Molecular Delineation Of BubR1-CENP-E-SKAP Signaling Cascade In Mitosis

In the process of cell division, chromosomes are distributed to each daughter cell by attachment to fibrous tracks assembled from tubulin making up microtubules. The chromosomes attach to these tracks through a specialized region called the kinetochore. Faithful attachment is essential for cell health, but very little is known about how this works normally and what goes wrong in diseases such as cancers. Genomic instability, including chromosome loss and translocation, plays a significant role in the progression of many human cancers. The spindle assembly checkpoint, biochemical machinery governing chromosome stability and mitotic plasticity, plays a vital role in the aforementioned process. However, little is known regarding the signaling cascade underlying spindle checkpoint and its regulatory mechanism. Thus, the theme of my doctoral study stems from the molecular delineation of dynamic kinetochore-microtubule orchestration and spindle checkpoint signaling circuitry.Mounting evidence demonstrates that mitotic kinesin CENP-E plays an important role in linking kinetochore microtubules to chromosome biorientation. However, it has remained elusive as to how CENP-E orchestrates the dynamic interaction between congressing chromosome and kinetochore microtubules. Although previous studies show that the mitotic kinesin CENP-E forms a link between attachment of the spindle microtubule to the kinetochore and the mitotic checkpoint signaling cascade, the molecular mechanism underlying dynamic kinetochore-microtubule interactions in mammalian cells remains elusive. Here, we identify a novel interaction between CENP-E and SKAP that functions synergistically in governing dynamic kinetochore-microtubule interactions. SKAP binds to the C-terminal tail of CENP-E in vitro and is essential for an accurate kinetochore-microtubule attachment in vivo. Immunoelectron microscopic analysis indicates that SKAP is a constituent of the kinetochore corona fibers of mammalian centromeres. Depletion of SKAP or CENP-E by RNA interference results in a dramatic reduction of inter-kinetochore tension, which causes chromosome mis-segregation with a prolonged delay in achieving metaphase alignment. Importantly, SKAP binds to microtubules in vitro, and this interaction is synergized by CENP-E. Based on these findings, we propose that SKAP cooperates with CENP-E to orchestrate dynamic kinetochore-microtubule interaction for faithful chromosome segregation. During mitosis, the spindle assembly checkpoint becomes activated in response to aberrant kinetochore attachment, which extends time to allow all chromosomes to achieve metaphase alignment before anaphase onset. Thus, spindle checkpoint plays a vital role in maintaining chromosome stability and avoiding ancuploidy. BubRl, a critical component of the spindle checkpoint machinery, contains a serine/theronine kinase motif. However, its kinase activity and regulation have remained uncharacterized. Using a phenotype-based screen combined with an in vitro BubRl-mediated32P incorporation assay, we have identified a novel chemical inhibitor BICl (BubRl inhibitory compound). BIC1exhibits a specific inhibition toward BubRl at an IC50of3μM. Remarkably, BIC1displays no inhibition on other mitotic kinases even at10μM, further demonstrating its specificity. Chemical syntheses of BIC1derivates coupled with in vitro kinase assay defines the functional groups of BIC1and rational design has enabled us to synthesize BIC1analogue called BIC2. Careful examination of the mitotic phenotype elicited by treatment of BIC1/BIC2vs BubRl suppression by BubRl siRNA has led us to conclude that BubRl functions in aberrant kinetochore attachment correction and spindle checkpoint signaling as inhibition of BubRl promotes premature anaphase. Detailed characterization indicates that BubRl links tension development the kinetochores to timing of anaphase onset. Thus, I reason that BubRl is a coupling factor between dynamic kinetochore microtubules and checkpoint cascade via BubR1-CENP-E-SKAP axis, by which it guards genomic stability. Currently, I am employing BIC1coupled with a FRET-based BubRl activity reporter to illustrate the BubRl kinase dynamics and its downstream effectors. Molecular delineation of BubR1-CENP-E-SKAP axis will enable us to illustrate the spatiotemporal dynamics of mitotic chromosome segregation.In anaphase, central spindle, the anti-parallel microtubules, is generated for preparation and completion of cytokinesis. Mitotic kinesin CENP-E translocates from kinetochore to the central spindle at the beginning of anaphase onset. Although an early microinjection experiment suggests for the role of CENP-E in anaphase, the experimental demonstration of such a function has not been successfully demonstrated. Here we take the advantage of CENP-E inhibitory compound syntelin and firm the critical role of CENP-E in central spindle organization. We found that inhibition of CENP-E motor activity can influence central spindle localization of several proteins, which is important to central spindle organization. For example, in syntelin treated cell, MKLP1, CENP-E and PRC1failed to become highly concentrated at the center of central spindle, suggesting the central spindle assembly errors. Using biochemical characterization, we found the Thr602is a substrate of PLK1and phosphorylation of Thr602regulates the temporal dynamics of PRC1relocation. Our studies reveal a regulatory axis underlying PPly-CENP-E-PRC1-PLK1signaling axis, which also settle the2-decade long debate on the role of CENP-E in anaphase.Taken together, my study has illustrated the cooperative interaction between CENP-E and SKAP during cell division and outlined the functional regulation of BubRl-CENP-E-SKAP axis in dynamic kinetochore-microtubule interactions, and my work demonsrate that CENP-E motor activity is essential for central spindle assembly by interacting with PRC1and PPlγ.