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首页> 外文期刊>Toxicology: An International Journal Concerned with the Effects of Chemicals on Living Systems >Functional inactivation of triosephosphate isomerase through phosphorylation during etoposide-induced apoptosis in HeLa cells: potential role of Cdk2.
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Functional inactivation of triosephosphate isomerase through phosphorylation during etoposide-induced apoptosis in HeLa cells: potential role of Cdk2.

机译：依托泊苷诱导HeLa细胞凋亡过程中磷酸化磷酸三磷酸磷酸酯酶的功能失活：Cdk2的潜在作用。

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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 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.
机译：已经表明细胞周期蛋白依赖性蛋白激酶2（Cdk2）活性的上调是各种凋亡刺激诱导的细胞凋亡进展的前提。在这项研究中，我们应用了磷酸化蛋白质组学技术来筛选依托泊苷诱导的细胞凋亡过程中Cdk2的目标分子。为此，通过使用Fe（3）+-IMAC柱色谱富集来自细胞裂解液的磷蛋白，并在高分辨率2D PAGE凝胶上进行解析。我们通过使用MALDI-TOF技术鉴定了六种磷蛋白。在依托泊苷存在的情况下，在用Cdk2显性负突变构建体转染的HeLa细胞中，这些磷蛋白的细胞水平显着降低。在这六个候选磷蛋白中，人三糖磷酸异构酶（TPI）是一种糖酵解酶，在依托泊苷诱导的细胞凋亡期间，它是Cdk2的直接底物。在体外磷酸化测定中，通过重组杆状蛋白A / Cdk2激酶将通过杆状病毒表达系统纯化的TPI磷酸化。这导致3-磷酸葡萄糖醛转化为磷酸二羟基丙酮的酶活性降低。通过用Cdk2的特异性抑制剂olomoucine处理，可以防止TPI的这种磷酸化及其随后的酶活性降低。综合以上发现，TPI可作为Cyclin A / Cdk2的潜在靶蛋白。由于该糖酵解酶的磷酸化，TPI催化活性的损失可能会破坏依托泊苷处理的HeLa细胞中的能量产生，使这些细胞易于发生凋亡。

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