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Chemical Genetic Validation of CSNK2 Substrates Using an Inhibitor-Resistant Mutant in Combination with Triple SILAC Quantitative Phosphoproteomics.
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.
Afiliación
  • Gyenis L; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Menyhart D; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Cruise ES; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Jurcic K; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Roffey SE; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Chai DB; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Trifoi F; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Fess SR; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Desormeaux PJ; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Núñez de Villavicencio Díaz T; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Rabalski AJ; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Zukowski SA; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Turowec JP; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Pittock P; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Lajoie G; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
  • Litchfield DW; Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
Front Mol Biosci ; 9: 909711, 2022.
Article en En | MEDLINE | ID: mdl-35755813
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Front Mol Biosci Año: 2022 Tipo del documento: Article País de afiliación: Canadá Pais de publicación: Suiza
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Front Mol Biosci Año: 2022 Tipo del documento: Article País de afiliación: Canadá Pais de publicación: Suiza