Functional inactivation of triosephosphate isomerase through phosphorylation during etoposide-induced apoptosis in HeLa cells: potential role of Cdk2.
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 phospho-proteomic technique to screen target molecules of Cdk2 during etoposide-induced apoptosis. For this purpose, phosphoproteins from the cell lysates were enriched by using Fe³+-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.
Lee
Won-Hee
WH
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.
Choi
Joon-Seok
JS
Byun
Mi-Ran
MR
Koo
Kyo-Tan
KT
Shin
Soona
S
Lee
Seung-Ki
SK
Surh
Young-Joon
YJ
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism*; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D007701:Kinetin / Q000494:pharmacology; D010750:Phosphoproteins / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014305:Triose-Phosphate Isomerase / Q000037:antagonists & inhibitors*; D015854:Up-Regulation
D000972:Antineoplastic Agents, Phytogenic; D019925:Cyclin A; D010750:Phosphoproteins; D004099:Dihydroxyacetone Phosphate; C090046:olomoucine; D005047:Etoposide; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D014305:Triose-Phosphate Isomerase; D007701:Kinetin