Park Chi Hoon, Park Jung-Eun, Kim Tae-Sung, Kang Young Hwi, Soung Nak-Kyun, Zhou Ming, Kim Nam-Hyung, Bang Jeong Kyu, Lee Kyung S
From the Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892-4258.
the Immune and Vascular Cell Network Research Center, Department of Life Science and GT5 Program, Ewha Womans University, Seoul 120-750, Korea.
J Biol Chem. 2015 Mar 27;290(13):8569-81. doi: 10.1074/jbc.M114.623546. Epub 2015 Feb 10.
Mammalian Plk1 is critically required for proper M phase progression. Plk1 is self-recruited to prekinetochores/kinetochores by phosphorylating and binding to the Thr-78 motif of a kinetochore scaffold protein, PBIP1 (also called CENP-U/50), which forms a stable complex with another kinetochore component, CENP-Q. However, the mechanism regulating Plk1 localization to this site remains largely unknown. Here, we demonstrate that the PBIP1·CENP-Q complex became hyperphosphorylated and rapidly delocalized from kinetochores as cells entered mitosis. Plk1 phosphorylated the CENP-Q subunit of the PBIP1·CENP-Q complex at multiple sites, and mutation of nine Plk1-dependent phosphorylation sites to Ala (9A) enhanced CENP-Q association with chromatin and prolonged CENP-Q localization to kinetochores. Conversely, mutation of the nine sites to phospho-mimicking Asp/Glu (9D/E) residues dissociated CENP-Q from chromatin and kept the CENP-Q(9D/E) mutant from localizing to interphase prekinetochores. Strikingly, both the 9A and 9D/E mutants induced a defect in proper chromosome segregation, suggesting that both timely localization of the PBIP1·CENP-Q complex to prekinetochores and delocalization from kinetochores are critical for normal M phase progression. Notably, although Plk1 did not alter the level of PBIP1 and CENP-Q ubiquitination, Plk1-dependent phosphorylation and delocalization of these proteins from kinetochores appeared to indirectly lead to their degradation in the cytosol. Thus, we propose that Plk1 regulates the timing of the delocalization and ultimate destruction of the PBIP1·CENP-Q complex and that these processes are important not only for promoting Plk1-dependent mitotic progression, but also for resetting the timing of Plk1 recruitment to prekinetochores in the next cell cycle.
哺乳动物的Plk1对于正确的M期进程至关重要。Plk1通过磷酸化并结合动粒支架蛋白PBIP1（也称为CENP-U/50）的Thr-78基序，将自身招募到前动粒/动粒，PBIP1与另一个动粒成分CENP-Q形成稳定的复合物。然而，调节Plk1定位于该位点的机制在很大程度上仍然未知。在这里，我们证明随着细胞进入有丝分裂，PBIP1·CENP-Q复合物发生过度磷酸化并迅速从动粒上脱离。Plk1在多个位点磷酸化PBIP1·CENP-Q复合物的CENP-Q亚基，将九个Plk1依赖性磷酸化位点突变为丙氨酸（9A）增强了CENP-Q与染色质的结合，并延长了CENP-Q在动粒上的定位时间。相反，将这九个位点突变为模拟磷酸化的天冬氨酸/谷氨酸（9D/E）残基会使CENP-Q从染色质上解离，并使CENP-Q(9D/E)突变体无法定位于间期前动粒。令人惊讶的是，9A和9D/E突变体均诱导了正确染色体分离的缺陷，这表明PBIP1·CENP-Q复合物及时定位于前动粒以及从动粒上脱离对于正常的M期进程都至关重要。值得注意的是，尽管Plk1没有改变PBIP1和CENP-Q泛素化的水平，但Plk1依赖性磷酸化以及这些蛋白从动粒上的脱离似乎间接导致它们在细胞质中降解。因此，我们提出Plk1调节PBIP·CENP-Q复合物脱离和最终破坏的时间，并且这些过程不仅对于促进Plk1依赖性有丝分裂进程很重要，而且对于在下一个细胞周期中重置Plk1招募到前动粒的时间也很重要。
