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

• Applicant: Professor Dr. Ralph Gräf
• Subject Area: Cell Biology ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 463103716

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.

• DFG Programme: Research Grants