Inositol Hexakisphosphate Kinase-2, a Physiologic Mediator of Cell Death

Eiichiro Nagata,Hongbo R Luo,Adolfo Saiardi,Byoung-Il Bae,Norihiro Suzuki,Solomon H Snyder

doi:10.1074/jbc.m409416200

Eiichiro Nagata, Hongbo R Luo + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m409416200

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Abstract

Diphosphoinositol pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate contain pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by a family of three inositol hexakisphosphate kinases (InsP6K). In this study we establish one of the InsP6Ks, InsP6K2, as a physiologic mediator of cell death. Overexpression of wild-type InsP6K2 augments the cytotoxic actions of multiple cell stressors in diverse cell lines, whereas transfection with a dominant negative InsP6K2 decreases cell death. During cell death, InsP6 kinase activity is enhanced, and intracellular InsP7 level is augmented. Deletion of InsP6K2 but not the other forms of InsP6K diminishes cell death, suggesting that InsP6K2 is the major InsP6 kinase involved in cell death. Cytotoxicity is associated with a translocation of InsP6K2 from nuclei to mitochondria, whereas the intracellular localization of the other isoforms of the enzyme does not change. The present study provides compelling evidence that endogenous InsP6K2, by generating InsP7, provides physiologic regulation of the apoptotic process.

Highlights

Diphosphoinositol pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate contain pyrophosphate bonds
Cytotoxicity is associated with a translocation of InsP6K2 from nuclei to mitochondria, whereas the intracellular localization of the other isoforms of the enzyme does not change
The present study provides compelling evidence that endogenous InsP6K2, by generating InsP7, provides physiologic regulation of the apoptotic process

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Cell Death Assays—HeLa cells, PC12 cells, Jurkat T cells, and OVCAR-3 cells were transfected with indicated constructs using a Lipofectamine 2000 transfection kit (Invitrogen) and a protocol provided by the manufacturer [17]. For staining of dead cells by terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay, the cells were fixed in 4% paraformaldehyde/phosphate-buffered saline and stained using a TUNEL assay kit following protocols provided by the. InsP6 kinase enzymatic activity was assayed as preciously described [11]. Radiolabeling and Detection of Inositol Phosphates in Intact Cells— The OVCAR-3 cells were plated at a density of 0.5 ϫ 106 cells/35-mm plate and incubated with [3H]inositol (final concentration, 100 ␮Ci/ml) for 4 days. The cell pellets were lysed in 0.2 ml of ice-cold lysis buffer (2 M perchloric acid, 0.1 mg/ml InsP6, 2 mM EDTA).

Inositol Pyrophosphate and Cell Death

RESULTS

DISCUSSION