Regulation of centrosome duplication and function by Cdc6

Abstract

The centrosome functions as major microtubule organizing center. Microtubules play critical roles in chromosome segregation, cell shape and motility, vesicular transport, and signal transduction. The Cdc6 protein has been primarily investigated as a component of the pre-replicative complex for the initiation of chromosome replication, which contributes to maintenance of chromosomal integrity. I demonstrated a novel function of Cdc6 in controlling microtubule formation at the centrosomes. A conserved centrosome localization signal allowed Cdc6 to localize to S- and G2-phase centrosomes in a cell cycle-dependent manner. Cdc6 depletion increased the amounts of the integral proteins Cep215/CDK5RAP2 and Cep192 of the pericentriolar material (PCM), promoting the gamma-tubulin ring complex (gamma-TuRC) recruitment and microtubule formation, suggesting that Cdc6 modulates the PCM to control gamma-TuRC recruitment. Furthermore, Cdc6 depletion increased cell adhesion and spreading, and Cdc6 overexpression had the opposite effect. These results demonstrate that Cdc6 contributes to control of cell cycle-regulated microtubule formation. Centrosome is duplicated through cell cycle and its integrity is critical for chromosome segregation that is tightly linked to chromosome stability. I addressed that Cdc6 negatively regulated centrosome amplification and Grp78 was required for centrosome amplification. Whereas Cdc6 depletion induced centrosome amplification, Cdc6 overexpression suppressed hydroxyurea (HU)-induced centrosome amplification. The centrosome amplification arising by Cdc6 depletion was caused by centrosome over-duplication rather than centriole fragmentation. Amino acid residues 197 to 214 of Cdc6 (Cdc6-SCA: suppression of centrosome amplification) was responsible for suppression of centrosome amplification. The Cdc6-SCA contained a FHA domain protein binding motif and mediated interaction with Grp78, which is a molecular chaperon of HSP70 family. Grp78 localized to interphase centrosomes and was necessary for centrosome amplification by Cdc6 depletion or HU treatment. My results suggest that interaction between Cdc6 and Grp78 is involved in controlling centrosome duplication. Through my graduate works, I have demonstrated novel functions of Cdc6 on the regulation of microtubule formation and centrosome duplication. My achievements will contribute to understanding centrosome function and cell cycle control.