The Functional Delineation Of Aurora Kinase Cascade In Mitosis Dynamics And Plasticity

The cell division cycle is orchestrated by a cascade of spatiotemporal regulated protein interaction, which ensures a faithful segregation of parent genome into two identical copies for equal distribution into two daughter cells. Any perturbation of this highly coordinated temporal and spatial control of cell cycle machinery will lead to a compromised genomic stability and cellular plasticity, which contributes to the tumorigenesis.Mitosis is driven by kinase cascades that govern mitotic spindle dynamics and plasticity to ensure accurate chromosome segregation. The key switch for the onset of mitosis is the archetypal cyclin-dependent kinase, Cdc2. In addition to the master mitotic kinase Cdc2, there are three protein serine/threonine kinase families, the Polo kinases, Aurora kinases and the NIMA-related kinases. Aurora kinases regulate centrosome maturation, spindle assembly, chromosome segregation and cytokinesis, which overexpress in a variety of tumors. My doctoral project is centered on the functional delineation of Aurora kinase cascade in mitotic spindle dynamics and plasticity.Here, we present my identification and functional characterization of a novel centrosome-associated protein ATIP3 essential for mitotic spindle plasticity. Using mono-specific antibody, my immunofluorescence microscopic analysis demonstrated that endogenous ATIP3 is localized at centrosome at the onset of mitosis, and relocated to the midbody during cytokinesis, while exogenous ATIP3 localized to MTs. My deletion analysis revealed that the region containing 489-762aa is essential for ATIP3 binding to microtubule. Inhibition of ATIP3 by RNA interference resulted in severe mitotic defects: multi-polar spindles, abnormal centrioles, mitotic delays, and apoptosis. Our biochemical characterization revealed that ATIP3 is a substrate of Aurora-A in vitro and in vivo, indicating the phospho-regulation of ATIP3 plays an important role in the assembly of a bipolar spindle and in orchestration of mitotic progression.Survivin is an inner centromere protein implicated in cell division and apoptotic decision. Survivin is overexpressed in most solid tumor, raising the possibility of its anti-apoptotic function in tumorigenesis. To clarify the confusion in the literature as to the functional relevance of survivin in cell fate decision when it is associated with the mitotic spindle, I conducted a systemic mutational analysis. To my surprise, its distribution to the mitotic apparatus is independent of its role in cell fate decision. In addition, those survivin mutants bear Aurora-B-binding activity and did not perturb the localization of Aurora-B and CENP-E to either kinetochore of midbody. In addition, cells overexpressing these survivin mutants progressed throughout mitosis completely, and committed apoptosis in the next interphase. We believe that the function of survivin in mitosis is spatio-temporally separated from its role in apoptosis.My pursuit for molecular function of Aurora-B led to the identification of a novel Aurora-B-binding partner p53. The tumor suppressor p53 plays an important role in the decision of cell growth or death in response to a variety of stimuli. My biochemical characterizeation shows that p53 is phosphorylated by Aurora-B at Ser315. Interestingly, the non-phosphorylatable mutant of p53^S315A bears an increased stability compared to that of phosphor-mimicking mutant p53^S315D when they were expressed in 293T cells. Thus, my studies provide the first line evidence in which tumor suppressor p53 is regulated by CDK2, Aurora-A and Aurora-B, in a spatial and temporal manner.Taken together, my studies illustrate functional interplay between Aurora kinases and their cognate substrates such as ATIP3, p53 and survivin, which provide novel insights into a better understanding of Aurora kinase cascade in mitotic spindle dynamics and plasticity.