Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK; Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK.
Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
Cell Rep. 2023 Mar 28;42(3):112139. doi: 10.1016/j.celrep.2023.112139. Epub 2023 Feb 24.
Ordered protein phosphorylation by CDKs is a key mechanism for regulating the cell cycle. How temporal order is enforced in mammalian cells remains unclear. Using a fixed cell kinase assay and phosphoproteomics, we show how CDK1 activity and non-catalytic CDK1 subunits contribute to the choice of substrate and site of phosphorylation. Increases in CDK1 activity alter substrate choice, with intermediate- and low-sensitivity CDK1 substrates enriched in DNA replication and mitotic functions, respectively. This activity dependence is shared between Cyclin A- and Cyclin B-CDK1. Cks1 has a proteome-wide role as an enhancer of multisite CDK1 phosphorylation. Contrary to the model of CDK1 as an exclusively proline-directed kinase, we show that Cyclin A and Cks1 enhance non-proline-directed phosphorylation, preferably on sites with a +3 lysine residue. Indeed, 70% of cell-cycle-regulated phosphorylations, where the kinase carrying out this modification has not been identified, are non-proline-directed CDK1 sites.
CDK 有序的蛋白磷酸化是调节细胞周期的关键机制。在哺乳动物细胞中，时间顺序是如何被强制执行的仍不清楚。通过固定细胞激酶测定和磷酸蛋白质组学，我们展示了 CDK1 活性和非催化 CDK1 亚基如何有助于选择底物和磷酸化位点。CDK1 活性的增加改变了底物的选择，中敏和低敏 CDK1 底物分别在 DNA 复制和有丝分裂功能中富集。这种活性依赖性在 Cyclin A 和 Cyclin B-CDK1 之间是共享的。Cks1 在广泛的蛋白质组范围内作为多位点 CDK1 磷酸化的增强子。与 CDK1 作为一个专门的脯氨酸导向激酶的模型相反，我们表明 Cyclin A 和 Cks1 增强了非脯氨酸导向的磷酸化，特别是在带有+3 赖氨酸残基的位点上。事实上，70%的细胞周期调控磷酸化，其中进行这种修饰的激酶尚未被确定，是非脯氨酸导向的 CDK1 位点。
