Department of Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Mol Cell Proteomics. 2010 Jun;9(6):1167-81. doi: 10.1074/mcp.M900335-MCP200. Epub 2010 Jan 23.
Reversible protein phosphorylation is a key regulatory mechanism of mitotic progression. Importantly, protein kinases themselves are also regulated by phosphorylation-dephosphorylation processes; hence, phosphorylation dynamics of kinases hold a wealth of information about phosphorylation networks. Here, we investigated the site-specific phosphorylation dynamics of human kinases during mitosis using synchronization of HeLa suspension cells, kinase enrichment, and high resolution mass spectrometry. In biological triplicate analyses, we identified 206 protein kinases and more than 900 protein kinase phosphorylation sites, including 61 phosphorylation sites on activation segments, and quantified their relative abundances across three specific mitotic stages. Around 25% of the kinase phosphorylation site ratios were found to be changed by at least 50% during mitotic progression. Further network analysis of jointly regulated kinase groups suggested that Cyclin-dependent kinase- and mitogen-activated kinase-centered interaction networks are coordinately down- and up-regulated in late mitosis, respectively. Importantly, our data cover most of the already known mitotic kinases and, moreover, identify attractive candidates for future studies of phosphorylation-based mitotic signaling. Thus, the results of this study provide a valuable resource for cell biologists and provide insight into the system properties of the mitotic phosphokinome.
蛋白质可逆磷酸化是有丝分裂进程的关键调控机制。重要的是，蛋白激酶本身也受到磷酸化-去磷酸化过程的调节；因此，激酶的磷酸化动力学包含了丰富的关于磷酸化网络的信息。在这里，我们使用 HeLa 悬浮细胞的同步化、激酶富集和高分辨率质谱法，研究了人类激酶在有丝分裂过程中的特定位点磷酸化动力学。在生物学三重分析中，我们鉴定了 206 种蛋白激酶和超过 900 个蛋白激酶磷酸化位点，包括激活片段上的 61 个磷酸化位点，并定量分析了它们在三个特定有丝分裂阶段的相对丰度。大约 25%的激酶磷酸化位点比率在有丝分裂过程中至少变化了 50%。对共同调控的激酶群进行的进一步网络分析表明，细胞周期蛋白依赖性激酶和丝裂原激活的蛋白激酶为中心的相互作用网络分别在晚期有丝分裂中协调地下调和上调。重要的是，我们的数据涵盖了大多数已被发现的有丝分裂激酶，并且还鉴定了基于磷酸化的有丝分裂信号的未来研究的有吸引力的候选者。因此，这项研究的结果为细胞生物学家提供了宝贵的资源，并深入了解了有丝分裂磷酸激酶组的系统特性。
