Background

Cyclin-B-Cyclin dependent kinase 1 (CDK1), which is a master regulator of mitosis, phosphorylates numerous targets to ensure mitotic progression (Nurse, 1990; Malumbres and Barbacid, 2005). During mitosis, chromosomes carrying the genetic information are equally divided into two daughter cells. The kinetochore is a key large protein complex ensuring the faithful chromosome segregation by bridging between chromosomes and spindle microtubules (Fukagawa and Earnshaw, 2014). Various proteins composing the kinetochore are phosphorylated by CDK1 (Gascoigne et al., 2013; Nishino et al., 2013; Hara et al., 2018b; Watanabe et al., 2019). The CDK1 phosphorylation plays critical roles in kinetochore assembly (Gascoigne et al., 2013) and also in the correct microtubule attachment (Nishino et al., 2013; Hara et al., 2018a).

In vitro kinase assays and phospho-protein analyses are important ways, which provide us understanding of how phospho-regulations are achieved in kinetochore assembly and function. A classical way to detect phospho-proteins is a radiolabeled assay using [γ-³²P]-ATP. However, although the radiolabeled assay gives high sensitivity, it has potential hazards. To use radioactivity, we would need to take a radiation safety training course, and prepare controlled area and safety protection equipment. Phospho-specific antibodies can be alternative ways. However, the antibodies are not always available for targets of interest.

Here, we describe a method to phosphorylate a kinetochore protein, CENP-C, by CDK1 in vitro and to detect the phosphorylated CENP-C using Phos-tag SDS-PAGE (Kinoshita et al., 2006). Phos-tag is 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex that has a vacancy on two metal ions and binds to phenyl phosphate dianion on the target protein via two metal ions. Phospho-proteins migrate slower in the SDS-PAGE containing Phos-tag. This method magnifies mobility shifts of the phospho-proteins, which even show no migration changes in conventional SDS-PAGE. The protocol can be applicable to detect phospho-proteins by other kinases. Given that mobility of the phospho-protein on Phos-tag SDS-PAGE changes with number of phosphorylation-sites as well as location of the phosphorylation-sites, the procedure could be also utilized for analysis of phosphorylation-sites. The protocol is easy and safe, and can be finished within a few hours including kinase reaction and detection, saving time compared with the conventional radiolabeled assays.