Regulatory Mechanisms Of Post-translational Modifications On Plk1 During Cell Cycle

In the year of 1988,Polo kinase family was first discovered in Drosophila.From that time on,Plks have emerged as a family of kinases that executes several essential functions to promote cell division.Among five members of family,The best-studied member is Plkl,which has been reported in various crucial cell-cycle-related.The activity of Plkl is regulated by its own conformation,because the PBD interacts with the kinase domain and suppresses its activity when Thr210(T210)is not phosphorylated in interphase cells.A Plkl-interacting protein,named Bora,is found to accumulate in the G2 phase and relieve the auto-inhibition by the PBD in Plkl.The Plkl activity is strictly controlled in the cell cycle.The kinase activity of Plk1 starts to increase during G2 phase and peaks in mitotic cells.One of the most important functions of Plkl in late G2/prophase is to control activation of CDK1/CyclinB to ensure timely entry into mitosis.Plkl-inhibited cells will eventually enter mitosis,although entry will be significantly delayed and they will arrest in prophase with immature centrosomes and unresolved chromosome arm cohesion.In mitosis,Plkl activity has also been reported to suppresses kinetochore-microtubule dynamics to stabilize initial attachments in prometaphase,and Plkl removal from kinetochores is necessary to maintain dynamic microtubules in metaphase.In this study,we reported a novel acetylated site Lys208(K208)on Plkl.We showed that the highly conserved N-terminal K208 of Plkl was a substrate of p300/CBP-associated factor(PCAF).SIRT1 competed with PCAF for deacetylation at K208.The acetylation at K208 took place after the binding of Bora but before phosphorylation at T210 by Aurora A.Also,acetylation on K208 impaired Plkl kinase activity by regulating T210 phosphorylation.Cells with high level of K208-acetylated Plkl exhibited defects during G2/M transition.Thus,deacetyaltion of K208 was becoming essential for Plkl to regain its maximal kinase activity and reached the threshold that gates mitotic entry.Also in our study,We found out that Plkl was methylated.We detected the interaction between Plkl and SET7/9 in vitro and in vivo.SET7/9 di-methylated Plkl at Lys191(K191).Methylation at K191 on Plkl regulated Plkl kinase activity negatively.We observed that Plk1 KI91R mutant showed defects in chromosome alignment.Knock down of SET7/9 in HeLa cells and inhibition of SET7/9 in RPE1 cells also exhibited defects in chromosome alignment.We then found out that these phenotypes were caused by persistent phosphorylation at Ser676(S676)on BubRl by unmethylated Plkl,which led to unstable kinetochore-microtube attachment,unaligned chromosome,and cells arrested in metaphase.In conclustion,Our study unraveled relative functional mechanisms of regulatory modification on Plkl in mitosis.We pointed out the fact that modification such as acetylation and methylation participated in mitotic progression through regulating kinase activity and phosphorylated substrates of important kinase in mitosis,providing experimental and theoretical basis for tumorigenesis caused by kinase modification dysfunction.