Phosphorylation-dependency of centrosomal central core layer proteins during Dictyostelium centrosome duplication

盘基网柄菌中心体复制过程中中心体中央核心层蛋白的磷酸化依赖性

• 批准号: 463103716
• 负责人: Professor Dr. Ralph Gräf
• 金额: --
• 依托单位: Arbeitsgruppe Zellbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/463103716?language=en
• 关键词: Phosphorylation; dependency; centrosomal; central; core

Dictyostelium amoebae are useful to study basic, centriole-independent processes in centrosome biology, because they are the only non-opisthokont model possessing centriole-free centrosomes. They consist of a three-layered core structure surrounded by a microtubule-nucleating corona. Our goal is to elucidate the molecular mechanisms involved in the duplication of these centrosomes. A key event at the G2/M transition is the splitting of the three-layered core structure, whereby the central layer disappears. The latter consists of three components, CP39, CP75 and CP91, which are potential targets for regulation by mitotic kinases at the onset of mitosis. Based on results from other organisms and on known BioID-protein-protein interactions, the three kinases Plk, CDK1 and Nek2, are candidates as regulators. In this project we will elucidate which of these kinases phosphorylates which of the three central layer proteins, and we will provide insight into the consequences of these phosphorylations for their specific protein-protein interactions.- We will perform point mutation studies of CP39, CP75 and CP91, focusing on the subdomains known to be involved in mitotic regulation of the respective proteins. Mutants will be expressed as GFP-fusion proteins. Using multiple phosphorylation-relevant point mutations, we expect to reveal which phosphorylation sites in the three proteins are required for their dissociation from mitotic centrosomes.- With regard to CDK1 we will also analyze the effects of point mutations in the cyclin B centrosomal targeting domain. - Plk, CDK1 and Nek2, respectively, will be verified as true modifiers of identified phosphorylation sites by in vitro kinase assays using the recombinant kinases expressed in the autologous host. In this context we will aim at establishing a new method for M-phase cell-cycle synchronization in Dictyostelium, in which endogenous CDK1 will be replaced by a CDK1 mutant than can reversibly be inhibited by an ATP analogon. - Mass spectrometric identification of in vivo phosphorylation sites: Control cells and strains expressing dominant-negative variants of the three kinases will be compared.- Effect of phosphomimetic point mutations of CP39, CP75 and CP91 on protein-protein interactions: The effect of point mutations on BioID interactions and on co-precipitation of GFP-tagged CP39, CP75 and CP91 proteins with known co-expressed His-Myc-tagged binding partners will be studied. For studies on the microscopic level we will use a mis-localization assay whereby the three candidates and their phosphomimetic mutants are targeted to the cell cortex. Mutation-dependent changes in cortical co-localization of their binding partners will be analyzed. Furthermore, we will investigate whether expression of the point-mutated variants causes alterations in centrosomal localization of their binding partners during mitosis, which would also indicate a dependence of mutual interactions on the phosphorylation state.

在中心体生物学中，Dictyostelialamobae是唯一具有中心粒非中心粒非中心粒的模型，因此对研究中心体生物学中基本的、中心粒非依赖性的过程是有用的。它们由三层核心结构组成，周围环绕着微管成核的日冕。我们的目标是阐明复制这些中心体的分子机制。G2/M转变的一个关键事件是三层核心结构的分裂，从而使中心层消失。后者由CP39、CP75和CP91三个组分组成，在有丝分裂开始时，CP39、CP75和CP91是有丝分裂酶调节的潜在靶点。根据其他生物的结果和已知的BioID-蛋白质-蛋白质相互作用，PLK、CDK1和Nek2这三个激酶是候选的调节者。在这个项目中，我们将阐明这些激酶中的哪一种使三种中心层蛋白质中的哪一种磷酸化，并深入了解这些磷酸化对它们特定的蛋白质-蛋白质相互作用的后果。-我们将进行CP39、CP75和CP91的点突变研究，重点放在参与各自蛋白质有丝分裂调控的亚域上。突变体将以GFP融合蛋白的形式表达。利用多个与磷酸化相关的点突变，我们希望揭示这三种蛋白质中的哪些磷酸化位点是它们从有丝分裂中心体解离所必需的。-关于CDK1，我们还将分析周期蛋白B中心体靶向结构域中的点突变的影响。-PLK、CDK1和Nek2将分别被证实为已鉴定的磷酸化位点的真正修饰物，通过使用在自体宿主中表达的重组激酶进行体外激酶检测。在这种背景下，我们将致力于建立一种新的方法来实现网柄苔藓M期细胞周期的同步，其中内源性CDK1将被一个可被ATP类似物可逆抑制的CDK1突变体所取代。-体内磷酸化位点的质谱学鉴定：将比较对照细胞和表达这三种酶的显性-阴性变体的菌株。-CP39、CP75和CP91的仿磷酸点突变对蛋白质-蛋白质相互作用的影响：将研究点突变对BioID相互作用的影响，以及对GFP标记的CP39、CP75和CP91蛋白与已知共表达的His-Myc标记结合伙伴的共沉淀的影响。对于微观水平的研究，我们将使用错误定位试验，即三个候选及其仿磷突变体定位于细胞皮质。将分析其结合伙伴的皮质共定位的突变依赖的变化。此外，我们将研究点突变变体的表达是否会导致其结合伙伴的中心体定位在有丝分裂期间发生变化，这也表明相互作用依赖于磷酸化状态。