PMID- 35755813 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20230328 IS - 2296-889X (Print) IS - 2296-889X (Electronic) IS - 2296-889X (Linking) VI - 9 DP - 2022 TI - Chemical Genetic Validation of CSNK2 Substrates Using an Inhibitor-Resistant Mutant in Combination with Triple SILAC Quantitative Phosphoproteomics. PG - 909711 LID - 10.3389/fmolb.2022.909711 [doi] LID - 909711 AB - 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. CI - Copyright (c) 2022 Gyenis, Menyhart, Cruise, Jurcic, Roffey, Chai, Trifoi, Fess, Desormeaux, Nunez de Villavicencio Diaz, Rabalski, Zukowski, Turowec, Pittock, Lajoie and Litchfield. FAU - Gyenis, Laszlo AU - Gyenis L AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Menyhart, Daniel AU - Menyhart D AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Cruise, Edward S AU - Cruise ES AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Jurcic, Kristina AU - Jurcic K AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Roffey, Scott E AU - Roffey SE AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Chai, Darren B AU - Chai DB AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Trifoi, Flaviu AU - Trifoi F AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Fess, Sam R AU - Fess SR AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Desormeaux, Paul J AU - Desormeaux PJ AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Nunez de Villavicencio Diaz, Teresa AU - Nunez de Villavicencio Diaz T AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Rabalski, Adam J AU - Rabalski AJ AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Zukowski, Stephanie A AU - Zukowski SA AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Turowec, Jacob P AU - Turowec JP AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Pittock, Paula AU - Pittock P AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Lajoie, Gilles AU - Lajoie G AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. FAU - Litchfield, David W AU - Litchfield DW AD - Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. AD - Department of Oncology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada. LA - eng PT - Journal Article DEP - 20220609 PL - Switzerland TA - Front Mol Biosci JT - Frontiers in molecular biosciences JID - 101653173 PMC - PMC9225150 OTO - NOTNLM OT - CSNK2 OT - CX-4945 OT - SILAC OT - chemical genetics OT - kinase-substrate relationship validation OT - mass spectrometry OT - phosphoproteomics OT - protein kinase CK2 COIS- The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. EDAT- 2022/06/28 06:00 MHDA- 2022/06/28 06:01 PMCR- 2022/01/01 CRDT- 2022/06/27 03:55 PHST- 2022/03/31 00:00 [received] PHST- 2022/05/02 00:00 [accepted] PHST- 2022/06/27 03:55 [entrez] PHST- 2022/06/28 06:00 [pubmed] PHST- 2022/06/28 06:01 [medline] PHST- 2022/01/01 00:00 [pmc-release] AID - 909711 [pii] AID - 10.3389/fmolb.2022.909711 [doi] PST - epublish SO - Front Mol Biosci. 2022 Jun 9;9:909711. doi: 10.3389/fmolb.2022.909711. eCollection 2022.