Polo-like kinase 1 (Plk1) is an essential protein kinase that promotes faithful mitotic progression in eukaryotes. The subcellular localization and substrate interactions of Plk1 are tightly controlled and require its binding to phosphorylated residues. To identify phosphorylation-dependent interactions within the Plk1 network in human mitotic cells, we performed quantitative proteomics on HeLa cells cultured with kinase inhibitors or expressing a Plk1 mutant that was deficient in phosphorylation-dependent substrate binding. We found that many interactions were abolished upon kinase inhibition; however, a subset was protected from phosphatase opposition or was unopposed, resulting in persistent interaction of the substrate with Plk1. This subset includes phosphoprotein phosphatase 6 (PP6), whose activity toward Aurora kinase A (Aurora A) was inhibited by Plk1. Our data suggest that this Plk1-PP6 interaction generates a feedback loop that coordinates and reinforces the activities of Plk1 and Aurora A during mitotic entry and is terminated by the degradation of Plk1 during mitotic exit. Thus, we have identified a mechanism for the previously puzzling observation of the Plk1-dependent regulation of Aurora A.

Polo 样激酶 1 (Plk1) 是一种重要的蛋白激酶，可促进真核生物忠实的有丝分裂进展。 Plk1 的亚细胞定位和底物相互作用受到严格控制，需要其与磷酸化残基结合。为了确定人类有丝分裂细胞中 Plk1 网络内磷酸化依赖性相互作用，我们对用激酶抑制剂培养或表达缺乏磷酸化依赖性底物结合的 Plk1 突变体的 HeLa 细胞进行了定量蛋白质组学。我们发现许多相互作用在激酶抑制后被消除。然而，有一个子集受到保护，免受磷酸酶对抗或未受到对抗，导致底物与 Plk1 持续相互作用。该子集包括磷蛋白磷酸酶 6 (PP6)，其对极光激酶 A (Aurora A) 的活性被 Plk1 抑制。我们的数据表明，这种 Plk1-PP6 相互作用产生一个反馈环，在有丝分裂进入期间协调和加强 Plk1 和 Aurora A 的活性，并在有丝分裂退出期间通过 Plk1 的降解而终止。因此，我们已经确定了 Aurora A 的 Plk1 依赖性调节的先前令人费解的观察机制。