The DNA replication protein Cdc6 inhibits the microtubule-organizing activity of the centrosome

Abstract The centrosome serves as a major microtubule-organizing center (MTOC). The Cdc6 protein is a component of the pre-replicative complex and a licensing factor for the initiation of chromosome replication and localizes to centrosomes during the S and G2 phases of the cfell cycle of human cells. This cell cycle-dependent localization of Cdc6 to the centrosome motivated us to investigate whether Cdc6 negatively regulates MTOC activity and to determine the integral proteins that comprise the pericentriolar material (PCM). Time-lapse live-cell imaging of microtubule regrowth revealed that Cdc6 depletion increased microtubule nucleation at the centrosomes and that expression of Cdc6 in Cdc6-depleted cells reversed this effect. This increase and decrease in microtubule nucleation correlated with the centrosomal intensities of PCM proteins such as γ-tubulin, pericentrin, CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), and centrosomal protein 192 (Cep192). The regulation of microtubule nucleation and the recruitment of PCM proteins to the centrosome required Cdc6 ATPase activity, as well as a centrosomal localization of Cdc6. These results suggest a novel function for Cdc6 in coordinating centrosome assembly and function.