Phosphorylation remodels the mitotic centrosome matrix to generate bipartite γ-tubulin complex docking sites.

Mitotic centrosomes consist of centrioles surrounded by a proteinaceous matrix that docks and activates γ-tubulin complexes (γTuCs) to nucleate microtubules for spindle assembly. During mitotic entry, phosphorylation at centrosomes remodels CDK5RAP2 family matrix proteins to generate γTuC docking sites. We address the mechanism of this conversion using C. elegans SPD-5 as a model. We show that SPD-5 contains two regions, PRGB1 and PRGB2, that are each sufficient for Polo-Like Kinase 1 (PLK1) phosphorylation-regulated γTuC binding. We define key phosphosites in each region and uncover autoinhibition mediated by interactions within and between them. PRGB2 is dimeric and requires γTuCs containing the Mozart family microprotein MZT-1 for binding, whereas PRGB1 is monomeric and binds independently of MZT-1. Our results support PLK1 phosphorylation inducing a conformational change that enables MZT-1-dependent PRGB2 binding, which in turn relieves PRGB1 inhibition. Such a multi-step mechanism would ensure robust spindle assembly by restricting microtubule nucleation in space and time.