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: 8845980
• 负责人: Jennine M Dawicki McKenna
• 金额: $ 5.6万
• 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/8845980
• 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(申请人提供)：未能正确地将姐妹染色单体的动点连接到有丝分裂纺锤体的微管上会导致错误的染色体分离，并是非整倍体的来源，这是癌症和出生缺陷的常见特征。极光B激酶在纠正动粒-微管附着错误中起着关键作用。极光B定位于着丝粒内，这是姐妹着丝点之间的一个染色质区域，是染色体乘客复合体(CPC)的一部分。不正确的连接导致姐妹动粒之间的张力降低，这被认为增加了动粒上Aurora B底物的磷酸化，破坏了微管连接的稳定。最近的一个重要发现是着丝粒的极光B水平是受调控的。在二倍体细胞中，极光B在染色体着丝粒上的含量高于基础水平，而不是在中期平板上正确排列。然而，在非整倍体细胞中，对齐和错位的染色体在极光B水平上没有差异，纠错效率低于二倍体细胞。二倍体细胞如何动态 调节极光B水平是一个重要的悬而未决的问题。动粒定位的蛋白水解酶Polo-like kinase1(Plk1)和不受苯并咪唑抑制的萌发(Bub1)可能参与其中。PLK1活性是增强Aurora B招募所必需的。在二倍体细胞中，依赖于Bub1的组蛋白修饰，即Thr120上磷酸化的H_2A(H_2A-pT120)的水平，在错位的染色体上丰富，但不是非整倍体细胞。然而，这些激酶调节极光B水平的机制尚不清楚。翻译后修饰调控CPC的功能和本地化。组蛋白修饰，如H2A-pT120，直接或通过接头蛋白如Shugoshin(Sgo1)将CPC招募到核小体。这个项目的假设是，Aurora B水平的动态调节是通过着丝粒的翻译后修饰发生的。首先，一种有针对性的细胞生物学方法将被用来通过改变激酶定位或调节蛋白质水平来扰乱Plk1和Bub1的功能。将评估对Aurora B招募到对齐和错位染色体着丝粒的影响。其次，将使用一种无偏见的生化方法来确定CPC-核小体复合体的翻译后修饰，该修饰介导着丝粒上Aurora B水平的动态调节。将从二倍体RPE和非整倍体HeLa细胞中分离出CPC-核小体复合体，并将使用细胞培养中氨基酸稳定同位素标记(SILAC)与质谱联用来定量比较复合体的翻译后修饰。该项目的成功完成将提供对动粒如何调节着丝粒Aurora B水平的更详细的了解，从而有效地纠正动粒-微管附着错误。

项目成果

The structural basis of the multi-step allosteric activation of Aurora B kinase.

• DOI: 10.7554/elife.85328
• 发表时间: 2023-05-25
• 期刊: eLife
• 影响因子: 7.7
• 作者: Segura-Peña D;Hovet O;Gogoi H;Dawicki-McKenna J;Hansen Wøien SM;Carrer M;Black BE;Cascella M;Sekulic N
• 通讯作者: Sekulic N