Chemical Genetic Validation of CSNK2 Substrates Using an Inhibitor-Resistant Mutant in Combination with Triple SILAC Quantitative Phosphoproteomics

Abstract

Casein Kinase 2 (CSNK2) is an extremely pleiotropic, ubiquitously expressed protein kinase involved in the regulation of numerous key biological processes. Mapping the CSNK2-dependent phosphoproteome is necessary for better characterization of its fundamental role in cellular signalling. While ATP-competitive inhibitors have enabled the identification of many putative kinase substrates, compounds targeting the highly conserved ATP-binding pocket often exhibit off-target effects limiting their utility for definitive kinase-substrate assignment. To overcome this limitation, we devised a strategy combining chemical genetics and quantitative phosphoproteomics to identify and validate CSNK2 substrates. We engineered U2OS cells expressing exogenous wild type CSNK2A1 (WT) or a triple mutant (TM, V66A/H160D/I174A) with substitutions at residues important for inhibitor binding. These cells were treated with CX-4945, a clinical-stage inhibitor of CSNK2, and analyzed using large-scale triple SILAC (Stable Isotope Labelling of Amino Acids in Cell Culture) quantitative phosphoproteomics. In contrast to wild-type CSNK2A1, CSNK2A1-TM retained activity in the presence of CX-4945 enabling identification and validation of several CSNK2 substrates on the basis of their increased phosphorylation in cells expressing CSNK2A1-TM. Based on high conservation within the kinase family, we expect that this strategy can be broadly adapted for identification of other kinase-substrate relationships.

Scoping Pleiotropy of CK2 in Musculoskeletal Disorders for a Novel Targeting Approach

“…This allowed the study of interaction when the cells performed their normal function. The disparity was observed within popular CK2 inhibitors, with only a proportion of inhibitors showing dose-dependent inhibition of CK2 activity [95].…”

CK2 Chemical Probes: Past, Present, and Future

“…In a study aimed at refining the list of bona fide CK2 substrates, SGC-CK2-1 and CX-4945 were once again used side-by-side and compared. Using triple stable isotope labeling by amino acids in cell culture (SILAC) in combination with inhibitor-resistant CK2, a reliable method was established for the identification and validation of CK2 substrates [57]. This methodology was employed to evaluate the selectivity of CX-4945 versus SGC-CK2-1.…”