14 B.Studies on human cells show that human Cdc14 A phosphatase regulates the G2/M transition of cells by controlling the activity of Cdc25 B,overexpression of Cdc14 A causes Cdc25 dephosphorylation,inhibits the activity of Cdc2 kinase and thus blocks cells in the G2 phase.Our previous studies on mouse one-cell zygote have confirmed that Cdc25 B is phosphorylated in G1 and S phase,dephosphorylated in G2 and M phase.However,it is not clear whether Cdc14 A has regulatory effect on Cdc25 B in one-cell zygote and by what means it is regulated and overexpressed Cdc14A(WT,wild-type has activity and C278 S,mutant has no activity),and whether it can block mouse one-cell stage zygote in G2 stage.In our study,with the one-cell fertilized mouse eggs as the research object,through microinjection of the mRNA of Cdc25B-WT,Cdc25B-S15 A,Cdc25B-S186 A,Cdc25B-S15 D,Cdc14A-WT and Cdc14A-C278 S,the author observed their effects on the cleavage rate,death rate and G2/M transition of one-cell fertilized eggs;then,through microinjection of the mRNA of Cdc25B-WT,Cdc25B-S15 A,Cdc25B-S186 A,Cdc25B-S15 D and Cdc14A-WT,the author observed their effects on the cleavage rate,death rate and G2/M transition of one-cell fertilized mouse eggs,thereby exploring the interaction between Cdc14 A and Cdc25 B in one-cell fertilized mouse eggs.Methods:1.After mouse were superovulated then we collect one-cell fertilized mouse eggs at G1 、S 、G2、M phase.2.Construction of pcDNA3.1-MYC-Cdc14A(C278S)、pcDNA3.1-MYC-Cdc14AWT、pcDNA3.1-3xflag-Cdc25B-WT、 pcDNA3.1-3xflag-Cdc25B(S15A)、pcDNA3.1-3xflag-Cdc25B(S15D)、 pcDNA3.1-3xflag-Cdc25B(S186A)eukaryotic ex pression vectors;3.The constructed expression plasmid was transcribed into mRNA for microinjection in vitro;4.Observe the effects on the development of fertilized mouse eggs after the microinjection of mRNA.5.Western Blotting was used to detect the phosphorylation status of Cdc2-Tyr15.6.Radioimmunoassay of MPF activity;Results:1.There were no differences in the initiation cleavage time,MPF activity and phosphorylation signal of Cdc2-Tyr15 between the Cdc14A-C278 S and Cdc25B-S15 A solely injection groups and the control group.2.The Cdc25B-WT,Cdc25B-S15 D and Cdc25B-S186 A solely injection groups,there were no differences in the initiation cleavage time,MPF activity and phosphorylation signal of Cdc2-Tyr15.3.Compared with the control group,the Cdc14A-WT solely injection group and Cdc14A-WT +Cdc25B-S15 A co-injection group,the cleavage time was delayed,the cleavage rate was decreased,the peak time of MPF activity was delayed,and the phosphorylation signal of Cdc2-Tyr15 was detected.4.There were no differences in the initiation cleavage time,MPF activity and phosphorylation signal of Cdc2-Tyr15 between the Cdc14A-WT + Cdc25B-WT co-injection group and the f Cdc14A-WT + Cdc25B-S186 A co-injection group.5.Compared with the control group,the Cdc14A-WT +Cdc25B-S15 D co-injection group showed earlier cleavage time,higher cleavage rate,the peak time of MPF activity and the phosphorylation signal of Cdc2-Tyr15 were detected earlier.Conclusion:1.Overexpression of Cdc14A-WT delayed the entry M phase of one-cell fertilized mouse eggs;Cdc14A-C278 S had no effect on G2/M transition of one-cell fertilized mouse eggs.2.Cdc25B-S186 A,Cdc25B-S15 D and Cdc25B-WT have the same or similar activity,which can accelerate the cleavage of one-cell fertilized mouse eggs and accelerate the transition of G2/M phase.As Cdc25B-S15 was mutated into alanine and could not be phosphorylated and inactivated,it had no effect on G2/M phase transformation of one-cell fertilized mouse eggs.3.There may be interaction between Cdc14 A and Cdc25 B in fertilized mouse eggs,and the specific site of interaction may be Cdc25B-S15,rather than Cdc25B-S186.4.Cdc14 A can dephosphorylate Cdc25B-S15 and inactivation,and fertilized mouse eggs is blocked in G2/M phase.Therefore,Cdc14 A is considered as a negative regulator of cell cycle. |
