AUTHOR=Gyenis Laszlo , Menyhart Daniel , Cruise Edward S. , Jurcic Kristina , Roffey Scott E. , Chai Darren B. , Trifoi Flaviu , Fess Sam R. , Desormeaux Paul J. , Núñez de Villavicencio Díaz Teresa , Rabalski Adam J. , Zukowski Stephanie A. , Turowec Jacob P. , Pittock Paula , Lajoie Gilles , Litchfield David W. TITLE=Chemical Genetic Validation of CSNK2 Substrates Using an Inhibitor-Resistant Mutant in Combination with Triple SILAC Quantitative Phosphoproteomics JOURNAL=Frontiers in Molecular Biosciences VOLUME=9 YEAR=2022 URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2022.909711 DOI=10.3389/fmolb.2022.909711 ISSN=2296-889X 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.