Characterization of a Selective Inhibitor of Inositol Hexakisphosphate Kinases

Inositol hexakisphosphate kinases (IP6Ks) phosphorylate inositol hexakisphosphate (InsP6) to yield 5-diphosphoinositol pentakisphosphate (5-PP-InsP5 or InsP7). In this study, we report the characterization of a selective inhibitor, N2-(m-(trifluoromethy)lbenzyl) N6-(p-nitrobenzyl)purine (TNP), for these enzymes. TNP dose-dependently and selectively inhibited the activity of IP6K in vitro and inhibited InsP7 and InsP8 synthesis in vivo without affecting levels of other inositol phosphates. TNP did not inhibit either human or yeast Vip/PPIP5K, a newly described InsP6/InsP7 1/3-kinase. Overexpression of IP6K1, -2, or -3 in cells rescued TNP inhibition of InsP7 synthesis. TNP had no effect on the activity of a large number of protein kinases, suggesting that it is selective for IP6Ks. TNP reversibly reduced InsP7/InsP8 levels. TNP in combination with genetic studies was used to implicate the involvement of two pathways for synthesis of InsP8 in yeast. TNP induced a fragmented vacuole phenotype in yeast, consistent with inhibition of Kcs1, a Saccharomyces cerevisiae IP6K. In addition, it also inhibited insulin release from Min6 cells in a dose-dependent manner further implicating InsP7 in this process. TNP thus provides a means of selectively and rapidly modulating cellular InsP7 levels, providing a new and versatile tool to study the biological function and metabolic relationships of inositol pyrophosphates. Inositol hexakisphosphate kinases (IP6Ks) phosphorylate inositol hexakisphosphate (InsP6) to yield 5-diphosphoinositol pentakisphosphate (5-PP-InsP5 or InsP7). In this study, we report the characterization of a selective inhibitor, N2-(m-(trifluoromethy)lbenzyl) N6-(p-nitrobenzyl)purine (TNP), for these enzymes. TNP dose-dependently and selectively inhibited the activity of IP6K in vitro and inhibited InsP7 and InsP8 synthesis in vivo without affecting levels of other inositol phosphates. TNP did not inhibit either human or yeast Vip/PPIP5K, a newly described InsP6/InsP7 1/3-kinase. Overexpression of IP6K1, -2, or -3 in cells rescued TNP inhibition of InsP7 synthesis. TNP had no effect on the activity of a large number of protein kinases, suggesting that it is selective for IP6Ks. TNP reversibly reduced InsP7/InsP8 levels. TNP in combination with genetic studies was used to implicate the involvement of two pathways for synthesis of InsP8 in yeast. TNP induced a fragmented vacuole phenotype in yeast, consistent with inhibition of Kcs1, a Saccharomyces cerevisiae IP6K. In addition, it also inhibited insulin release from Min6 cells in a dose-dependent manner further implicating InsP7 in this process. TNP thus provides a means of selectively and rapidly modulating cellular InsP7 levels, providing a new and versatile tool to study the biological function and metabolic relationships of inositol pyrophosphates. Inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) 2The abbreviations used are: Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetraphosphate; InsP6, inositol hexakisphosphate; IP6K, inositol hexakisphosphate kinase; 5-PP-InsP5 or InsP7, bisdiphosphoinositol pentakisphosphate; TNP, N2-(m-(trifluoromethy)lbenzyl) N6-(p-nitrobenzyl)purine; TG, thapsigargin; PEI, polyethyleneimine; PP2-InsP4 or InsP8, bisdiphosphoinositol tetrakisphosphate; GFP, green fluorescent protein; BisTris, 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid; PPIP5K, diphosphoinositol pentakisphosphate kinase; HPLC, high pressure liquid chromatography; IP, inositol phosphate; IP3-3K, Ip3-3 kinase, inositol(1,4,5)trisphosphate-3kinase; Ip3-3KA, isoform A of Ip3-3K; IPMK, inositol polyphosphate multikinase; InsP3, inositol trisphosphate; InsP4, inositol tetraphosphate; InsP5, inositol pentaphosphate. is the cytosolic product of inositol phospholipid-specific phospholipase Cs and serves multiple biological functions. In higher eukaryotes, it regulates Ca2+ release from intracellular stores via binding to Ins(1,4,5)P3-specific receptors located in the endoplasmic reticulum. In addition, in Saccharomyces cerevisiae and all other eukaryotes that have been studied, Ins(1,4,5)P3 also undergoes complex metabolism to generate a series of inositol polyphosphates with diverse functions (1Irvine R.F. J. Physiol. (Lond.).. 2005; 566: 295-300Google Scholar, 2Shears S.B. Biochem. J... 2004; 377: 265-280Google Scholar). Inositol hexakisphosphate (InsP6) can be synthesized in inositol phospholipid-specific phospholipase C/Ins(1,4,5)P3-mediated pathways in most, if not all, eukaryotes. InsP6 is further metabolized by the inositol hexakisphosphate kinases (IHPKs or IP6Ks), which add a pyrophosphate moiety at the 5-position to generate 5-PP-InsP5 or 5-InsP7. Dictyostelium discoideum and S. cerevisiae, each possess one IP6K gene product designated Kcs1 in yeast (3Luo 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: 559-572Google Scholar, 4Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem... 2000; 275: 24686-24692Google Scholar). In mammals, three IP6Ks have been identified (5Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Curr. Biol... 1999; 9: 1323-1326Google Scholar, 6Saiardi A. Nagata E. Luo H.R. Snowman A.M. Snyder S.H. J. Biol. Chem... 2001; 276: 39179-39185Google Scholar). In addition, a second InsP6/5-InsP7 kinase, designated Vip/PPIP5K, has been identified in yeast (7Mulugu S. Bai W. Fridy P.C. Bastidas R.J. Otto J.C. Dollins D.E. Haystead T.A. Ribeiro A.A. York J.D. Science.. 2007; 316: 106-109Google Scholar) and mammalian cells (8Fridy P.C. Otto J.C. Dollins D.E. York J.D. J. Biol. Chem... 2007; 282: 30754-30762Google Scholar, 9Choi J.H. Williams J. Cho J. Falck J.R. Shears S.B. J. Biol. Chem... 2007; 282: 30763-30765Google Scholar). This kinase is distinct from IP6K/Kcs1 in that it phosphorylates the 1/3-position of InsP6 and 5-PP-InsP5 (10Lin H. Fridy P.C. Ribeiro A.A. Choi J.H. Barma D.K. Vogel G. Falck J.R. Shears S.B. York J.D. Mayr G.W. J. Biol. Chem... 2009; 284: 1863-1872Google Scholar). Insights into the biological functions of inositol pyrophosphates have come from genetic studies in yeast and by manipulating expression of inositol polyphosphate kinases in mammalian cells. More recently, yeast mutants failing to synthesize inositol pyro phosphate molecules have been found to be impaired in several cellular functions, such as DNA repair, chromatin remodeling, and telomere length maintenance (11Huang K.N. Symington L.S. Genetics.. 1995; 141: 1275-1285Google Scholar, 12York S.J. Armbruster B.N. Greenwell P. Petes T.D. York J.D. J. Biol. Chem... 2005; 280: 4264-4269Google Scholar, 13Steger D.J. Haswell E.S. Miller A.L. Wente S.R. O'Shea E.K. Science.. 2003; 299: 114-116Google Scholar-14Shen X. Xiao H. Ranallo R. Wu W.H. Wu C. Science.. 2003; 299: 112-114Google Scholar). Production of 1/3-InsP7 is necessary for regulating cellular phosphate starvation responses through the inhibition of the cyclin/cyclin-dependent kinase (7Mulugu S. Bai W. Fridy P.C. Bastidas R.J. Otto J.C. Dollins D.E. Haystead T.A. Ribeiro A.A. York J.D. Science.. 2007; 316: 106-109Google Scholar, 10Lin H. Fridy P.C. Ribeiro A.A. Choi J.H. Barma D.K. Vogel G. Falck J.R. Shears S.B. York J.D. Mayr G.W. J. Biol. Chem... 2009; 284: 1863-1872Google Scholar, 15Lee Y.S. Mulugu S. York J.D. O'Shea E.K. Science.. 2007; 316: 109-112Google Scholar). In D. discoideum, InsP7 has been reported to be important in chemotaxis (3Luo 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: 559-572Google Scholar). In mammals, overexpression of the IP6K1 increases insulin release from pancreatic β cells, whereas overexpression of IP6K2 increases apoptosis in a variety of cell lines (16Illies C. Gromada J. Fiume R. Leibiger B. Yu J. Juhl K. Yang S.N. Barma D.K. Falck J.R. Saiardi A. Barker C.J. Berggren P.O. Science.. 2007; 318: 1299-1302Google Scholar, 17Nagata E. Luo H.R. Saiardi A. Bae B.I. Suzuki N. Snyder S.H. J. Biol. Chem... 2005; 280: 1634-1640Google Scholar). As a means to further study the roles of inositol pyrophosphate messengers, we set out to develop pharmacological tools that permit acute inhibition of specific inositol polyphosphate kinases. Here we report the characterization of an IP6K inhibitor, which appears to be selective in vitro and in vivo and its use in furthering our understanding of the role of InsP7 synthesis in yeast and in insulin secretion in mammalian cells. Ip3-3 kinase inhibitor (TNP; see "Results”) and thapsigargin (TG) were from Calbiochem. 2-3HInositol and γ-32PATP were from GE Healthcare. HeLa cells were from ATCC. PEI-cellulose TLC sheets were from Merck. Reduced glutathione was from Sigma. Oligonucleotides were from the University of Dundee in-house DNA synthesis center. Cloning—The human IP6K1 and the catalytic fragment of IP3-3KA were from the IP6K1, and and the for and were the IP6K1 was into the mammalian expression green protein whereas the catalytic fragment of IP3-3KA was into as were in the in-house center. cells were in with at in a HeLa cell lines were by cells on with of DNA of green protein or green the at by this the was by as were of in were identified by and by protein expression by Min6 cells were in and at in a cells were into each of a were for in of the were with of DNA and of and of IP6K1 IP6K1 was into HeLa cells on cells were in and were by at at for of was with of the with of protein on a for at was and the were with of and with of and for or activity activity the were in of of activity and of TNP or of was was by the of at a of no were at on a for were by the of of were and the was to a was from the was the of the in the to the in was the TNP for and of and of the of catalytic fragment of IP3-3KA was as a protein in the of E. was to an of at expression was induced by the of and of the to were with by at for at was in and inhibitor from were by with of two on an at the was at for at on an was with of for at the was and were with of with no the protein was from with reduced was on a in and the was for the of the protein was and at a of in at IP3-3KA was with of γ-32PATP and Ins(1,4,5)P3 in of the was PEI-cellulose TLC were in to out and phosphate sheets were and a was by to as a of the of γ-32PATP of was TNP to the was the were from the by the of were the the from is the for and is the of used in the or IP6K1 of fragment or S. cerevisiae was with or TNP for on the γ-32PATP and InsP6 was to the and the was out at for as the reported by Fridy J. H. M. P. Biochem. J... 2003; Scholar). and the were by PEI-cellulose in a and and 2-3HInositol of cells were at cells on in of with and of for the of TNP or the were TNP of cells was out as from a of TNP was to cells with as and of was to cells. was of the of in all with was also out a with from a of was to cells with or without TNP at for at for of TNP the cells were with TNP, was and cells were three with of with were to for the inositol were and of Inositol by or cells were with and with cells were and the was from the by in a for at was by the of was as the was a inositol were from the and was as at for in the by in the by a high of for the and a with A for the was the and were This was for of InsP6, which at InsP7 at and InsP8 at of and Inositol and were in with Inositol were as TNP at a of was to and cells were to at of were to the and the of was cells through in were by at for in a were with and by with on for was by at in a was by the of and the inositol were by as and of yeast, and to were in at TNP at a of was to and cells were to for of were to the and the of was the yeast vacuole from was of yeast in were by and in were with and yeast vacuole to the was and cells were a with from Min6 was and cells were with and to of and were with of TNP for in of the of and as with TNP, the was with and TNP at the of cell were out and used for of insulin was the insulin as the each a was insulin in the of cell the were with to a the of the of insulin was from the were with IP6Ks to the of inositol polyphosphate kinases, also and by of in A has been reported for IP3-3KA from and from S. cerevisiae, the and of the and the of the protein the inositol phosphate binding B. R.F. Williams Cell.. 2004; Scholar, J.H. Cell.. 2004; Scholar). IP6Ks in the and inositol phosphate binding with the other of the it is that a of the and inositol phosphate binding from human and in IP3-3KA and that not in the IP6Ks or all of the of the high levels of in binding and binding B. R.F. Williams Cell.. 2004; Scholar, W. G. J. Biol. Chem... Scholar). each the and in and the important for binding and the for A of molecules a with either a (TNP; or at the and an at the were as for from a of TNP was found to have the for Choi G. Bae Y.S. M. Choi S.H. Scholar). have this as a inhibitor for IP6Ks. TNP a of the IP6Ks in and in if IP6Ks and inhibited by TNP, we in vitro activity with in the of of this IP6K1 was and from HeLa cells. of the is in was with and and of was of InsP7 was inhibited by the of TNP with an of from and of the for and the in the this to a of of by TNP of IP3-3KA IP6K1 of for IP3-3KA were in our and in other and for and IP6K1 have been reported in this were the for IP3-3KA were in our and in other A. K. A.M. S.J. C. C. 2005; and for and IP6K1 have been reported (5Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Curr. Biol... 1999; 9: 1323-1326Google Scholar, 6Saiardi A. Nagata E. Luo H.R. Snowman A.M. Snyder S.H. J. Biol. Chem... 2001; 276: 39179-39185Google Scholar, A. Nagata E. Luo H.R. A. Luo X. Snowman A.M. Snyder S.H. S. 2001; Scholar, G.W. S. K. J. Biol. Chem... 2005; 280: in this were the in a new in vitro studies with a fragment of the kinase the catalytic was to a J.H. Cell.. 2004; Scholar). that the of the fragment of IP3-3KA fragment was with γ-32PATP and Ins(1,4,5)P3, and of was in the of of TNP by of and and of with TNP, and the from was from two of protein and In this the and to the in the of is higher the reported by other Choi G. Bae Y.S. M. Choi S.H. Scholar, A. K. A.M. S.J. C. C. 2005; Scholar). in vitro to an in the for TNP IP6K1 and IP3-3KA whereas the for were these the were the is in the as that the that the IP3-3KA be at the binding whereas the IP6K1 be at the TNP is a and is an these to the that the in vivo of TNP be in of the IP6K1 was by HeLa cells with of TNP and in inositol phosphate levels. the of the inositol from cells. InsP6 levels the of TNP used in this we used the in the InsP6 to the levels of the other inositol phosphates. levels of and a of were the of TNP levels were also whereas InsP7 levels in to the of TNP an of for the InsP7 the from the in vitro to be by the in vivo levels further for in vivo of TNP for is the product of the of the that has a for A. Nagata E. Luo H.R. A. Luo X. Snowman A.M. Snyder S.H. S. 2001; Scholar, G.W. S. K. J. Biol. Chem... 2005; 280: Miller A.L. Y. Wente S.R. J. Biol. Chem... Scholar). if the in cellular InsP7 by TNP be rescued by overexpression of IP6Ks. HeLa cells with IP6K1 were with to and InsP7 levels were as a function of TNP the of InsP6 and InsP7 from cells IP6K1 and cells, with of cells had InsP7 cells TNP InsP7 levels in in the two cell As were not and at the cells had InsP7 levels with cells, and at TNP, the InsP7 was of that in IP6K1 overexpression InsP7 synthesis in the of with TNP had a effect on InsP7 levels in cells IP6K2 and -3 as IP6K1 all three mammalian IP6Ks with each other and all of have for the in InsP7 levels in cells IP6K1 or IP6K2 or -3 that the effect of TNP on InsP7 via inhibition of one or all three kinases. studies reported an in levels and a in the of release in cells with TNP Choi G. Bae Y.S. M. Choi S.H. Scholar). an in intracellular to the in InsP7 through this InsP7 levels in cells with TNP were with in cells with TG, which increases intracellular levels of by the Ca2+ on the B. S.B. Scholar). of the inositol from cells with TNP and in of InsP3, InsP4, and InsP7 as a of InsP6 that in InsP7 levels and cells. cells a in InsP7, whereas cells in InsP7 with InsP7 levels in was no in levels in cells of and this serves as a it has been that to increases intracellular without in Ins(1,4,5)P3 levels. further the that in InsP7 levels TNP to a specific inhibition of IP6Ks. we to the of TNP on the of kinases (7Mulugu S. Bai W. Fridy P.C. Bastidas R.J. Otto J.C. Dollins D.E. Haystead T.A. Ribeiro A.A. York J.D. Science.. 2007; 316: 106-109Google Scholar, P.C. Otto J.C. Dollins D.E. York J.D. J. Biol. Chem... 2007; 282: 30754-30762Google J.H. Williams J. Cho J. Falck J.R. Shears S.B. J. Biol. Chem... 2007; 282: 30763-30765Google Scholar). In vitro were with an fragment of H. or S. cerevisiae in the and of either or PP2-InsP4 InsP6 was used as the for the and its activity was by the of TNP activity of S. cerevisiae was also 5-PP-InsP5 was used as a not that not of we the of TNP on a of protein kinases. In vitro were in the of and TNP, described C.J. P. Scholar, J. H. M. P. Biochem. J... 2003; H. M. P. Biochem. J... 2000; Scholar). of the kinases in the were inhibited by TNP at all in this study were out at this the that TNP is specific for the TNP is a and IP6Ks via binding to the binding the effect of TNP be of TNP inhibition was by HeLa cells with TNP and to the of this with TNP, InsP7 levels and cells were to for the of TNP, InsP7 levels to levels, that TNP inhibition of IP6Ks is TNP a for the and of Inositol and that InsP8 is by the of IP6K and pathways have been In InsP6 is at the 5-position by IP6K and at the 1/3-position by Vip/PPIP5K, whereas is the the selective of TNP on the IP6K we that the be a for the of each InsP8 TNP inhibition of InsP7 and InsP8, whereas InsP8 levels if TNP were to through levels of InsP8 in cell or the we its as reported X. Choi K. Shears S.B. J. Biol. Chem... 2004; Scholar). this HeLa cells to with were by with in the or of TNP, and inositol polyphosphates were and by a in the to InsP8 and TNP reduced the levels of InsP7 and InsP8 in a dose-dependent manner with of and of inhibition at the of TNP used was consistent with the that for the of InsP8 synthesis the studies have that of the inositol or S.J. Armbruster B.N. Greenwell P. Petes T.D. York J.D. J. Biol. Chem... 2005; 280: 4264-4269Google Scholar, A.M. Bastidas R.J. York J.D. J. Biol. Chem... 2005; 280: which is by we TNP inhibition of InsP7 metabolism in a variety of yeast In cells, the of InsP7 is of that of of cells with TNP, we a of InsP7 Kcs1 is this of InsP7 is consistent with inhibition by TNP of In further of we an in the of InsP7 in cells for and found that with TNP or Kcs1 InsP7 that be by for and a in InsP7 levels of the with TNP or of Kcs1 further InsP7 levels as with consistent with a of metabolism through which be by was our for the of and in yeast. In to study the of TNP on cellular functions for we two biological In S. cerevisiae, of the IP6K, Kcs1, in vacuole and A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem... 2000; 275: 24686-24692Google Scholar, E. B. Shears S.B. J. Biol. Chem... Scholar). yeast were with TNP for and were the fluorescent which selectively the with TNP rapidly the of in yeast with a that is to that which is in and and that TNP the phenotype in these the of TNP as a for the biological functions of inositol pyrophosphates and for the that a acute of InsP7 rapidly used a second biological from that in InsP7 the of pancreatic β cells and cell lines to insulin secretion (16Illies C. Gromada J. Fiume R. Leibiger B. Yu J. Juhl K. Yang S.N. Barma D.K. Falck J.R. Saiardi A. Barker C.J. Berggren P.O. Science.. 2007; 318: 1299-1302Google Scholar). insulin release from Min6 cells with cells a to that insulin release by with a that was dose-dependently inhibited by Overexpression of IP6K1 insulin secretion in this and these cells from the of TNP, suggesting that TNP to inhibit secretion via its inhibition of one or IP6Ks A and the of implicating InsP7 in insulin secretion and that these acute and not to responses to in the cellular of this In the study, we that a TNP, as an inhibitor Choi G. Bae Y.S. M. Choi S.H. is a selective and inhibitor of IP6Ks. of TNP for from two in that TNP has an for IP6K1 that for that the of these two for also in the with a that is that of IP6Ks. TNP is an inhibitor of these cellular levels in the the for these in vivo of This appears to be out by inositol phosphate levels in HeLa cells TNP dose-dependently reduced InsP7 and InsP8 levels by at without affecting other inositol phosphate the several molecules that were for in to TNP, two other molecules had of and Choi G. Bae Y.S. M. Choi S.H. Scholar). that these be as of IP6Ks. from TNP and the in a inhibitor, was reported to inhibit of J. Biol. Chem... Scholar). In our did not inhibition of IP3-3KA in vitro not which be with that its of inhibition was have also been reported to be of and G.W. S. K. J. Biol. Chem... 2005; 280: Scholar). this inhibition also to have other cellular that for intracellular of InsP7 in S. cerevisiae can pathways of InsP7 in was to be the IP6K the phosphate on the 5-position of the inositol to generate cells levels of an This to the and of Vip/PPIP5K, a InsP6 kinase to a of cellular InsP7 two that is in metabolic by the (7Mulugu S. Bai W. Fridy P.C. Bastidas R.J. Otto J.C. Dollins D.E. Haystead T.A. Ribeiro A.A. York J.D. Science.. 2007; 316: 106-109Google Scholar). A that a of InsP7/InsP8 synthesis in mammalian cells was by X. Choi K. Shears S.B. J. Biol. Chem... 2004; Scholar) and is in our in the InsP8 was at the of the InsP7 This that the in InsP7 and InsP8 synthesis were and (8Fridy P.C. Otto J.C. Dollins D.E. York J.D. J. Biol. Chem... 2007; 282: 30754-30762Google Scholar, 9Choi J.H. Williams J. Cho J. Falck J.R. Shears S.B. J. Biol. Chem... 2007; 282: 30763-30765Google which the human of the yeast have been which synthesize as in IP6Ks and to the pathways from InsP6 to TNP of HeLa cells the pathways into TNP is the for IP6Ks and be to the cellular of its high for this of TNP be to out all cellular InsP7, InsP7 be This can be either to inhibition of the cellular IP6K or to the of other InsP7 by the enzymes. to the the the which from the binding of in and in it is that to be of TNP, and this was by a in vitro InsP7 which the of IP6Ks and 5-PP-InsP5 and (10Lin H. Fridy P.C. Ribeiro A.A. Choi J.H. Barma D.K. Vogel G. Falck J.R. Shears S.B. York J.D. Mayr G.W. J. Biol. Chem... 2009; 284: 1863-1872Google Scholar) be to be by the used in our the that were to and InsP8 levels, appears to be the be in yeast for in which InsP7 levels were these TNP further InsP7 levels, the of as a in cells In mammalian cells, it is that if all of the of which have been identified Caffrey J.J. Yang X. Shears S.B. J... Scholar, Falck J.R. Shears S.B. J. Biol. Chem... 1999; the of InsP7 by be in a manner to that in or cells in S. of which for in binding of was by B. Williams R. D. A. B. Williams Cell.. of the and of enzymes. that this can be to the of inositol polyphosphate kinases. these the of kinase for such to as of biological function and in as have a kinases, a is the of large of enzymes. kinases by the of kinases with in the human the of TNP a of protein and kinases were inhibited at the used in the studies described This the of TNP as a selective pharmacological tool in studies of the functions of inositol yeast were with TNP yeast either or the of this in the biological functions of inositol pyrophosphates in a of cell and acute of an inhibitor of IP6K implicate a an effect of of InsP7 and further studies of the of vacuole and that not be genetic inhibition of insulin release from Min6 cells the of TNP, that the function of InsP7 in this is to be and the to study the of the insulin in have in our understanding of the cellular and functions of inositol polyphosphates by studies in yeast and in Overexpression of has also been used to new functions, the of the that for these of the has not been of a inhibitor of IP6Ks the to the of inositol pyrophosphate and genetic TNP rapidly InsP7 levels in mammalian cells, and of the and to the it for providing Min6 cells. and of the for the IP6K1 and for the protein kinase also of and and and and of University of for and with