KO of 5-InsP ₇ kinase activity transforms the HCT116 colon cancer cell line into a hypermetabolic, growth-inhibited phenotype

Abstract

The inositol pyrophosphates 5-InsP (diphosphoinositol pentakisphosphate) and 1,5-InsP (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-InsP and raises 5-InsP 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-InsP] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP] provides direct support for an energy-sensing attribute (i.e., 1 mM for ATP) of the 5-InsP-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP and 5-InsP as drivers of the hypermetabolic and p53-elevated phenotypes, we used RNAi and the pan-IP6K inhibitor,2-(-trifluorobenzyl), 6-(-nitrobenzyl) purine (TNP), to return 5-InsP levels in cells to those of WT cells without rescuing 1,5-InsP levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP regulates p53 expression, whereas 1,5-InsP regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP to bioenergetic homeostasis.

Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis

“…This hypothesis is supported by our observation that the vih1-2 vih2-4 mutant can be complemented with the human enzyme carrying mutations in its phosphatase domain ( Figure 5I). In line with our biochemical experiments, it has been previously shown that in vivo InsP 8 levels decrease and that InsP 7 accumulates in vih2-4 mutant plants (Laha et al, 2015) and that InsP 8 levels are reduced in PPIP5K1 and 2 double mutants (Gu et al, 2017b). Importantly, the 1PP-InsP5 kinase activity of VIHs is essential for plant growth and development, as a mutations targeting the kinase active site cannot rescue the vih1-2 vih2-4 mutant phenotypes.…”

“…It has been previously suggested for human PPIP5K that the kinase and phosphatase domains may respond to changes in cellular ATP and Pi levels, respectively (Gu et al, 2017b;Shears, 2018). To test if the regulation of plant Pi homeostasis may be similar to the human system, we complemented our vih1-2 vih2-4 mutant with the HsVIP2, codon-optimized for Arabidopsis and expressed under the control of the Arabidopsis VIH2 promoter ( Figure 5I,J).…”

“…How these enzymes may sense changes in cellular Pi concentration remains to be elucidated in mechanistic detail. Currently it is known that Kcs1/IP6Ks have a K m for ATP in the low millimolar range and may therefore generate 5PP-InsP5 in response to changes in cellular ATP levels (Saiardi et al, 1999;Gu et al, 2017aGu et al, , 2017b. Vip1/PPIP5Ks are bifunctional enzymes harboring an N-terminal InsP kinase and and a C-terminal phosphatase domain (Figure 2A) (Mulugu et al, 2007;Fridy et al, 2007;Wang et al, 2015).…”

“…The phosphatase domain has been characterized as a specific 1PP-InsP 5 / InsP 8 phosphatase in yeast . Deletion of PPIP5K in human cells results in lower InsP 8 and elevated ATP levels, due to enhanced mitochondrial oxidative phosphorylation and increased glycolysis (Gu et al, 2017b).…”

Identity and functions of inorganic and inositol polyphosphates in plants

“…In addition, PP-InsPs have also been implicated in the regulation of target of rapamycin (TOR)-mediated carbon metabolism in Chlamydomonas (Couso et al, 2016), and in the control of mitochondrial function and ATP homoeostasis in yeast and human cells (Gu et al, 2017a). How PP-InsPs are able to integrate such a diversity of signaling pathways is far from understood.…”