Quantitative Analysis of the Human Spindle Phosphoproteome at Distinct Mitotic Stages

During mitosis, phosphorylation of spindle associated proteins is a key regulatory mechanism for spindle formation, mitotic progression, and cytokinesis. In the recent past, mass spectrometry has been applied successfully to identify spindle proteomes and phosphoproteomes, but did not address their dynamics. Here, we present a quantitative comparison of spindle phosphoproteomes prepared from different mitotic stages. In total, we report the identification and SILAC based relative quantitation of 1940 unique phosphorylation sites and find that late mitosis (anaphase, telophase) is correlated with a drastic alteration in protein phosphorylation. Further statistical cluster analyses demonstrate a strong dependency of phosphorylation dynamics on kinase consensus patterns, thus, linking subgroups of identified phosphorylation sites to known key mitotic kinases. Surprisingly, we observed that during late mitosis strong dephosphorylation occurred on a significantly larger fraction of phospho-threonine than phospho-serine residues, suggesting a substrate preference of phosphatases for phospho-threonine at this stage. Taken together, our results constitute a large quantitative data resource of phosphorylation abundances at distinct mitotic stages and they provide insight into the systems properties of phosphorylation dynamics during mitosis.

在有丝分裂进程中，纺锤体相关蛋白的磷酸化是调控纺锤体组装、有丝分裂进程与胞质分裂的核心机制。近年来，质谱技术（mass spectrometry）已被成功应用于纺锤体蛋白质组（spindle proteomes）与磷酸化蛋白质组（phosphoproteomes）的鉴定，但尚未能阐明其动态变化规律。本研究针对不同有丝分裂时期的纺锤体磷酸化蛋白质组开展了定量比较分析。本研究共鉴定出1940个独特磷酸化位点，并完成了基于SILAC的相对定量分析；结果显示，有丝分裂晚期（后期、末期）的蛋白质磷酸化水平发生了显著改变。进一步的统计聚类分析表明，磷酸化动态变化与激酶共识基序存在显著依赖关系，由此将鉴定出的磷酸化位点亚群与已知的核心有丝分裂激酶建立了关联。令人意外的是，本研究观察到，在有丝分裂晚期，磷酸化苏氨酸残基的去磷酸化比例显著高于磷酸化丝氨酸残基，这提示该阶段的磷酸酶对磷酸化苏氨酸底物具有偏好性。综上，本研究的结果构建了覆盖不同有丝分裂时期磷酸化丰度的大型定量数据集资源，并为解析有丝分裂过程中磷酸化动态变化的系统特性提供了新的研究视角。