RESUMO
Centrioles organize centrosomes, the cell's primary microtubule-organizing centers (MTOCs). Centrioles double in number each cell cycle, and mis-regulation of this process is linked to diseases such as cancer and microcephaly. In C. elegans, centriole assembly is controlled by the Plk4 related-kinase ZYG-1, which recruits the SAS-5-SAS-6 complex. While the kinase activity of ZYG-1 is required for centriole assembly, how it functions has not been established. Here we report that ZYG-1 physically interacts with and phosphorylates SAS-5 on 17 conserved serine and threonine residues in vitro. Mutational scanning reveals that serine 10 and serines 331/338/340 are indispensable for proper centriole assembly. Embryos expressing SAS-5S10A exhibit centriole assembly failure, while those expressing SAS-5S331/338/340A possess extra centrioles. We show that in the absence of serine 10 phosphorylation, the SAS-5-SAS-6 complex is recruited to centrioles, but is not stably incorporated, possibly due to a failure to coordinately recruit the microtubule-binding protein SAS-4. Our work defines the critical role of phosphorylation during centriole assembly and reveals that ZYG-1 might play a role in preventing the formation of excess centrioles.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteínas de Ciclo Celular , Centríolos , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Centríolos/metabolismo , Fosforilação , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Serina/metabolismo , Sequência de Aminoácidos , Proteínas QuinasesRESUMO
Centrosomes are composed of a centriolar core surrounded by a pericentriolar material (PCM) matrix that docks microtubule-nucleating γ-tubulin complexes. During mitotic entry, the PCM matrix increases in size and nucleating capacity in a process called centrosome maturation. Polo-like kinase 1 (PLK1) is recruited to centrosomes and phosphorylates PCM matrix proteins to drive their self-assembly, which leads to PCM expansion. Here, we show that in addition to controlling PCM expansion, PLK1 independently controls the generation of binding sites for γ-tubulin complexes on the PCM matrix. Selectively preventing the generation of PLK1-dependent γ-tubulin docking sites led to spindle defects and impaired chromosome segregation without affecting PCM expansion, highlighting the importance of phospho-regulated centrosomal γ-tubulin docking sites in spindle assembly. Inhibiting both γ-tubulin docking and PCM expansion by mutating substrate target sites recapitulated the effects of loss of centrosomal PLK1 on the ability of centrosomes to catalyze spindle assembly.