Polo-like kinase 1 (Plk1)-dependent centrosome maturation and spindle bipolarity

纺锤体双极性的建立是染色体正确分离和有丝分裂进程的关键。哺乳动物polo样激酶1（Plk 1）被认为在这一过程中发挥了核心作用。然而，Plk 1如何促成这一事件仍不清楚。在这里，我们证明了Cdc 2依赖性磷酸化的γ-TuRC招募蛋白，Nedd 1/GCP-WD，在两个不同的网站提供了时间和空间线索，及时带来Plk 1功能的中心体和纺锤体通过磷酸化依赖性的相互作用与Polo盒结构域的Plk 1。在中心体，Plk 1与Nedd 1的C-末端磷酸化基序相互作用，使后者磷酸化，这一步骤对基于中心体的微管成核至关重要。紧接着这一事件，Plk 1也相互作用的N-末端磷酸化基序Nedd 1沿着纺锤体和磷酸化Augmin亚基，Hice 1，以促进微管为基础的微管成核。Nedd 1介导的Hice 1磷酸化Plk 1的损失导致受损的Augmin与微管的相互作用和减少γ-微管蛋白招聘的纺锤体，最终导致缺陷，在适当的双极纺锤体形成和染色体分离。总之，这里提供的数据表明，两个不同的Plk 1-Nedd 1相互作用的中心体和纺锤体的潜在机制被破译成不同的生化和细胞的结果，以实现正常的双极纺锤体的形成和有丝分裂的进展。这项研究可能使我们了解Plk 1如何与其他有丝分裂组分合作，以促进在不同的亚细胞结构的特定蛋白质复合物的及时激活的潜在机制。除了Plk 1在中心体调节Nedd 1中的作用外，我们一直专注于了解Cep 192-Plk 1相互作用的生理意义。我们的研究结果表明Plk 1与Cep 192的磷酸化S995基序结合。为了更好地理解这一事件的意义，我们已经产生了表达磷酸化缺陷型Cep 192 S995 A突变体的细胞。对这种突变体的鉴定正在进行中。初步结果表明，p-S995依赖Cep 192和Plk 1之间的相互作用是重要的纺锤体双极性在M期的细胞周期的正确建立。深入分析Cep 192 S995 A突变体表达细胞可以让我们理解Aurora A如何影响Plk 1的功能，反之亦然。

Establishment of spindle bipolarity is pivotal for proper chromosome segregation and mitotic progression. Mammalian polo-like kinase 1 (Plk1) has been thought to play a central role in this process. However, how Plk1 contributes to this event remains unknown. Here we demonstrated that Cdc2-dependent phosphorylation on a gamma-TuRC recruitment protein, Nedd1/GCP-WD, at two distinct sites provides both temporal and spatial cues to timely bring about Plk1 functions to the centrosomes and spindles via phosphorylation-dependent interaction with the polo-box domain of Plk1. At centrosomes, Plk1 interacted with a C-terminal phosphorylated motif of Nedd1 to phosphorylate the latter, a step suggested to be important for centrosome-based microtubule nucleation. Immediately following this event, Plk1 also interacted with an N-terminal phosphorylated motif of Nedd1 along the spindles and phosphorylated an Augmin subunit, Hice1, to promote microtubule-based microtubule nucleation. Loss of Nedd1-mediated Hice1 phosphorylation by Plk1 resulted in impaired Augmin interaction with microtubules and diminished gamma-tubulin recruitment to the spindles that ultimately led to defects in proper bipolar spindle formation and chromosome segregation. Taken together, the data provided here demonstrate the underlying mechanisms of how the two distinct Plk1-Nedd1 interactions at the centrosomes and spindles are deciphered into different biochemical and cellular outcomes to achieve normal bipolar spindle formation and mitotic progression. This study may allow us to understand the underlying mechanism of how Plk1 cooperates with other mitotic components to promote timely activation of specific protein complexes at distinct subcellular structures. Aside from the role of Plk1 in the regulation of Nedd1 at centrosomes, we have been focusing on understanding the physiological significance of the Cep192-Plk1 interaction. Our results showed that Plk1 binds to the phosphorylated S995 motif of Cep192. To better understand the significance of this event, we have generated cells expressing a phosphorylation-defective Cep192 S995A mutant. Characterization of this mutant is underway. Preliminary results suggests that the p-S995-dependent interaction between Cep192 and Plk1 is important for proper establishment of spindle bipolarity during M-phase of the cell cycle. In depth analysis of the Cep192 S995A mutant-expressing cells may allow us to comprehend how Aurora A influences the function of Plk1, vice versa.