Cui Heying, Loftus Kyle M, Noell Crystal R, Solmaz Sozanne R
Department of Chemistry, State University of New York at Binghamton.
Department of Chemistry, State University of New York at Binghamton;
J Vis Exp. 2018 May 3(135):57674. doi: 10.3791/57674.
Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.
细胞周期蛋白依赖性激酶1（Cdk1）是所有真核生物细胞周期的主要调控因子，估计能磷酸化蛋白质组中8%-13%的蛋白质；然而，已确定的Cdk1靶点数量仍然很少，尤其是在人类细胞中。确定Cdk1特异性磷酸化位点很重要，因为它们能为Cdk1如何控制细胞周期提供机制上的见解。细胞周期调控对于准确的染色体分离至关重要，这一复杂过程中的缺陷会导致染色体畸变和癌症。在这里，我们描述了一种用于鉴定Cdk1特异性磷酸化位点的体外激酶测定法。在该测定法中，一种纯化的蛋白质在体外被市售的人Cdk1/细胞周期蛋白B磷酸化。通过SDS-PAGE确认磷酸化成功，随后通过质谱鉴定磷酸化位点。我们还描述了纯化方案，可得到适用于激酶测定的高纯度和均一性的蛋白质制剂，以及用于对已鉴定的磷酸化位点进行功能验证的结合测定法，该方法可探测经典核定位信号（cNLS）与其核转运受体核转运蛋白α之间的相互作用。为了辅助实验设计，我们回顾了从蛋白质序列预测Cdk1特异性磷酸化位点的方法。这些方案共同提供了一种非常强大的方法，可产生Cdk1特异性磷酸化位点，并能够对Cdk1如何控制细胞周期进行机制研究。由于该方法依赖于纯化的蛋白质，它可应用于任何模式生物并产生可靠的结果，特别是与细胞功能研究相结合时。
