Gu C, Nguyen HN, Ganini D, Chen Z, Jessen HJ, Gu Z, Wang H, Shears SB.
Proc Natl Acad Sci U S A. 2017 Nov 7;114(45):11968-11973. doi: 10.1073/pnas.1702370114. Epub 2017 Oct 25. Abstract
The inositol pyrophosphates 5-InsP7 (diphosphoinositol pentakisphosphate) and 1,5-InsP8
(bis-diphosphoinositol tetrakisphosphate) are highly energetic cellular
signals interconverted by the diphosphoinositol pentakisphosphate
kinases (PPIP5Ks).
Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP8 and raises 5-InsP7 levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP7] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP7] provides direct support for an energy-sensing attribute (i.e., 1 mM Km for ATP) of the 5-InsP7-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP7 levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in PPIP5K-/- HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP8 and 5-InsP7 as drivers of the hypermetabolic and p53-elevated phenotypes, we used IP6K2 RNAi and the pan-IP6K inhibitor, N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine (TNP), to return 5-InsP7 levels in PPIP5K-/- cells to those of WT cells without rescuing 1,5-InsP8 levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP7 regulates p53 expression, whereas 1,5-InsP8 regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP8 to bioenergetic homeostasis.
Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP8 and raises 5-InsP7 levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP7] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP7] provides direct support for an energy-sensing attribute (i.e., 1 mM Km for ATP) of the 5-InsP7-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP7 levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in PPIP5K-/- HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP8 and 5-InsP7 as drivers of the hypermetabolic and p53-elevated phenotypes, we used IP6K2 RNAi and the pan-IP6K inhibitor, N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine (TNP), to return 5-InsP7 levels in PPIP5K-/- cells to those of WT cells without rescuing 1,5-InsP8 levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP7 regulates p53 expression, whereas 1,5-InsP8 regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP8 to bioenergetic homeostasis.
Published under the PNAS license.
2.
Fairfax BP, Makino S, Radhakrishnan J, Plant K, Leslie S, Dilthey A, Ellis P, Langford C, Vannberg FO, Knight JC.
Nat Genet. 2012 Mar 25;44(5):502-10. doi: 10.1038/ng.2205.
-- Using paired purified primary monocytes and B cells, we identify new predominantly cell
type-specific cis and trans expression quantitative trait loci (eQTLs),
including multi-locus trans associations to LYZ and KLF4 (znf) in monocytes
and B cells, respectively. Additionally, we observe a B cell-specific trans association of rs11171739 at 12q13.2, a known autoimmune disease locus, with IP6K2 (P = 5.8 × 10(-15)), PRIC285 (P = 3.0 × 10(-10)) and an upstream region of CDKN1A (P = 2 × 10(-52)), suggesting roles for cell cycle regulation and peroxisome proliferator-activated receptor γ (PPARγ) signaling in autoimmune pathogenesis.Free PMC Article
3.
Koldobskiy MA, Chakraborty A, Werner JK Jr, Snowman AM, Juluri KR, Vandiver MS, Kim S, Heletz S, Snyder SH.
Proc Natl Acad Sci U S A. 2010 Dec 7;107(49):20947-51. doi: 10.1073/pnas.1015671107. Epub 2010 Nov 15.
Inositol pyrophosphates (PP-InsPx) have been implicated in numerous biological processes.
Inositol hexakisphosphate kinase-2 (IP6K2), which generates the inositol pyrophosphate , PP-IP5, diphosphoinositol pentakisphosphate (IP7), influences apoptotic cell death. The tumor suppressor p53 responds to genotoxic stress by engaging a transcriptional program leading to cell-cycle arrest or apoptosis.
We demonstrate that IP6K2 is required for p53-mediated apoptosis and modulates the outcome of the p53 response. Gene disruption of IP6K2 in colorectal cancer cells selectively impairs p53-mediated apoptosis, instead favoring cell-cycle arrest. IP6K2
acts by binding directly to p53 and decreasing expression of proarrest
gene targets such as the cyclin-dependent kinase inhibitor p21.Free PMC Article
4.
Morrison BH, Bauer JA, Kalvakolanu DV, Lindner DJ.
J Biol Chem. 2001 Jul 6;276(27):24965-70. Epub 2001 May 3. Abstract
Interferons (IFNs)
regulate the expression of genes that mediate their antiviral,
antitumor, and immunomodulatory actions. We have previously shown that
IFN-beta suppresses growth of human ovarian carcinoma xenografts in vivo
and induces apoptosis of ovarian carcinoma cells in vitro. To
investigate mechanisms of IFN-beta-induced apoptosis we employed an
antisense technical knockout approach to identify gene products that
mediate cell
death and have isolated several regulators of interferon-induced death
(RIDs). In this investigation, we have characterized one of the RIDs,
RID-2. Sequence analysis revealed that RID-2 was identical to human
inositol hexakisphosphate kinase 2 (IP6K2). IP6K2 is post-transcriptionally induced by IFN-beta in ovarian carcinoma cells. A mutant IP6K2
with substitutions in the putative inositol phosphate binding domain
abrogates IFN-beta-induced apoptosis. These studies identify a novel
function for IP6K2 in cell growth regulation and apoptosis.
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