Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signaling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilization. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely non-canonical substrates. Surprisingly, sequence interrogation of ~300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signaling.

中文翻译：

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使用基于SILAC的磷酸化蛋白质组学研究Polo样激酶4（PLK4）抑制剂孕酮的研究细胞内信号网络

Polo样激酶4（PLK4）是后生生物中中心粒重复的主要调控因子。PLK4的催化活性和蛋白质更新在人类细胞中紧密耦合，因为PLK4浓度和催化作用的改变对与非整倍性和癌症相关的中心粒复制和多余的中心体具有深远的影响。最近，PLK4已被多种以小分子酮为代表的小分子激酶抑制剂靶向，这些抑制剂可迅速诱导对PLK4的抑制作用并导致靶点中心体耗竭。尽管如此，在人类细胞中已经明确地鉴定出相对较少的PLK4底物，而且在中心体网络之外的PLK4信号传递的特征仍然很差。我们报告了稳定的PLK4表达U2OS人类细胞暴露于孕酮的细胞蛋白磷酸化的无偏质谱（MS）为基础的定量分析。PLK4磷酸化本身对短暂暴露于该化合物敏感，从而导致PLK4稳定。分析异步细胞群体后，我们报告了数百种受孕酮调节的细胞磷蛋白，包括中心体蛋白和细胞周期蛋白以及各种可能的非经典底物。出乎意料的是，对约300个显着下调的磷蛋白的序列询问揭示了一个广泛的网络，该网络由对雌激素敏感的[Ser / Thr] Pro磷酸化序列基序组成，根据我们的分析，其可能是PLK4的直接或间接靶标。此外，我们确认NMYC和PTPN12在体外和人细胞中均为PLK4底物。