title: Unbiased functional proteomics strategy for protein kinase inhibitor validation and identification of bona fide protein kinase substrates: application to identification of EEF1D as a substrate for CK2.
abstract: Protein kinases have emerged as attractive targets for treatment of several diseases prompting large-scale phosphoproteomics studies to elucidate their cellular actions and the design of novel inhibitory compounds. Current limitations include extensive reliance on consensus predictions to derive kinase-substrate relationships from phosphoproteomics data and incomplete experimental validation of inhibitors. To overcome these limitations in the case of protein kinase CK2, we employed functional proteomics and chemical genetics to enable identification of physiological CK2 substrates and validation of CK2 inhibitors including TBB and derivatives. By 2D electrophoresis and mass spectrometry, we identified the translational elongation factor  EEF1D  as a protein exhibiting CK2 inhibitor-dependent decreases in phosphorylation in (32)P-labeled HeLa cells. Direct phosphorylation of  EEF1D  by CK2 was shown by performing CK2 assays with  EEF1D -FLAG from HeLa cells. Dramatic increases in  EEF1D  phosphorylation following λ-phosphatase treatment and phospho- EEF1D  antibody recognizing  EEF1D  pS162 indicated phosphorylation at the CK2 site in cells. Furthermore, phosphorylation of  EEF1D  in the presence of TBB or TBBz is restored using CK2 inhibitor-resistant mutants. Collectively, our results demonstrate that  EEF1D  is a bona fide physiological CK2 substrate for CK2 phosphorylation. Furthermore, this validation strategy could be adaptable to other protein kinases and readily combined with other phosphoproteomic methods.
authors.affiliation: Department of Biochemistry, The University of Western Ontario , London, Ontario, N6A 5C1, Canada.
authors.forename: Laszlo
authors.initials: L
authors.lastname: Gyenis
authors.forename: James S
authors.initials: JS
authors.lastname: Duncan
authors.forename: Jacob P
authors.initials: JP
authors.lastname: Turowec
authors.forename: Maria
authors.initials: M
authors.lastname: Bretner
authors.forename: David W
authors.initials: DW
authors.lastname: Litchfield
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D047390:Casein Kinase II / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D020134:Catalytic Domain; D015180:Electrophoresis, Gel, Two-Dimensional; D057075:Enzyme Assays; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009842:Oligopeptides / Q000737:chemistry*; D020648:Peptide Elongation Factor 1 / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D009842:Oligopeptides; D020648:Peptide Elongation Factor 1; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D047390:Casein Kinase II