Functional Analysis Of Lysine Acetylation Of Mitotic Kinase Aurora B And PLK1

The fundamental event in life is cell division in which parental genome is equally segregated into two daughter cells.Faithful cell division is essential for cell health and tissue homeostasis.Any error in chromosome segregation will result in genomic instability which is a hallmark of human cancer.Genomic instability has been proposed to play an essential role in the tumorigenesis.There are four mechanisms that maintains genomic stability,the fidelity of DNA replication in S phase,the accurate segregation of chromosome in M phase,the DNA repair and the checkpoints across the cell cycle.Errors in any of the four mechanisms can lead to the imbalance of progression in cell cycle and tumorigenesis.Kinetochore is a super-macromolecular structure located to centromeres of the chromosome.The essential function of kinetochore is to orchestrate accurate interactions between chromosomes and spindle microtubules and serve as a quality control device to determine the time for sister chromatid separation,which is under a tight control of protein kinase cascades including CDK1,Aurora B,PLK1,and BUB1/BUBR1.However,the precise mechanisms underlying those signaling cascades and their cross-talks have remained elusive.I came to the notion of acetyl-phosphorylation as I learned the fact that the acetyltransferase PCAF controls microtubule dynamics via acetylation of microtubule end-binding protein EB1 and mitotic kinase such as BubR1,which inspired my exploration of acetyl-phosphorylation cross-talk and its underlying mechanisms.Using a combination of siRNA-based knock-down and chemical inhibitors of TIP60,my work has demonstrated the regulatory role of TIP60 acetyl-transferase activity in accurate chromosome segregation as suppression of TIP60 led to the misalignment and lagging chromosomes in mitosis.Mass spectrometric analysis of mitotic acetylation revealed that Aurora B kinase is acetylated.My biochemical characterization demonstrates that TIP60 interacts with and acetylates Aurora B at lysine 215.Interestingly,acetylation-mimicking mutant of Aurora B K215Q exhibits enhanced kinase activity,suggesting that the aceylation at K215 promotes Aurora B activity.Mechanistically,TIP60 acetylation of Aurora B at K215 protects the phosphorylation of its activation loop from PP2A-mediated dephosphorylation.These findings define an important signaling axis that integrates protein phosphorylation and acetylation to connect cell cycle progression to genomic stability maintenance,which highlights a key role for CDK1 in such processes.The second aspect of my dissertation study involves characterization of acetylation of PLK1 by another acetyltransferase PCAF as mass spectrometric analyses revealed that the lysine 208 of PLK1 is acetylated in cells.Using the genetic encoding methods,I also purified lysine 208 acetylated PLK1 from E.coli by addition of N-ε-acetyl-L-lysine and the HDAC inhibitor nicotinamide to the expression system.Interestingly,the modification of lysine 208 suppressed PLK1 activation by phosphorylation at threonine of 210,which is catalyzed by Aurora A at the G2/M transition.Thus,our study suggests that temporal order of acetylation and phosphorylation of PLK1 in G2/M transition provides a novel checkpoint control of mitotic entry.In sum,my dissertation provided molecular analyses of acetyl-regulation of mitotic kinases in cell cycle control.Further delineation of acetyl-regulation of Aurora B and PLK1 kinase activity in live cell mitosis will enable us to consolidate the acetyl-phosphorylation cross-talk into the dynamics underlying systems biology of mitosis,which will provide foundation for the development of next generation therapeutics in clinic oncology.