Unbiased Functional Proteomics Strategy for Protein Kinase Inhibitor Validation and Identification ofbona fideProtein 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 32P-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.

Identification and Validation of Inhibitor-Responsive Kinase Substrates Using a New Paradigm To Measure Kinase-Specific Protein Phosphorylation Index

“…Gyenis et al 56 developed a strategy combining metabolic labeling with 32 P-orthophosphate with transfection of inhibitor-resistant CK2 mutants to identify and validate inhibitor-responsive proteins as true physiological substrates. Interestingly, the translation factor EF-1-delta was shown to become hypo-phosphorylated on S162 in response to the CK2 inhibitor TBBz.…”

“…Our studies independently identified the orthologous serine residue (S106) in EF1-β as responsive to CX-4945, in good agreement with these data. While the approach developed by Gyenis et al 56 can readily detect inhibitor-responsive kinase substrates in cultured cells, it would be difficult to apply to pre-clinical animal models and is not amenable to analysis of patient samples.…”

Loss of Pannexin 1 Attenuates Melanoma Progression by Reversion to a Melanocytic Phenotype

“…Proteomic Analysis and Validation-Four 10-cm plates of scrambled control or shRNA knockdown (sh21) cells were lysed in lysis buffer (8 M urea, 2% CHAPS, 1 mM benzamidine, 25 g/ml leupeptin, 20 g/ml pepstatin A, 20 g/ml aprotinin, 1 M okadaic acid, 1 M microcystin, 1 mM sodium orthovanadate, 0.5% ampholyte, and 40 mM DTT) on ice according to previously established methods (26). The cell lysates were cleared by centrifugation and the protein concentration was measured with a Bradford Protein Assay (Bio-Rad).…”

“…Samples were processed using the MASSPrep Automated Digestor (Waters/Micromass) in the Functional Proteomics Facility at the University of Western Ontario. Peptides were spotted on MALDI plates and MALDI-TOF analysis was performed using a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA) as described (26). Mass spectra were searched against the NCBInr data base of Mus musculus using the MASCOT search engine with carbamidomethyl (C) fixed and oxidation (M) plus phosphorylation (ST) variable modification.…”

EEF1D (eukaryotic translation elongation factor 1 delta)

“…Some posttranslational modifications are observed, such as phosphorylation, acetylation and succinylation (https://www.ncbi.nlm.nih.gov). eEF1D can be hyperphosphorylated and the phosphorylations are made by some protein kinases, including casein kinase 2 (Gyenis et al, 2011;Browne and Proud, 2002) and cyclin-dependent kinase 1 ( CDK1) (Kawaguchi et al, 2003). In particular, CDK1 phosphorylates EEF1D at Ser-133 (Kawaguchi et al, 2003).…”