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. 2025 May 28;147(21):17626-17641.
doi: 10.1021/jacs.4c16206. Epub 2025 May 15.

Pools of Independently Cycling Inositol Phosphates Revealed by Pulse Labeling with 18O-Water

Affiliations

Pools of Independently Cycling Inositol Phosphates Revealed by Pulse Labeling with 18O-Water

Geun-Don Kim et al. J Am Chem Soc. .

Abstract

Inositol phosphates control many central processes in eukaryotic cells including nutrient availability, growth, and motility. Kinetic resolution of a key modulator of their signaling functions, the turnover of the phosphate groups on the inositol ring, has been hampered by slow uptake, high dilution, and constraining growth conditions in radioactive pulse-labeling approaches. Here, we demonstrate a rapid (seconds to minutes) and nonradioactive labeling strategy of inositol polyphosphates through 18O-water in yeast, human cells, and amoeba, which can be applied in any media. In combination with capillary electrophoresis and mass spectrometry, 18O-water labeling simultaneously dissects the in vivo phosphate group dynamics of a broad spectrum of even rare inositol phosphates. The good temporal resolution allowed us to discover vigorous phosphate group exchanges in some inositol polyphosphates and pyrophosphates, whereas others remain remarkably inert. We propose a model in which the biosynthetic pathway of inositol polyphosphates and pyrophosphates is organized in distinct, kinetically separated pools. While transfer of compounds between those pools is slow, each pool undergoes rapid internal phosphate cycling. This might enable the pools to perform distinct signaling functions while being metabolically connected.

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Figures

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Pathways for synthesis of lipidic and generic soluble inositol phosphates. Inositol can either be taken up by cells or synthesized from glucose after uptake and phosphorylation. Cytosolic inositol phosphates are generated from PtdInsP2 (PIP2) through phospholipase C (PLC), generating diacylglycerol and InsP3. In mammalian cells, an additional lipid-independent pathway can generate InsP3 directly from glucose. Forward phosphorylation by kinases up to InsP8 is possible while phosphatases remove the phosphate groups, generating lower phosphorylated forms. The balance between kinases and phosphatases will likely occur at different rates controlled by metabolic and/or signaling inputs. Therefore, we can envisage an InsP network constantly turning over, harmonizing the steady-state concentrations of the different metabolites.
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Workflow for the analysis of cellular 18O labeled ATP and InsP pools through 18O-water labeling, combined with CE-MS analysis.
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(A) qTOF MS analysis of ATP. The analysis reveals the kinetics of 18O incorporation into ATP at different exchangeable positions. Wild-type yeast cells were grown in SC medium at 20 °C to slow down the incorporation. At the 0 min time point, the medium was changed to SC medium prepared with 50% 18O-water. Samples were harvested at different time points (1 and 60 min), extracted with perchloric acid and TiO2, and analyzed by qTOF mass spectrometry. Theoretical [M-H] for ATP, [18O] ATP, [18O2] ATP, [18O3] ATP, [18O4] ATP, [18O5] ATP, [18O6] ATP, and [18O7] ATP are 505.9885, 507.9927, 509.9970, 512.0012, 514.0055, 516.0097, 518.0139, and 520.0182, respectively. (B) The incorporation of 18O into ATP was analyzed at 0, 1, and 60 min using CE-qTOF (MS1) and CE-QQQ (MRM) methods. The means of 3 samples with standard deviation are shown.
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Kinetics of 18O entry into ATP of yeast under steady-state conditions. Time-dependent formation of ATP isotopologues in yeast with different numbers of 18O atoms studied by CE-QQQ with wide mass resolution. Wild-type yeast cells were grown logarithmically in SC medium at 20 °C. At the 0 min time point, the medium was changed to SC medium prepared with A 50% or B 100% of 18O-labeled water (99% enrichment). After further incubation at 20 °C for the indicated periods of time, cells were extracted with perchloric acid and TiO2. The graphs show the fractions of ATP that carried the indicated numbers of 18O at any position. The means of 3 samples with standard deviation are shown.
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Kinetics of 18O entry into soluble InsPs of yeast under steady-state conditions. Wild-type yeast cells were grown logarithmically in SC medium at 20 °C. The medium was changed to SC medium prepared with 50% of 18O-labeled water. After further incubation at 20 °C for the indicated periods of time, cells were extracted with perchloric acid and TiO2 and analyzed by CE-QQQ or qTOF. Note that the levels of 1-InsP7 in these samples were too low to be reliably detected. The means of 3 samples with standard deviation is shown, representing (A) 1,5-InsP8, (B) 5-InsP7, (C) InsP6, (D) 2-OH-InsP5, (E) Ins­(1,3,4,5,)­P4, (F) unassigned InsP4, (G) Ins­(2,4,6)­P3, (H) Ins­(1,4,5)­P3, (I,J) higher numbers of 18O incorporation into InsP3 were observed by CE-qTOF of samples as in (G,H,K). Proposed pathways of interconversion. Rapid interconversion of metabolites is indicated by red arrows, and slow interconversion by black arrows. CE-QQQ with wide mass resolution was used in (A,C,D,E,F,G,H), and CE-QQQ with unit mass resolution was used in (B).
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Kinetics of 18O entry into soluble InsPs and ATP of yeast under different Pi conditions and in vip1Δ mutant. Yeast cells grown in Pi-rich medium were transferred to Pi-rich or Pi-free media containing 50% 18O-labeled water, and aliquots were analyzed at the indicated time points following the transfer. Cells were extracted with perchloric acid and TiO2 and analyzed by CE-QQQ. The means of 3 samples with standard deviation is shown. (A) 18O labeled 5-InsP7 (sum of 18O1 and 18O2), 1-InsP7 (sum of 18O1 and 18O2), InsP6 (sum of 18O1 and 18O2), and ATP (sum of 18O1 to 18O7) in the wild-type yeast under different Pi conditions. ATP was measured by CE-QQQ with unit mass resolution, whereas the others were measured by CE-QQQ with wide mass resolution. (B) 18O labeled 5-InsP7 (sum of 18O1 and 18O2), InsP6 (sum of 18O1 and 18O2), and ATP (sum of 18O1 to 18O7) in the wild-type and vip1Δ mutant under Pi-rich condition. ATP and 5-InsP7 were measured by CE-QQQ with unit mass resolution, whereas the others were measured by CE-QQQ with wide mass resolution.
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Kinetics of 18O entry into ATP and cytosolic InsPs from human cells. Kinetics of 18O entry into 1,5-InsP8 (A), 5-InsP7 (B), 1-InsP7 (C), InsP6 (D), 2-OH InsP5 (E), Ins­(1,4,5,6)­P4 (F), unassigned InsP4 (G), Ins­(1,3,4,5)­P4 (H), Ins­(1,4,5)­P3: Ins­(1,4,5)­P3 (I), and ATP (J) were monitored in mammalian cells. HCT116 cells were grown in DMEM medium, detached, and collected by centrifugation. The cell pellet was resuspended in DMEM medium prepared with 50% of 18O-labeled water. After the indicated periods of further incubation at 37 °C, aliquots of the cells were extracted with perchloric acid. CE-MS analyses of the extracts were performed and rare analytes (PP-InsPs) were measured in wide mass resolution. The means of three replicates are shown with standard deviations.
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Kinetics of 18O entry into soluble InsPs from D. discoideum. Kinetics of 18O entry into 5,6-InsP8 (A,) 6-InsP7 (B), and InsP6 (C) monitored in Dictyostelium. Cells were precultured in SIH medium and transferred to SIH media made of 50% of 18O-labeled water with Pi. After further incubation for the indicated periods of time, samples were harvested and extracted. Extracts were measured by CE-QQQ with wide mass resolution. The means of four replicates with standard deviations are shown.

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