Dephoure Noah, Zhou Chunshui, Villén Judit, Beausoleil Sean A, Bakalarski Corey E, Elledge Stephen J, Gygi Steven P
Department of Cell Biology, Harvard University Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10762-7. doi: 10.1073/pnas.0805139105. Epub 2008 Jul 31.
The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated. Using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, we examined protein phosphorylation in a human cell line arrested in the G(1) and mitotic phases of the cell cycle. We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.
真核细胞分裂周期的特点是一系列有序且高度调控的事件，这些事件导致所有细胞物质复制并分离到两个新形成的子细胞中。细胞周期蛋白依赖性激酶（CDK）介导的蛋白质磷酸化驱动这一周期。为了更深入了解磷酸化如何调控细胞周期，我们试图鉴定其磷酸化受细胞周期调控的蛋白质。我们采用稳定同位素标记以及两步法富集磷酸肽和高精度质谱技术，研究了处于细胞周期G1期和有丝分裂期的人细胞系中的蛋白质磷酸化情况。我们报告了超过14,000种不同磷酸化事件的鉴定结果，据我们所知，其中一半以上在文献中未曾描述，同时还给出了这些位点中大多数的相对定量数据。我们观察到有丝分裂过程中磷酸化增加的蛋白质超过1000种，其中包括许多已知的细胞周期调节因子。受调控的磷酸肽上的大多数位点位于[S/T]P基序中，这是CDK作用的最小必需序列，表明许多蛋白质可能是CDK的底物。对不含脯氨酸位点的磷酸肽的分析鉴定出两个独特的基序，这表明至少存在两种未被发现的有丝分裂激酶。
