Investigating the regulation of Aurora B levels at the centromere

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DESCRIPTION (provided by applicant): Failure to properly attach kinetochores of sister chromatids to microtubules of the mitotic spindle leads to mistakes in chromosome segregation and is a source of aneuploidy, which is a common feature of cancer and birth defects. Aurora B kinase plays a critical role in correcting kinetochore-microtubule attachment errors. Aurora B localizes to the inner centromere, a region of chromatin between sister kinetochores, as part of the chromosomal passenger complex (CPC). Incorrect attachment leads to reduced tension across sister kinetochores, which is thought to increase phosphorylation of Aurora B substrates at the kinetochore, destabilizing microtubule attachment. An important recent discovery is that centromeric Aurora B levels are regulated. Aurora B is enriched above basal levels on the centromeres of chromosomes not properly aligned at the metaphase plate in diploid cells. In aneuploid cells, however, aligned and misaligned chromosomes show no difference in Aurora B levels, and error correction is less efficient than in diploid cells. How diploid cells dynamically regulate Aurora B levels is an important unanswered question. Kinetochore-localized kinases Polo-like kinase-1 (Plk1) and Budding uninhibited by benzimidazoles 1 (Bub1) may be involved. Plk1 activity is required for enhanced Aurora B recruitment. Levels of a Bub1-dependent histone modification, H2A phosphorylated on Thr120 (H2A-pT120), are enriched on misaligned chromosomes in diploid but not aneuploid cells. However, the mechanisms by which these kinases regulate Aurora B levels are unclear. Post-translational modification regulates CPC function and localization. Histone modifications, such as H2A-pT120, recruit the CPC to nucleosomes directly or through adaptor proteins such as Shugoshin (Sgo1). The hypothesis motivating this project is that the dynamic regulation of Aurora B levels occurs through post-translational modification at the centromere. First, a targeted cell biological approach will be used to disrupt Plk1 and Bub1 function by altering kinase localization or by modulating protein levels. The impact on Aurora B recruitment to the centromeres of aligned and misaligned chromosomes will be assessed. Second, an unbiased biochemical approach will be used to identify post-translational modifications of the CPC-nucleosome complex that mediate the dynamic regulation of Aurora B levels at the centromere. CPC-nucleosome complexes will be isolated from diploid RPE and aneuploid HeLa cells, and post-translational modifications of the complexes will be compared in a quantitative fashion using stable isotope labeling by amino acids in cell culture (SILAC) coupled to mass spectrometry. Successful completion of this project will provide a more detailed understanding of how the kinetochore regulates Aurora B levels at the centromere so that kinetochore-microtubule attachment errors can be efficiently corrected.