Phosphorylation of the centrosomal protein, Cep169, by Cdk1 promotes its dissociation from centrosomes in mitosis.

Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle.

Microtubule-bundling activity of the centrosomal protein, Cep169, and its binding to microtubules.

CDK5RAP2 is a centrosomal protein that regulates the recruitment of a γ-tubulin ring complex (γ-TuRC) onto centrosomes and microtubules (MTs) dynamics as a member of MT plus-end-tracking proteins (+TIPs). In our previous report, we found mammalian Cep169 as a CDK5RAP2 binding partner, and Cep169 accumulates at the distal ends of MTs and centrosomes, and coincides with CDK5RAP2. Depletion of Cep169 induces MT depolymerization, indicating that Cep169 targets MT tips and regulates stability and dynamics of MTs. However, how Cep169 contributes to the stabilization of MT remains unclear. Here we show that Cep169 is able to stabilize MTs and induces formation of long MT bundles with intense acetylation of MTs with CDK5RAP2, when expressed at higher levels in U2OS cells. In addition, we demonstrated that Cep169 forms homodimers through its N-terminal domain and directly interacts with MTs through its C-terminal domain. Interestingly, Cep169 mutants, which lack each domains, completely abolished the activity, respectively. Therefore, Cep169 bundles MTs and induces solid structure of MTs by crosslinking each adjacent MTs as a homodimer.