Mechanisms of Plk1 at the mitotic centromere

有丝分裂着丝粒 Plk1 的机制

• 批准号: 10381722
• 负责人: Mark E Burkard
• 金额: $ 30.57万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10381722
• 关键词: Affect; Binding; Biochemical; Biological Assay; Biological Process; Bloom Syndrome; Bloom syndrome protein; Case Study; Cells; Centromere; Chemicals; Chromatin; Chromosome Arm; Chromosome Segregation; Chromosomes; Complex; DNA-Directed RNA Polymerase; Disease; Dislocations; Environment; Enzymes; Event; Genetic Transcription; Genome; Genomic Instability; Goals; Human; Kinetochores; Learning; Location; Mammalian Cell; Maps; Mediating; Microscopic; Microtubules; Mitosis; Mitotic; Mitotic spindle; PLK1 gene; Phenotype; Phosphorylation; Phosphorylation Site; Production; RNA; Regulation; Resolution; Role; Signal Transduction; Structure; System; Techniques; Testing; Transcript; Transcriptional Regulation; Work; arm; base; centromere protein C; chemical genetics; experimental study; genome integrity; helicase; human disease; innovation; insight; recruit

SUMMARY Our goal is to identify how Polo-like kinase 1 (Plk1) signals at the human centromere to maintain genome integrity. In mitosis, the kinetochore (KT) is assembled at the centromere and this complex is a major focus of Plk1 activity. The KT is a 100 nm-structure assembled on the centromere that attaches chromosomes to the mitotic spindle. Using super-resolution techniques, we have discovered that the bulk of Plk1 localizes to the centromere, directly on chromatin >50 nm from the outer KT. Our long-term goal is to delineate Plk1 partners, substrates, and timing of its activities to maintain faithful chromosome segregation. We are currently focused on activities at the centromere, building on our key findings: Bloom Syndrome RecQ Helicase (BLM) directly or indirectly mediates chromosome arm dislocation and centromere unwinding that occur with loss of Plk1 activity; Plk1 regulates nascent transcripts on chromatin in mitosis and phosphorylates the N-terminus of Centromere Protein C (CENP-C), both known to maintain KT integrity. Our central hypothesis is that a discrete centromere pool of Plk1 stabilizes the mitotic centromere and operates through inactivating helicases, by supporting transcription, and by maintaining CENP-C function. In Aim 1, we will map the spatial environments of Plk1 that contribute to its functions along the KT-centromere axis. We will test the idea that a chromatin-localized pool of Plk1 targets substrates and mediates activities separately from a KT-localized pool, to regulate faithful chromosome segregation. Aim 2 will test how Plk1 dampens BLM helicase function to maintain centromere integrity against mitotic pulling forces. To do this, we will identify Plk1 phosphorylation sites on the BLM protein, determine the role of Plk1 on BLM localization, and evaluate how phosphorylation modulates its helicase activities in biochemical assays and cells. Aim 3 will identify the role of Plk1 on CENP-C stabilization via direct phosphorylation and transcriptional regulation. We have already mapped Plk1 phosphorylation sites on CENP- C and have discovered that Plk1 regulates mitotic transcription. Towards this end, we will functionally analyze the CENP-C phosphorylation events and identify the functional effect of Plk1 on RNA polymerases and mitotic RNAs. Together, this work will reveal how Plk1 operates regionally in the KT to maintain genomic integrity.

摘要 我们的目标是确定Polo-like kinase1(Plk1)是如何在人类着丝粒上发出信号来维持基因组的 正直。在有丝分裂中，着丝粒(KT)组装在着丝粒上，这个复合体是 PLK1活性。KT是组装在着丝粒上的100 nm结构，将染色体连接到 有丝分裂纺锤体。使用超分辨率技术，我们发现Plk1的大部分定位于 着丝粒，直接在染色质上，距离外部KT；50 nm。我们的长期目标是确定Plk1合作伙伴， 底物，以及其活动的时间，以保持忠实的染色体分离。我们目前专注于 着丝粒的活动，基于我们的主要发现：Bloom综合征RecQ螺旋酶(BLM)直接或 间接介导因Plk1活性丧失而发生的染色体臂错位和着丝粒解离； PLK1在有丝分裂中调节染色质上的新生转录物，并磷酸化着丝粒的N端 蛋白C(CENP-C)，两者都能维持KT的完整性。我们的中心假设是一个离散的着丝粒 Plk1池稳定有丝分裂着丝粒，并通过失活解旋酶进行操作，通过支持 转录，并维持CENP-C的功能。在目标1中，我们将绘制Plk1的空间环境图 促进其沿KT着丝粒轴线的功能。我们将测试一个染色质定位的池 PLK1以底物为靶标，并与KT定位池分开调节活动，以调节信徒 染色体分离。Aim 2将测试Plk1如何抑制BLM解旋酶维持着丝粒的功能 针对有丝分裂拉力的完整性。为此，我们将确定BLM蛋白上的Plk1磷酸化位点， 确定Plk1在BLM定位中的作用，并评估磷酸化如何调节其解旋酶 生化分析和细胞中的活动。目标3将确定Plk1在CENP-C稳定中的作用 磷酸化和转录调控。我们已经定位了CENP上的Plk1磷酸化位点- C，并发现Plk1调控有丝分裂转录。为此，我们将从功能上分析 CENP-C的磷酸化事件及Plk1对RNA聚合酶和有丝分裂的功能影响 RNA。总之，这项工作将揭示Plk1如何在KT中区域性地运作，以维持基因组的完整性。