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

哺乳动物polo样激酶1（Plk 1）在M期进展中起关键作用。Plk 1定位于特定的亚细胞结构，如中心体，动粒，和中间体，通过polo-box结构域（PBD），特异性地与各种细胞内蛋白质中发现的S-pS/pT表位相互作用。该过程的失败导致不适当的双极纺锤体形成、染色体错误分离和胞质分裂缺陷，从而导致非整倍体的产生。PBD依赖的定位和从特定的亚细胞位置去定位的分子机制在很大程度上仍然难以捉摸。在这里，我们表明，Plk 1的催化活性自我调节的时间，不仅其本地化的间期着丝粒，但也从有丝分裂动粒离域。在分裂间期，Plk 1在T78磷酸化PBIP 1，并与PBIP 1-Cenp-Q复合物相互作用，以促进其自身向着丝粒的募集。然而，在有丝分裂中，Plk 1活性升高通过使Plk 1-PBIP 1-Cenp-Q复合物从该位置过度磷酸化和解离而触发其自身从动粒的离域。有趣的是，PBIP 1的T78突变为Ala或PBIP 1-Cenp-Q复合物的破坏足以消除Plk 1依赖性Cenp-Q磷酸化，这表明P-T78依赖性Plk 1-PBIP 1相互作用和PBIP 1-Cenp-Q复合物的预先形成都是该事件所需的。Plk 1的耗竭增加了与染色质相关的PBIP 1-Cenp-Q复合物的分数，并且Plk 1活性的急性抑制诱导复合物重新定位于动粒。PBIP 1的T78突变为Ala或Cenp-Q上Plk 1依赖的9个磷酸化位点突变为Ala促进了它们与染色质的结合，而9个残基突变为磷酸化模拟的Asp和Glu残基则触发Cenp-Q从染色质上解离。两者合计，我们的研究结果表明，Plk 1自主调节其本地化的间期着丝粒和有丝分裂动粒的解离时间，通过直接调节PBIP 1-Cenp-Q复合物的磷酸化水平。目前正在进一步研究PBIP 1-Cenp-Q复合物在染色体分离和基因组稳定性调节中的作用。

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.