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

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.Inositol phosphates are major intracellular signaling molecules with the best known of these, inositol 1,4,5-trisphosphate, releasing intracellular calcium (1, 2). Inositol pyrophosphates occur physiologically, the most prominent being diphosphoinositol pentakisphosphate (InsP7) 1 and bis-diphosphoinositol tetrakisphosphate (InsP8) (3, 4). Inositol pyrophosphates have been implicated in diverse functions including vesicular trafficking (5-7), DNA recombination and repair (8, 9), and chemotaxis (10). Molecular mechanisms whereby they influence these functions are not well established, although there is evidence that InsP7 can compete for PH domain binding with PtdIns(3,4,5)P3 (10) and serve as a phosphate donor to proteins. 2The formation of InsP7 and InsP8 is mediated by a family of three inositol hexakisphosphate kinases (InsP6Ks) (11)(12)(13)(14). Differential functions of the enzymes are implied by their varying intracellular localizations with InsP6K2 being exclusively nuclear, whereas the other two enzymes are both cytosolic and nuclear (12). A role for InsP6K2 in cell death is suggested by the findings of Lindner and co-workers (15, 16) that deletion of InsP6K2 prevents apoptotic actions of interferon ␤ (15, 16) and ␥-irradiation (15, 16) in ovarian carcinoma cells, whereas transfection of InsP6K2 augments cell death, and interferon ␤ treatment leads to enhanced InsP6K2 activity.In the present study we show that transfection of InsP6K2 in multiple cell lines augments apoptotic actions of several cell stressors, accompanied by major increases in InsP7 formation. Although transfection of all three subtypes of InsP6K increases cell death, deleting InsP6K2, but not InsP6K1 or InsPK3, prevents apoptosis. Additionally, apoptotic stimuli elicit translocation of InsP6K2 from nuclei to damaged mitochondria, whereas no alteration in the intracellular localizations of InsP6K1 or InsP6K3 is demonstrable. EXPERIMENTAL PROCEDURESCell L...

Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas

“…Interactions between TOR signaling and InsP in yeast and animals have been suggested by several studies (Chakraborty et al, 2010;Kim and Guan, 2011) including roles for InsP kinases upstream (Nagata et al, 2005) and downstream of TOR (Worley et al, 2013). These findings are not mutually exclusive, but they do highlight the potential complexity of intracellular signaling pathways where feedback loops can connect signaling systems at different levels.…”

HSP90 regulates cell survival via inositol hexakisphosphate kinase-2

“…Cellular IP7 was measured as described previously (8,21). Details are found in SI Materials and Methods.…”

“…Lindner and coworkers (20) screened an antisense library and uncovered IP6K2 as a proapoptotic gene. Overexpression of IP6K2 sensitizes numerous cancer cell lines to apoptotic actions of various cell stressors (20)(21)(22)(23), whereas depletion of IP6K2 by RNAi prevents the apoptotic actions of several agents, indicating an association of IP6K2 with cell death (20,21). IP6K2-augmented cell death is associated with an increase in DR4 [TNF-related apoptosis-inducing ligand (TRAIL) receptor] and caspase-8 expression and subsequent activation (23).…”

“…Cell death in response to anticancer drugs has been shown to involve IP6K2 (21,23). Thus, cisplatin (CP) augments IP7(5) generation in OVCAR-3 cells, and the apoptotic influences of the drug are substantially augmented by IP6K2 overexpression (21).…”

“…Thus, cisplatin (CP) augments IP7(5) generation in OVCAR-3 cells, and the apoptotic influences of the drug are substantially augmented by IP6K2 overexpression (21). IP6K2-mediated cell death depends on its catalytic activity because its kinase-dead mutant prevents apoptosis.…”