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

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. 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 (1Berridge M.J. Lipp P. Bootman M.D. Science. 2000; 287: 1604-1605Crossref PubMed Scopus (164) Google Scholar, 2Irvine R.F. Schell M.J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 327-338Crossref PubMed Scopus (538) Google Scholar). Inositol pyrophosphates occur physiologically, the most prominent being diphosphoinositol pentakisphosphate (InsP7) 1The abbreviations used are: InsP7, diphosphoinositol pentakisphosphate; InsP8, bis-diphosphoinositol tetrakisphosphate; InsP6, inositol hexakisphosphate; InsP6K, inositol hexakisphosphate kinase; TUNEL, terminal deoxynucleotide transferase dUTP nick end labeling; HPLC, high pressure liquid chromatography; siRNA, small interfering RNA; MOPS, 4-morpholinepropanesulfonic acid; GFP, green fluorescent protein; PI, propidium iodide; HA, hemagglutinin. and bis-diphosphoinositol tetrakisphosphate (InsP8) (3Glennon M.C. Shears S.B. Biochem. J. 1993; 293: 583-590Crossref PubMed Scopus (97) Google Scholar, 4Stephens L. Radenberg T. Thiel U. Vogel G. Khoo K.H. Dell A. Jackson T.R. Hawkins P.T. Mayr G.W. J. Biol. Chem. 1993; 268: 4009-4015Abstract Full Text PDF PubMed Google Scholar). Inositol pyrophosphates have been implicated in diverse functions including vesicular trafficking (5Saiardi A. Sciambi C. McCaffery J.M. Wendland B. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14206-14211Crossref PubMed Scopus (149) Google Scholar, 6Luo H.R. Saiardi A. Nagata E. Ye K. Yu H. Jung T.S. Luo X. Jain S. Sawa A. Snyder S.H. Neuron. 2001; 31: 439-451Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 7Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), DNA recombination and repair (8Huang K.N. Symington L.S. Genetics. 1995; 141: 1275-1285Crossref PubMed Google Scholar, 9Luo H.R. Saiardi A. Yu H. Nagata E. Ye K. Snyder S.H. Biochemistry. 2002; 41: 2509-2515Crossref PubMed Scopus (67) Google Scholar), and chemotaxis (10Luo H.R. Huang Y.E. Chen J.C. Saiardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). 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 (10Luo H.R. Huang Y.E. Chen J.C. Saiardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) and serve as a phosphate donor to proteins. 2A. Saiardi and S. H. Snyder, unpublished data. The formation of InsP7 and InsP8 is mediated by a family of three inositol hexakisphosphate kinases (InsP6Ks) (11Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 12Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Schell M.J. Letcher A.J. Brearley C.A. Biber J. Murer H. Irvine R.F. FEBS Lett. 1999; 461: 169-172Crossref PubMed Scopus (74) Google Scholar, 14Saiardi A. Nagata E. Luo H.R. Snowman A.M. Snyder S.H. J. Biol. Chem. 2001; 276: 39179-39185Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). 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 (12Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). A role for InsP6K2 in cell death is suggested by the findings of Lindner and co-workers (15Morrison B.H. Bauer J.A. Hu J. Grane R.W. Ozdemir A.M. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu D.V. Lindner D.J. Oncogene. 2002; 21: 1882-1889Crossref PubMed Scopus (61) Google Scholar, 16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) that deletion of InsP6K2 prevents apoptotic actions of interferon β (15Morrison B.H. Bauer J.A. Hu J. Grane R.W. Ozdemir A.M. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu D.V. Lindner D.J. Oncogene. 2002; 21: 1882-1889Crossref PubMed Scopus (61) Google Scholar, 16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) and γ-irradiation (15Morrison B.H. Bauer J.A. Hu J. Grane R.W. Ozdemir A.M. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu D.V. Lindner D.J. Oncogene. 2002; 21: 1882-1889Crossref PubMed Scopus (61) Google Scholar, 16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) in ovarian carcinoma cells, whereas transfection of InsP6K2 augments cell death, and interferon β 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. 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 (17Luo H.R. Hattori H. Hossain M.A. Hester L. Huang Y. Lee-Kwon W. Donowitz M. Nagata E. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11712-11717Crossref PubMed Scopus (217) Google Scholar). We routinely obtained a transfection efficiency of over 70% in these cells. HL-60 cells, a promyelocytic cell line, were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 4 mm glutamine. HL-60 cells could not be transfected by Lipofectamine reagent, and we utilized a newly invented Nucleofection™ system (Amaxa, Inc.) to introduce DNA construct into these cells. We regularly obtained a transfection efficiency of over 50% in HL60 cells. The cells were plated at 5 × 106 cells/well in 6-well plastic plates 1 day before the experiment, and cell death was induced by the addition of cisplatin (30 μm), staurosporine (1 μm), etoposide (25 μm), or hydrogen peroxide (750 μm) (18Okuno S. Shimizu S. Ito T. Nomura M. Hamada E. Tsujimoto Y. Matsuda H. J. Biol. Chem. 1998; 273: 34272-34277Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). For generating hypoxia condition, the transfected cells were cultured at 37 °C for 20 h in a Gas-Pak anaerobic chamber (BD Biosciences, Cockeysville, MD). In parallel, cells grown for the same duration under normoxic conditions served as controls. Toxicity was assayed 12-14 h after drug exposure by microscopic examination with computer-assisted cell counting. The numbers of total and dead cells were determined by nuclear staining with 100 ng/ml 4′,6-diamidino-2-phenylindole and propidium iodide (10 μm), respectively. After 10 min of incubation, the cells were examined under a fluorescence microscope (Zeiss) with excitation at 360 nm. Cell death was determined as the ratio of dead-to-total cell number and quantified by counting 1,000 cells. 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 then stained using a TUNEL assay kit following protocols provided by the manufacturer (Molecular Probes, Eugene, OR). InsP6 Kinase Assay—Apoptosis of OVCAR-3 cells was initiated by treating cells with 30 μm cisplatin. The cells were lysed at indicated time points, and the resulting lysate was used for activity assay. InsP6 kinase enzymatic activity was assayed as preciously described (11Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Radiolabeling and Detection of Inositol Phosphates in Intact Cells— The OVCAR-3 cells were plated at a density of 0.5 × 106 cells/35-mm plate and incubated with 3Hinositol (final concentration, 100 μCi/ml) for 4 days. The cells were harvested and washed twice with ice-cold DB buffer (10Luo H.R. Huang Y.E. Chen J.C. Saiardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). 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). The Lysates were centrifuged for 5 min at 4 °C, and the supernatant was neutralized with K2CO3 as described previously (5Saiardi A. Sciambi C. McCaffery J.M. Wendland B. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14206-14211Crossref PubMed Scopus (149) Google Scholar). Inositol phosphates were resolved by HPLC as described above. Disruption of InsP6K Expression by RNA Interference—The double-stranded RNAs used in RNA interference experiment were synthesized using a Silencer™ siRNA mixture kit (Ambion, Inc.). The PCR amplification of each target site (InsP6K1, InsP6K2, and InsP6K3) was performed using primers that contained T7 promoter sequences. We used the following sequences: InsP6K1 (5′ primer, 5′-TAA TAC GAC TCA CTA TAG GGA TGT GTG TTT GTC AAA CC, and 3′ primer, 5′-TAA TAC GAC TCA CTA TAG GGA CAG GCT GGC TTT CTC), InsP6K2 (5′ primer, 5′-TAA TAC GAC TCA CTA TAG GGA TGA GCC CAG CCT TCA GGG, and 3′ primer, 5′-CTAA TAC GAC TCA CTA TAG GGC GGG AAG TCA GGT TTT CCA), and InsP6K3 (5′ primer, 5′-TAA TAC GAC TCA CTA TAG GGC AGA TGC CGG GGA CAT GAG, and 3′ primer, 5′-TAA TAC GAC TCA CTA TAG GGC TCC TAA TGA CAG AGA GG). After synthesis of each double-stranded RNA, siRNA were produced using RNase III, and each siRNA was transfected into HEK293 cells using siPORT™ XP-1 transfection agent (Ambion, Inc.). For Northern blotting analysis, total RNAs from cells transfected with InsP6K1, InsP6K2, and InsP6K3 siRNAs were extracted by TRIzol (Invitrogen) and prepared using LiCl precipitation methods. RNA (30 μg) was loaded and to for InsP6K1, InsP6K2, and InsP6K3 and were with using InsP6K2 cells were transfected with GFP, and After transfected cells were incubated with staurosporine (1 μm) a from for 4 The cells were then fixed with 4% for 2 h and stained with of the fluorescent cells were obtained Overexpression of Cell ovarian carcinoma cells Lindner and co-workers (15Morrison B.H. Bauer J.A. Hu J. Grane R.W. Ozdemir A.M. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu D.V. Lindner D.J. Oncogene. 2002; 21: 1882-1889Crossref PubMed Scopus (61) Google Scholar, 16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) that transfection of InsP6K2 cell death by interferon β (15Morrison B.H. Bauer J.A. Hu J. Grane R.W. Ozdemir A.M. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu D.V. Lindner D.J. Oncogene. 2002; 21: 1882-1889Crossref PubMed Scopus (61) Google Scholar, 16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) and γ-irradiation (15Morrison B.H. Bauer J.A. Hu J. Grane R.W. Ozdemir A.M. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu D.V. Lindner D.J. Oncogene. 2002; 21: 1882-1889Crossref PubMed Scopus (61) Google Scholar, 16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). We have these findings to multiple cell lines and apoptotic stimuli 1 and In ovarian carcinoma cells with InsP6K2 transfection augments cell death by staining for mitochondria, 4′,6-diamidino-2-phenylindole staining for nuclear propidium iodide staining for and TUNEL staining for DNA all InsP6K2 transfection increases cell death by endogenous InsP6K2 apoptotic actions of we transfected cells with InsP6K2, cell death, with a physiologic role for endogenous InsP6K2 in apoptosis. InsP6K2 transfection to influence the level of cell death in ovarian carcinoma cells. the of the role of InsP6K2 in we examined cell HEK293 cells, cells, PC12 cells, Jurkat T cells, and HL60 cells We multiple cell stressors including hydrogen and In all cell lines with all stressors, transfection of InsP6K2 increases apoptosis. the InsP6K2 prevents cell death in all is most for Jurkat T and HL60 cell lines with whereas the are in PC12 cells. Cell InsP7 and Deletion of InsP6K2 Cell and co-workers (15Morrison B.H. Bauer J.A. Hu J. Grane R.W. Ozdemir A.M. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu D.V. Lindner D.J. Oncogene. 2002; 21: 1882-1889Crossref PubMed Scopus (61) Google Scholar, 16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) enhanced InsP6K activity in of ovarian carcinoma cells with interferon We have the influence of cell stressors inositol pyrophosphate and the of the three InsP6K enzymes to cell death. We inositol pyrophosphate formation in ovarian carcinoma cells with cisplatin a major in InsP7 formation with a of InsP8 increases in InsP7 occur at and 30 the of both InsP7 and InsP6 to suggesting that the cell death regulation of enzyme activity. We in InsP7 from in cell h following cisplatin The InsP6K activity is the same with cell not that the of cisplatin is not from to of InsP6K1, InsP6K2, or InsP6K3 following cisplatin treatment not that the of enzyme activity a of the as Lindner and co-workers (15Morrison B.H. Bauer J.A. Hu J. Grane R.W. Ozdemir A.M. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu D.V. Lindner D.J. Oncogene. 2002; 21: 1882-1889Crossref PubMed Scopus (61) Google Scholar, 16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) no in InsP6K2 level following interferon β treatment of ovarian carcinoma cells. of the three InsP6K we transfected the isoforms and InsP7 formation in cells following staurosporine treatment A and In cells staurosporine a in InsP7 with each of the three InsP6K isoforms a in InsP7 formation with no following staurosporine The three isoforms are of cell death. of the three in cells leads to a in cell death. In cells, transfection of the three enzymes the cytotoxic actions of the same are with hypoxia as a mediator of in multiple cell and Jurkat cells were cultured and as previously described (17Luo H.R. Hattori H. Hossain M.A. Hester L. Huang Y. Lee-Kwon W. Donowitz M. Nagata E. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11712-11717Crossref PubMed Scopus (217) Google Scholar). cells were transfected with indicated constructs using the Lipofectamine 2000 in a construct was of transfection of InsP6K2 HL-60 cells, a promyelocytic cell 270: PubMed Scopus Google Scholar), were cultured in RPMI 1640 medium supplemented with 10% fetal serum and 4 mm glutamine. HL-60 cells could not be transfected by Lipofectamine reagent, and we utilized a newly invented Nucleofection™ system (Amaxa, Inc.) to introduce DNA construct into these cells. is and a of and to the DNA to the cell We regularly obtained a transfection efficiency of over 50% in HL60 cells. Cell death was as described in the to of these provided the same The of staining are in this cells were with μm and was assayed h after the HEK293 cells were with 1 μm and was assayed h after the HEK293 cells were cultured at 37 °C in a Gas-Pak anaerobic chamber (BD Toxicity was assayed h after the PC12 cells were with 1 μm and was assayed h after the Jurkat T cells were with μm and was assayed h after the HL60 cells were with μm and was assayed h after the of the were for at three and the are the of three The the in cells. OVCAR-3 cells were cultured in RPMI 1640 medium supplemented with 10% fetal The cells were transfected with indicated constructs using a Lipofectamine 2000 transfection kit (Invitrogen) and a protocol provided by the is a dominant negative A construct was ratio and constructs is of transfection of with that of cells not The cells were with 30 μm drug and was assayed h after the In each assay, at three were performed with a of cells Cell death was determined as the ratio of dead-to-total cell The are the of three The the cell by fluorescence following The cells were incubated with (Molecular for 20 is a The of this is the in apoptotic cells not be stained by For analysis, a cell was as 100 staining were in this cell by fluorescence following 4′,6-diamidino-2-phenylindole nuclear is and both and dead cells. were as cells. cell by is and can the cells. cell determined by the TUNEL assay. into the DNA was using assay DNA in apoptotic of InsP6K1 and InsP6K3 apoptosis. HEK293 cells were transfected with indicated constructs using the Lipofectamine 2000 We routinely obtained a transfection efficiency of over using this of was by blotting using were prepared h after the level of InsP7 in transfected cells. The labeling and of intracellular InsP7 was as described in the to The labeling of the cells with 3Hinositol before transfection and was by 1 μm and inositol phosphates were extracted h after the and cells were cell death determined by HEK293 cells were with 1 μm and was assayed h after the The transfected cells were by of The are the of three The the cell death determined by The of cell death using the Gas-Pak anaerobic chamber was performed as described in the to Toxicity was assayed h after the The are the of three The the Although the three forms of InsP6K have the to InsP7 formation and cell death, transfected not endogenous we the three InsP6K isoforms by RNA interference treatment in RNA for the three enzymes deletion of InsP6K2 cell death by deletion of the other two isoforms does not physiologically, InsP6K2 cell of InsP6K2 from to cell stressors the intracellular localization of InsP6K isoforms HEK293 cells with staurosporine and ovarian carcinoma cells with cisplatin A and previously (12Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), under conditions InsP6K2 is exclusively nuclear, whereas InsP6K1 and InsP6K3 are both cytosolic and The cell stressors not the intracellular localization of InsP6K1 or InsP6K2 but a of nuclear InsP6K2 associated with in the the of the we for of InsP6K2 with for and with fluorescent InsP6K2 does not with of these not We and apoptotic for and of InsP6K2 is with InsP6K2 with The of InsP6K2 with is well known to from to Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. Y. Hattori S. Shimizu S. Tsujimoto Y. K. N. Y. J. PubMed Scopus Google Scholar, A. J. Cell Biol. PubMed Scopus Google Scholar), of InsP6K2 with to the apoptotic process. The present study provides compelling evidence that endogenous InsP6K2, by generating InsP7, provides physiologic regulation of the apoptotic process. multiple cell stressors in diverse cell all InsP7 formation. Deletion of InsP6K2 but not the other forms of InsP6K diminishes 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. of InsP6K2 augments the cytotoxic actions of multiple cell stressors in diverse cell lines, whereas in cells, the transfection increases cell death in but not other cell We are to that the of InsP6K2 for of cells. with InsP6K2 decreases cell death in all cell examined with to was by Lindner and co-workers (15Morrison B.H. Bauer J.A. Hu J. Grane R.W. Ozdemir A.M. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu D.V. Lindner D.J. Oncogene. 2002; 21: 1882-1889Crossref PubMed Scopus (61) Google Scholar, 16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) in a cell with a a dominant negative associated with InsP7 formation. Cell leads to of InsP7 we both in cells and in cell with no associated in for of the three InsP6K are best by of one of the InsP6K by translocation of InsP6K2 from nuclei to to enzyme activity. The regulation of by InsP6K activity. InsP7 formation by a InsP6K2 diminishes cell death. of InsP7 by of the three isoforms augments cell death. the physiologic regulation of to InsP6K2, deletion by RNA interference treatment cell death, whereas deletion of InsP6K1 and InsP6K3 is with the of Lindner and co-workers (15Morrison B.H. Bauer J.A. Hu J. Grane R.W. Ozdemir A.M. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu D.V. Lindner D.J. Oncogene. 2002; 21: 1882-1889Crossref PubMed Scopus (61) Google Scholar, 16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) implicated InsP6K2 in this by that InsP6K2 for the of a of constructs to cell death by interferon a translocation of InsP6K2 from nuclei to InsP6K2 with stained for damaged the apoptotic but not with that cell InsP6K2 to from nuclei to mitochondria, a role in the apoptotic process. is with the of InsP6K2 deletion to apoptosis. We not for the translocation of InsP6K2 from the We have to nuclear in cell a to InsP6K2 and to InsP7 elicit actions of InsP7 with PtdIns(3,4,5)P3 for binding to PH of (10Luo H.R. Huang Y.E. Chen J.C. Saiardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) and of Although InsP7 multiple a have been and these are InsP7 with for binding to a of PH domain one of is (10Luo H.R. Huang Y.E. Chen J.C. Saiardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. 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