Characterization of PBIP1, a novel polo-box-binding protein at the kinetochores

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Mammalian polo-like kinase 1 (Plk1) plays a pivotal role during M-phase progression. Plk1 localizes to specific subcellular structures such as centrosomes, kinetochores, and midbody, through the polo-box domain (PBD) that specifically interacts with S-pS/pT epitopes found in various intracellular proteins. Failure in the process results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect, thus leading to the generation of aneuploidy. The molecular mechanism underlying PBD-dependent localization to and de-localization from specific subcellular locations remains largely elusive. Here we showed that the Plk1 catalytic activity self-regulates the timing of not only its localization to the interphase centromeres but also its delocalization from the mitotic kinetochores. During interphase, Plk1 phosphorylated PBIP1 at T78 and interacted with the PBIP1-Cenp-Q complex to promote its own recruitment to the centromeres. In mitosis, however, elevated Plk1 activity triggered its own delocalization from the kinetochores by hyperphosphorylating and dissociating the Plk1-PBIP1-Cenp-Q complex from this location. Interestingly, either mutation of the T78 of PBIP1 to Ala or disruption of the PBIP1-Cenp-Q complex was sufficient to abolish the Plk1-dependent Cenp-Q phosphorylation, suggesting that both p-T78-dependent Plk1-PBIP1 interaction and the prior formation of the PBIP1-Cenp-Q complex are required for this event. Depletion of Plk1 increased the fraction of the PBIP1-Cenp-Q complex associating with chromatin and acute inhibition of the Plk1 activity induced relocalization of the complex to the kinetochores. A single mutation of the T78 of PBIP1 to Ala or mutations of the Plk1-dependent 9 phosphorylation sites on Cenp-Q to Ala promoted their association with chromatin, whereas mutations of the 9 residues to phospho-mimicking Asp and Glu residues triggered dissociation of Cenp-Q from chromatin. Taken together, our results presented here demonstrate that Plk1 autonomously regulates the timing of its localization to the interphase centromeres and its dissociation from the mitotic kinetochores by directly regulating the level of phosphorylation on the PBIP1-Cenp-Q complex. Further investigation on the role of the PBIP1-Cenp-Q complex in the regulation of chromosome segregation and genomic stability is under way.