Département de Biochimie and Centre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal, Montréal, QC, Canada H3C 3J7.
Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15716-21. doi: 10.1073/pnas.1305420110. Epub 2013 Sep 9.
Cyclin-dependent kinases (Cdks) are regulatory enzymes with temporal and spatial selectivity for their protein substrates that are governed by cell cycle-regulated cyclin subunits. Specific cyclin-Cdk complexes bind to and phosphorylate target proteins, coupling their activity to cell cycle states. The identification of specific cyclin-Cdk substrates is challenging and so far, has largely been achieved through indirect correlation or use of in vitro techniques. Here, we use a protein-fragment complementation assay based on the optimized yeast cytosine deaminase to systematically identify candidate substrates of budding yeast Saccharomyces cerevisiae Cdk1 and show dependency on one or more regulatory cyclins. We identified known and candidate cyclin dependencies for many predicted protein kinase Cdk1 targets and showed elusory Clb3-Cdk1-specific phosphorylation of γ-tubulin, thus establishing the timing of this event in controlling assembly of the mitotic spindle. Our strategy can be generally applied to identify substrates and accessory subunits of multisubunit protein complexes.
周期蛋白依赖性激酶（Cdks）是调节酶，对其蛋白质底物具有时间和空间选择性，这些底物受细胞周期调节的周期蛋白亚基控制。特定的周期蛋白-Cdk 复合物与靶蛋白结合并使其磷酸化，将其活性与细胞周期状态偶联。特定的周期蛋白-Cdk 底物的鉴定具有挑战性，到目前为止，主要通过间接相关性或使用体外技术来实现。在这里，我们使用基于优化的酵母胞嘧啶脱氨酶的蛋白片段互补测定法，系统地鉴定出芽殖酵母酿酒酵母 Cdk1 的候选底物，并显示其对一种或多种调节性周期蛋白的依赖性。我们鉴定了许多预测蛋白激酶 Cdk1 靶标的已知和候选周期蛋白依赖性，并显示了 γ-微管蛋白的难以捉摸的 Clb3-Cdk1 特异性磷酸化，从而确定了该事件在控制有丝分裂纺锤体组装中的时间。我们的策略可以广泛应用于鉴定多亚基蛋白复合物的底物和辅助亚基。
