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

Abstract

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 CDK5 regulatory subunit associated protein 2 (CDK5RAP2) family matrix proteins to generate γTuC docking sites. We address the mechanism of this conversion using <i>Caenorhabditis elegans</i> SPindle Defective (SPD-5) as a model. We show that SPD-5 contains two regions, phospho-regulated γTuC binding region 1 (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 a model in which PLK1 phosphorylation induces a conformational change that enables MZT-1-dependent PRGB2 engagement, which in turn relieves PRGB1 inhibition. Such a multistep mechanism would ensure robust spindle assembly by restricting microtubule nucleation in space and time.