Cyclin-dependent kinase 1 (CDK1) plays a crucial role in various mitotic processes such as cytoskeletal reorganization, chromosome segregation, and cytokinesis. It is an essential regulatory factor. CDK1 is expressed stably throughout the cell cycle, and its activity is closely related to cyclin A and cyclin B, while being regulated by many post-translational modifications (PTMs), with phosphorylation being the most significant. Throughout the cell cycle, CDK1 can phosphorylate numerous mitotic substrates, thereby precisely controlling the mitotic process in both time and space. Nevertheless, the currently identified phosphorylated substrates are quite limited.
To better reveal the network structure of CDK1 phosphorylation, the authors utilized quantitative phosphoproteomics technology SILAC to quantitatively compare phosphorylation levels in HeLa cells. Due to the relatively high rate of the cell cycle in cancer cells, small molecule inhibitors targeting CDK1 are a very effective class of chemotherapy drugs. The authors selected two small molecule inhibitors of CDK1: Flavopiridol and RO-3306. The former is an ATP-competitive inhibitor and the first CDK inhibitor to enter clinical trials; the latter is a recently reported inhibitor with higher selectivity for CDK1. Under the presence of the inhibitors, the authors identified a total of 24,840 phosphorylation sites, of which the phosphorylation levels of 1,215 sites from 551 proteins decreased by at least 2.5 times. Through sequence analysis, these phosphorylation sites were found to be enriched before proline, consistent with the classic substrate sequence of CDK1, and these substrates are concentrated in proteins specifically expressed during the G2 and M phases of the cell cycle.
This article successfully applied quantitative proteomics technology to solve a critical issue in cell division, providing a vast database of CDK1 substrate distribution, which is a significant advantage of omics technology.