Investigating the regulation of Aurora B levels at the centromere

研究着丝粒 Aurora B 水平的调节

• 批准号: 8717918
• 负责人: Jennine M Dawicki McKenna
• 金额: $ 5.33万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8717918
• 关键词: Adaptor Signaling Protein; Amino Acids; Aneuploid Cells; Aneuploidy; Benzimidazoles; Biochemical; Biological; Cell Culture Techniques; Cell division; Cells; Centromere; Chromatin; Chromosome Segregation; Chromosomes; Complex; Congenital Abnormality; Coupled; Diploid Cells; Diploidy; Dominant-Negative Mutation; Epithelial; Failure; Hela Cells; Human; Kinetochores; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Metaphase Plate; Microtubules; Mitotic spindle; Mutagenesis; Nucleosomes; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Post-Translational Protein Processing; Prometaphase; Proteins; Recruitment Activity; Regulation; Retinal Pigments; Role; Sister; Sister Chromatid; Source; Stable Isotope Labeling; Testing; aurora B kinase; benzimidazole; histone modification; human PLK1 protein; polo-like kinase kinase 1; public health relevance; small hairpin RNA

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.

描述（由申请人提供）：未能将姐妹染色单体的着丝粒正确连接到有丝分裂纺锤体的微管上，导致染色体分离错误，是非整倍体的来源，这是癌症和出生缺陷的常见特征。极光B激酶在纠正运动舞蹈-微管附着错误中起关键作用。极光B定位于内部着丝粒，姐妹着丝粒之间的染色质区域，作为染色体乘客复合体（CPC）的一部分。不正确的附着导致姐妹动粒之间的张力降低，这被认为会增加动粒处Aurora B底物的磷酸化，使微管附着不稳定。最近的一个重要发现是，着丝粒极光B水平的调节。极光B富集在二倍体细胞中期板上未正确排列的染色体的着丝粒上的基础水平之上。然而，在非整倍体细胞中，对齐和未对齐的染色体在Aurora B水平上没有差异，并且错误校正的效率低于二倍体细胞。二倍体细胞如何动态地 调节极光B水平是一个重要的未回答的问题。可能涉及激动剂定位的激酶Polo样激酶1（Plk 1）和不受苯并咪唑1（Bub 1）抑制的出芽。Plk 1活性是增强Aurora B募集所必需的。Bub 1依赖性组蛋白修饰的水平，H2 A磷酸化的Thr 120（H2 A-pT 120），富集在二倍体，但不是非整倍体细胞的染色体错位。然而，这些激酶调节极光B水平的机制尚不清楚。翻译后修饰调节CPC的功能和定位。组蛋白修饰，如H2 A-pT 120，直接或通过接头蛋白如Shugoshin（Sgo 1）将CPC募集到核小体。激发该项目的假设是极光B水平的动态调节通过着丝粒的翻译后修饰发生。首先，靶向细胞生物学方法将用于通过改变激酶定位或通过调节蛋白质水平来破坏Plk 1和Bub 1功能。将评估对Aurora B募集至对齐和未对齐染色体的着丝粒的影响。第二，将使用无偏倚的生物化学方法来鉴定CPC-核小体复合物的翻译后修饰，所述CPC-核小体复合物介导着丝粒处极光B水平的动态调节。将从二倍体RPE和非整倍体HeLa细胞中分离CPC-核小体复合物，并使用细胞培养物中氨基酸的稳定同位素标记（SILAC）结合质谱法以定量方式比较复合物的翻译后修饰。这个项目的成功完成将提供一个更详细的了解着丝粒如何调节极光B水平的着丝粒，使着丝粒微管附着错误可以有效地纠正。