Global screening of CK2 kinase substrates by an integrated phosphoproteomics workflow

Due to its constitutive activity and ubiquitous distribution, CK2 is the most pleiotropic kinase among the individual members of the protein kinase superfamily. Identification of CK2 substrates is vital to decipher its role in biological processes. However, only a limited number of CK2 substrates were identified so far. In this study, we developed an integrated phosphoproteomics workflow to identify the CK2 substrates in large scale. First, in vitro kinase reactions with immobilized proteomes were combined with quantitative phosphoproteomics to identify in vitro CK2 phosphorylation sites, which leaded to identification of 988 sites from 581 protein substrates. To reduce false positives, we proposed an approach by comparing these in vitro sites with the public databases that collect in vivo phosphorylation sites. After the removal of the sites that were excluded in the databases, 605 high confident CK2 sites corresponding to 356 proteins were retained. The CK2 substrates identified in this study were based on the discovery mode, in which an unbiased overview of CK2 substrates was provided. Our result revealed that CK2 substrates were significantly enriched in the spliceosomal proteins, indicating CK2 might regulate the functions of spliceosome.