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

Laszlo Gyenis , James S. Duncan , Jacob P. Turowec , Maria Bretner , David W. Litchfield

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 ??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.
Author Laszlo Gyenis
Laszlo Gyenis,,
-
, James S. Duncan
James S. Duncan,,
-
, Jacob P. Turowec
Jacob P. Turowec,,
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, Maria Bretner ZTiBSL
Maria Bretner,,
- Department Of Drug Technology And Biotechnology
, David W. Litchfield
David W. Litchfield,,
-
Journal seriesJournal of Proteome Research, ISSN 1535-3893
Issue year2011
Vol10
No11
Pages4887-4901
Keywords in English 2D electrophoresis; CK2 inhibitor; functional proteomics; chemical genetics; protein kinase; inhibitor-resistant kinase; unbiased validation strategy
DOIDOI:10.1021/pr2008994
URL http://dx.doi.org/10.1021/pr2008994
Languageen angielski
File
Journal of Proteome Research.pdf (file archived - login or check accessibility on faculty) Journal of Proteome Research.pdf 3.09 MB
Score (nominal)40
Publication indicators WoS Impact Factor: 2011 = 5.113 (2) - 2011=5.413 (5)
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