Chemical genetic approach to identify substrates of CK2 kinase using Mass spectrometry

Protein kinase CK2 plays major roles in multiple biological processes, as has been reported in literature. Phosphoproteomic and bioinformatics data suggests that CK2 is involved in many cellular pathways however there is no report published on the unbiased global validation of its bona fine cellular substrates and how the CK2 dependent phosphorylation is modulating these pathways. Therefore, there is a need for methods that can do this validation process. Since the kinases have similar nature to phosphorylate their substrates, it is a great challenge to find or validate new substrates of a targeted kinase. In this study, a chemical genetics approach was used when the phenylalanine bulky residues were identified in CK2α as F113 and CK2α’ as F114. These residues were mutated to glycine to expand the ATP binding pocket, and create an analogues sensitive kinase which can utilize an ATP analogue nucleotide. The substitution did not alter the activity or specificity of the kinase. Furthermore, the analogue sensitive-CK2 (as-CK2) and wild type-CK2 (wt-CK2) α and α’ SILAC labeled stable cell lines were generated, then thiophosphrylation reactions were done using N6-(2-phenylethyl) ATPγS (2peATPγS) to label substrates in the cell lines. The thiopeptides were identified using mass spectrometry based proteomics approach. The thiopeptides found only in as-CK2 compared to control were considered as CK2 substrates. Our results highlight the potential of using analog-sensitive CK2 to further expand our knowledge of the cellular roles of CK2 and elucidate kinase-substrate relationships.