The Roles Of Protein Methylation On Plk1 Kinase

Protein lysine methylation as one of the most important modifications in epigenetics,has been attracted a lot of attention in life sciences.Similar to post-translational modifications on histones,lysine methylation on non-histone proteins also function in regulating protein stability,cellular localization and protein-protein interaction.These functions are widely involved in gene transcription,DNA replication and repair,tumor cell proliferation and migration.Furthermore,lysine methylation is capable to crosstalk with some other post-translational modifications to dynamically control cellular homeostasis.Therefore,investigating protein methylation would aid us better understanding a whole picture of various cellular events.Plk1(Polo-like kinase 1)is a protein kinase required for regulating cell cycle progression.It has been well documented that Plk1 participates in multiple transitions in mitosis such as kinetochore formation,chromatin condensation,and spindle formation.Some other studies showed that Plk1 is involved in DNA replication,maintaining genome stability after replication stress.In addition,elevated Plk1 levels and enhanced enzymatic activity have been found in many types of cancers.Knockdown of Plk1 leads to tumor cells more sensitive to anticancer drugs and radiation therapy.On the other hand,inhibition of Plk1 kinase activity by small molecular inhibitors suppresses tumor cell division and cell proliferation,and facilitates apoptosis.These results suggest that Plk1 could be a potential target of cancer therapy.Research on small molecule inhibitors of Plk1 has made a great progress.The main strategy is to competitively bind Plk1 with ATP by ATP analogs or to inhibit binding its Polo-box domain to substrate proteins.However,many Plk1 inhibitors so far are still in clinical trials,and more attention should be focused on develop new small molecules.Thus,the more detailed mechanism of Plk1 in cancer cell should be further investigated.During cell cycle,Plk1 phosphorylates different substrates to regulate various cellular events in a spatiotemporal-dependent manner.Previous studies showed that regulation of Plk1 kinase activity is the key to determine its functions,and its activity is regulated in a variety of ways.Full activation of Plk1 is dependent on phosphorylation of T210 by Aurora A after interaction with Bora.In addition,ubiquitination and SUMOylation of Plk1 can also regulate its function by altering the interaction of Plk1 with other proteins.Previous work by our group has been focused on methylation of Plk1 and found the Lys209 of Plk1 can be monomethylated.Since K209 is in close proximity to T210,we speculate that methylation on K209 may have important functions.By screening many methyltransferases using in vitro kinase assay,we found that the methyltransferase G9 a is the main enzyme responsible for the modification of K209.The level of K209me1 is regulated by changing the protein level of G9 a in cells.Furthermore,methylation of K209 in Plk1 is inhibited by phosphorylation of T210.The T210 phosphorylation modified peptide segment inhibits the occurrence of G9a-mediated methylation modification.Overexpression or knockdown of Aurora A in cells can also affect the levels of K209me1.However,sustained methylation modifications can also cause dysfunction of Plk1.using a CRISPR-Cas9 technique,we generated a methyl-mimetic(K209M)mutant in He La/RFP-H2 B cell lines.This cell lines showed defects in cell cycle progression.Real-time fluorescence experiments showed that the duration time required for Plk1-K209 M cells from metaphase to anaphase was significantly lengthened.We found that Plk1-K209 M mutation may lead to a decrease in Plk1 activity,which results in defects of the dissociation of interchromosomal adhesion protein complexes.The reduction of Plk1 K209me1 at mitosis is critical for cell cycle progression,especially for anaphase onset.However,the mechanism of the regulation of G9a-mediated K209 methylation are still not fully understood.Of note,the effect of K209me1 on Plk1 kinase activity,and the specific functions of this modification in cellular events are still unclear.To figure out the interplay of Plk1 methylation with Plk1 phosphorylation,we purified the methyltransferases G9 a,the kinase Aurora A and the substrate Plk1.In vitro asssay was carried out with sequential reactions,The results showed that phosphorylation of T210 by Aurora A was inhibited by methylation of K209 by G9 a.Furthermore,Plk1 kinase activity is regulated by K209me1.Moreover we have demonstrated that K209me1 mainly occurs at the S phase of the cell cycle,which maintains genomic stability under replication stress.The progression of S phase is slightly changed in Plk1-K209 A mutation cells.We also found that K209me1 is regulated by the DNA damage repair signaling pathway,and the damage signal increases K209me1 and decreases pT210,suggesting that K209me1 is involved in DNA damage repair.In Plk1-K209 A mutant cells,DNA damage repair process is delayed.Laser microirradiation experiments have demonstrated that Plk1 can be recruited to the DNA damage site.However,the recruitment of Plk1 is not affected by the K209 methylation,and the modification did not significantly change the recruitment of other repair proteins such as PARP1,NBS1 and RPA2.Further studies revealed that RPA2 and RAD51 accumulate at the DNA damage site after stimulation of DNA damage signals in Plk1-K209 A mutant cells,suggesting that methylation of Plk1 may be necessary for dissociation of these repair proteins.In summary,we conducted a study on the function of G9a-mediated methylation of K209 in Plk1.We proved that K209me1 and pT210 modifications antagonize each other,regulating the kinase activity of Plk1.We also revealed the mechanism of Plk1 methylation in S phase procession and DNA damage repair.The "crosstalk" between methylation and phosphorylation provides a new perspective to reveal the regulation of protein functions.Our research on Plk1 may also provides new insights for tumor therapy in targeting Plk1.