S-Space Graduate School of Convergence Science and Technology (융합과학기술대학원) Dept. of Molecular and Biopharmaceutical Sciences (분자의학 및 바이오제약학과) Journal Papers (저널논문_분자의학 및 바이오제약학과)
Functional inactivation of triosephosphate isomerase through phosphorylation during etoposide-induced apoptosis in HeLa cells: Potential role of Cdk2
- Lee, Won-Hee; Choi, Joon-Seok; Byun, Mi-Ran; Koo, Kyo-tan; Shin, Soona; Lee, Seung-Ki; Surh, Young-Joon
- Issue Date
- Toxicology, Vol.278 No.2, pp.224-228
- Up-regulation of cyclin-dependent protein kinase 2 (Cdk2) activity has been suggested to be prerequisite for progression of apoptosis induced by various apoptotic stimuli. In this study, we applied a phosphoproteomic technique to screen target molecules of Cdk2 during etoposide-induced apoptosis. For this purpose, phosphoproteins from the cell lysates were enriched by using Fe(3+)-IMAC column chromatography and resolved on a high resolution 2D PAGE gel. We identified six phosphoproteins by the use of MALDI-TOF technique. The cellular levels of these phosphoproteins were markedly reduced in the presence of etoposide in HeLa cells transfected with dominant negative mutant construct of Cdk2. Among the six candidate phosphoproteins, human triosephosphate isomerase (TPI), a glycolytic enzyme, was found to be a direct substrate of Cdk2 during etoposide-induced apoptosis. In an in vitro phosphorylation assay, TPI purified by use of a baculoviral expression system was phosphorylated by recombinant Cyclin A/Cdk2 kinase. This led to reduced enzyme activity for the conversion of glucose aldehyde-3-phosphate to dihydroxyacetone phosphate. Such phosphorylation of TPI and a subsequent decrease in its enzyme activity were prevented by treatment with olomoucine, a specific inhibitor of Cdk2. The above findings, taken together, suggest TPI as a potential target protein of Cyclin A/Cdk2. Loss of catalytic activity of TPI as a consequence of phosphorylation of this glycolytic enzyme may disrupt energy production in etoposide-treated HeLa cells, rendering these cells prone to undergo apoptosis. (C) 2010 Published by Elsevier Ireland Ltd.
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