Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics
Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics
复制标题
使用 Polo 样激酶 4 (PLK4) 抑制剂 centrinone 通过基于 SILAC 的磷酸蛋白质组学研究细胞内信号网络
DOI：
10.1101/2020.05.22.110767
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Byrne D
中科院分区：
文献类型：
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作者：
Byrne D
Polo样激酶4（PLK 4）是后生动物中心粒复制的主要调节因子。PLK 4的催化活性和蛋白质周转在人类细胞中紧密耦合，因为PLK 4浓度和催化的变化对中心粒复制和额外中心体具有深远影响，这与非整倍性和癌症相关。最近，PLK 4已经被以centrinone为例的各种小分子激酶抑制剂靶向，其快速诱导对PLK 4的抑制作用并导致靶向中心体耗竭。尽管如此，在人类细胞中已经明确鉴定了相对较少的PLK 4底物，并且中心粒网络外的PLK 4信号传导仍然很难表征。我们报告了一种基于无偏质谱（MS）的细胞蛋白磷酸化定量分析，该分析对暴露于Centrinone的稳定PLK 4表达U2 OS人类细胞中的细胞蛋白磷酸化进行了定量分析。PLK 4磷酸化本身对短暂暴露于化合物敏感，导致PLK 4稳定。分析异步细胞群体，我们报告了数百个centrinone调节的细胞磷蛋白，包括中心体和细胞周期蛋白和各种可能的“非经典”底物。令人惊讶的是，对PLK 300显著下调的磷蛋白的序列询问揭示了一个广泛的中心蛋白敏感的[Ser/Thr]Pro磷酸化序列基序网络，根据我们的分析，这可能是PLK 4的直接或间接靶点。此外，我们证实NMYC和PTPN 12是PLK 4的底物，无论是在体外还是在人细胞中。我们的研究结果表明，PLK 4催化输出直接控制一组不同的细胞蛋白质的磷酸化，包括可能在PLK 4介导的细胞信号传导中很重要的Pro定向靶点。
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 signalling 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 stabilisation. 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, bothin vitroand 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 signalling.