Dissmeyer Nico, Schnittger Arp
Leibniz Institute of Plant Biochemistry (IPB), Independent Junior Research Group on Protein Recognition and Degradation, Halle (Saale), Germany.
Methods Mol Biol. 2011;779:93-138. doi: 10.1007/978-1-61779-264-9_6.
Biological information is often transmitted by phosphorylation cascades. However, the biological relevance of specific phosphorylation events is often difficult to determine. An invaluable tool to study the effect of kinases and/or phosphatases is the use of phospho- and dephospho-mimetic substitutions in the respective target proteins. Here, we present a generally applicable procedure of how to design, set-up, and carry out phosphorylation modulation experiments and subsequent monitoring of protein activities, taking -cyclin-dependent kinases (CDKs) as a case study. CDKs are key regulators of cell cycle progression in all eukaryotic cells. Consequently, CDKs are controlled at many levels and phosphorylation of CDKs -themselves is used to regulate their kinase activity. We describe in detail complementation experiments of a mutant in CDKA;1, the major cell cycle kinase in Arabidopsis, with phosphorylation-site variants of CDKA;1. CDKA;1 versions were generated either by mimicking a phosphorylated amino acid by replacing the respective residue with a negatively charged amino acid, e.g., aspartate or glutamate, or by mutating it to a non-phoshorylatable amino acid, such as alanine, valine, or phenylalanine. The genetic complementation studies were accompanied by the isolation of these kinase variants from plant extract and subsequent kinase assays to determine changes in their activity levels. This work allowed us to judge the importance of -posttranslational regulation of CDKA;1 in plants and has shown that the molecular mechanistics of CDK function are apparently conserved across the kingdoms. However, the regulatory wiring of CDKs is -strikingly different between plants, animals, and yeast.
生物信息通常通过磷酸化级联反应进行传递。然而，特定磷酸化事件的生物学相关性往往难以确定。研究激酶和/或磷酸酶作用的一个宝贵工具是在各自的靶蛋白中使用磷酸化模拟和去磷酸化模拟替代物。在这里，我们以细胞周期蛋白依赖性激酶（CDK）为例，介绍一种普遍适用的程序，用于设计、设置和进行磷酸化调节实验以及随后对蛋白质活性进行监测。CDK是所有真核细胞中细胞周期进程的关键调节因子。因此，CDK在多个水平受到调控，其自身的磷酸化被用于调节激酶活性。我们详细描述了拟南芥中主要的细胞周期激酶CDKA;1突变体与CDKA;1磷酸化位点变体的互补实验。通过用带负电荷的氨基酸（如天冬氨酸或谷氨酸）取代相应残基来模拟磷酸化氨基酸，或者将其突变为不可磷酸化的氨基酸（如丙氨酸、缬氨酸或苯丙氨酸）来产生CDKA;1变体。遗传互补研究伴随着从植物提取物中分离这些激酶变体以及随后的激酶测定以确定其活性水平的变化。这项工作使我们能够判断植物中CDKA;1翻译后调控的重要性，并表明CDK功能的分子机制在整个生物界显然是保守的。然而，植物、动物和酵母中CDK的调控方式明显不同。
