Quantitative Analysis of the Human Spindle Phosphoproteome at Distinct Mitotic Stages
Quantitative Analysis of the Human Spindle Phosphoproteome at Distinct Mitotic Stages
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DOI：
10.1021/pr9003773
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发表时间：
2009-10-01
影响因子：
4.4
通讯作者：
Koerner, Roman
中科院分区：
文献类型：
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作者：
Malik, Rainer;Lenobel, Rene;Koerner, Roman
在有丝分裂过程中，纺锤体相关蛋白的磷酸化是纺锤体形成、有丝分裂进程和胞质分裂的关键调控机制。近期，质谱分析已成功应用于鉴定纺锤体蛋白质组和磷酸化蛋白质组，但并未涉及它们的动态变化。在此，我们对从不同有丝分裂阶段制备的纺锤体磷酸化蛋白质组进行了定量比较。我们总共报道了1940个独特磷酸化位点的鉴定以及基于稳定同位素标记氨基酸的细胞培养（SILAC）相对定量，并且发现有丝分裂后期（后期、末期）与蛋白质磷酸化的剧烈改变相关。进一步的统计聚类分析表明，磷酸化动态变化对激酶共有模式有很强的依赖性，从而将鉴定出的磷酸化位点亚组与已知的关键有丝分裂激酶联系起来。令人惊讶的是，我们观察到在有丝分裂后期，磷酸化苏氨酸的去磷酸化程度比磷酸化丝氨酸残基显著更高，这表明在此阶段磷酸酶对磷酸化苏氨酸具有底物偏好。总之，我们的研究结果构成了不同有丝分裂阶段磷酸化丰度的大量定量数据资源，并为深入了解有丝分裂过程中磷酸化动态的系统特性提供了依据。
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.