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

网骨藻是研究中心体生物学中基本的中心粒独立过程的有用工具，因为它们是唯一具有无中心粒中心体的非后鞭毛模型。它们由一个三层的核心结构所包围的微管成核冠。我们的目标是阐明这些中心体复制的分子机制。G2/M转变的关键事件是三层核心结构的分裂，从而中心层消失。后者由三种组分组成，CP 39，CP 75和CP 91，它们是有丝分裂开始时由有丝分裂激酶调节的潜在靶点。基于来自其他生物的结果和已知的BioID-蛋白质-蛋白质相互作用，三种激酶Plk、CDK 1和Nek 2是调节剂的候选者。在这个项目中，我们将阐明这些激酶中的哪一个磷酸化三个中心层蛋白中的哪一个，我们将深入了解这些磷酸化对其特定蛋白质-蛋白质相互作用的影响。我们将进行CP 39，CP 75和CP 91的点突变研究，重点是已知参与相应蛋白质有丝分裂调控的亚结构域。突变体将表达为GFP融合蛋白。使用多个磷酸化相关的点突变，我们希望揭示这三种蛋白质中的磷酸化位点是它们从有丝分裂中心体解离所必需的。关于CDK 1，我们还将分析细胞周期蛋白B中心体靶向结构域点突变的影响。- Plk、CDK 1和Nek 2将分别通过使用自体宿主中表达的重组激酶的体外激酶测定法被验证为鉴定的磷酸化位点的真正修饰剂。在这种情况下，我们的目标是建立一个新的方法，M期细胞周期同步在Dictyosteoblasts，其中内源性CDK 1将被替换的CDK 1突变体比可以可逆地抑制ATP类似物。- 体内磷酸化位点的质谱鉴定：将比较对照细胞和表达三种激酶的显性阴性变体的菌株。CP 39、CP 75和CP 91的磷酸化模拟点突变对蛋白质-蛋白质相互作用的影响：将研究点突变对BioID相互作用以及对GFP标记的CP 39、CP 75和CP 91蛋白与已知共表达的His-Myc标记的结合配偶体的共沉淀的影响。对于显微镜水平的研究，我们将使用错误定位测定，其中三个候选人和他们的拟磷酸化突变体靶向细胞皮层。将分析其结合配偶体的皮质共定位的突变依赖性变化。此外，我们将研究点突变变体的表达是否会导致有丝分裂期间其结合伴侣的中心体定位的改变，这也表明相互作用对磷酸化状态的依赖性。

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.