细胞周期蛋白依赖性激酶1对着丝粒蛋白C的磷酸化作用
CENP-C Phosphorylation by CDK1 .
作者信息
Watanabe Reito, Hara Masatoshi, Ariyoshi Mariko, Fukagawa Tatsuo
机构信息
Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
出版信息
Bio Protoc. 2021 Jan 5;11(1):e3879. doi: 10.21769/BioProtoc.3879.
Accurate chromosome segregation during mitosis requires the kinetochore, a large protein complex, which makes a linkage between chromosomes and spindle microtubes. An essential kinetochore component, CENP-C, is phosphorylated by Cyclin-B-Cyclin dependent kinase 1 (CDK1) that is a master kinase for mitotic progression, promoting proper kinetochore assembly during mitosis. Here, we describe an CDK1 kinase assay to detect CENP-C phosphorylation using Phos-tag SDS-PAGE without radiolabeled ATP. Our protocol has advantages in ease and safety over conventional phosphorylation assays using [γ-P]-ATP, which has potential hazards despite their better sensitivity. The protocol described here can be applicable to other kinases and be also useful for analysis of phospho-sites in substrates .
有丝分裂期间准确的染色体分离需要动粒，这是一种大型蛋白质复合体，它在染色体与纺锤体微管之间建立联系。动粒的一个关键组成部分CENP-C，会被细胞周期蛋白B-细胞周期蛋白依赖性激酶1（CDK1）磷酸化，CDK1是有丝分裂进程的主要激酶，在有丝分裂期间促进动粒的正确组装。在这里，我们描述了一种使用Phos-tag SDS-PAGE检测CENP-C磷酸化的CDK1激酶测定方法，无需使用放射性标记的ATP。与使用[γ-P]-ATP的传统磷酸化测定方法相比，我们的方案在操作简便性和安全性方面具有优势，尽管传统方法灵敏度更高，但存在潜在危害。这里描述的方案可适用于其他激酶，也有助于分析底物中的磷酸化位点。
Zotero 插件