Inositol Hexakisphosphate Kinase 1 (IP6K1) Regulates Inositol Synthesis in Mammalian Cells

Abstract

myo-Inositol, the precursor of all inositol compounds, has pivotal roles in cell metabolism and signaling pathways. Although physiological studies indicate a strong correlation between abnormal intracellular inositol levels and neurological disorders, very little is known about the regulation of inositol synthesis in mammalian cells. In this study, we report that IP6K1, an inositol hexakisphosphate kinase that catalyzes the synthesis of inositol pyrophosphate, regulates inositol synthesis in mammalian cells. Ip6k1 ablation led to profound changes in DNA methylation and expression of Isyna1 (designated mIno1), which encodes the rate-limiting enzyme inositol-3-phosphate synthase. Interestingly, IP6K1 preferentially bound to the phospholipid phosphatidic acid, and this binding was required for IP6K1 nuclear localization and the regulation of mIno1 transcription. This is the first demonstration of IP6K1 as a novel negative regulator of inositol synthesis in mammalian cells.

Keywords: ISYNA1; Opi1; genetics; inositol; inositol phosphate; inositol pyrophosphate; lipid; metabolism; phosphatidic acid.

FIGURE 1.

IP6K1 rescues kcs1Δ inositol auxotrophy. Serial dilutions of yeast kcs1Δ cells transformed with indicated plasmids were spotted on synthetic complete Ura⁻ medium in the presence or absence or 75 μm inositol. Plates were incubated at the indicated temperatures for 3 days.

FIGURE 2.

Inositol synthesis is up-regulated in IP6K1-KO cells. A, mIno1 mRNA levels are increased in IP6K1-KO cells. WT and IP6K1-KO MEF cells were grown in DMEM with 10% FBS and harvested for mRNA extraction. mIno1 mRNA levels were determined by RT-PCR (two independent experiments with two replicates each) (*, p < 0.05). B, mINO1 protein levels were profoundly increased in IP6K1-KO cells (left). The expression of Ip6k1 in IP6K1-KO cells restored mINO1α and mINO1γ levels (right). MEF cells were grown in DMEM with 10% FBS in the presence or absence of 4 μg/ml blasticidin. Cells were harvested and lysed for protein extraction. 30 μg of total cell extract from each sample were subjected to Western blot analysis using 10% SDS-polyacrylamide gel. C, IP6K1-KO cells exhibited increased intracellular inositol levels. Intracellular inositol levels in MEF cells were determined as described under “Experimental Procedures” (three independent experiments with two replicates each) (*, p < 0.05). D, intracellular Glc-6-P levels were decreased in IP6K1-KO cells. Intracellular Glc-6-P levels in MEF cells were determined by enzyme-coupled fluorescence assay as described under “Experimental Procedures” (three independent experiments with two replicates each) (*, p < 0.05). Values are mean ± S.E. Statistical significance was determined by unpaired t test.

FIGURE 3.

Methylation pattern of mIno1 DNA is altered in IP6K1-KO cells. A, MEF cells were grown in DMEM with 10% FBS and harvested for DNA isolation. DNA methylation levels were determined as described under “Experimental Procedures.” CpG islands are depicted as balloons. Methylated cytosines are filled, and unmethylated cytosines are unfilled. B, raw data of mIno1 DNA methylation experiment. CpG islands are depicted in bold. Methylated cytosines are depicted in red, and unmethylated cytosines are depicted in black. CpG islands with altered methylation in IP6K1-KO cells are highlighted with yellow shading, and the start codon is highlighted with green shading.

FIGURE 4.

IP6K1 binds preferentially to PA. A, IP6K1 protein was purified from E. coli cells expressing the Ip6k1 gene on the pGEX-6-P2 overexpression vector. Serial dilutions of the indicated lipids were spotted on a nitrocellulose membrane, which was incubated overnight in buffer containing 25 μg of IP6K1 protein. Interactions between IP6K1 and lipids were determined by immunoblotting, as described under “Experimental Procedures.” B, IP6K1 exhibits sequence homology to yeast Opi1 (upper panel). The IP6K1 HOPA domain, which exhibits homology to the PA-binding domain of yeast Opi1, is highlighted in red. The catalytic motif of IP6K1 is highlighted in blue. The PA-binding domain of yeast Opi1 is highlighted in gray. The NLS of yeast Opi1 is underlined in purple. IP6K1 deletion mutants were constructed by site-directed mutagenesis according to the schematic figure (lower panel). Residues deleted are indicated by numbers above the bar. C, deletion of the Q2 domain of IP6K1 decreased binding to PA. WT and mutant IP6K1 proteins were overexpressed and purified from E. coli. Interactions between protein and PA were determined as described previously. Figure represents three independent experiments. D, deletion of the HOPA domain of IP6K1 did not affect binding to PA. WT and HOPAΔ IP6K1 proteins were expressed and purified from E. coli. Interactions between protein and PA were determined as described previously.

FIGURE 5.

PA binding is required for nuclear localization of IP6K1. IP6K1-KO cells were transfected with plasmids harboring GFP-tagged WT or mutant IP6K1. Intracellular localization of IP6K1 (upper panel) was determined by fluorescence microscopy. The number of cells showing each phenotype (lower panel) was determined in three independent experiments (n > 150). Values are mean ± S.E.

FIGURE 6.

PA binding to IP6K1 is required for repression of mIno1 transcription. IP6K1-KO cells were transfected with plasmids harboring the indicated GFP-tagged IP6K1 mutants. The mIno1 mRNA levels were determined by RT-PCR (two independent experiments with two replicates) (*, p < 0.05). Values are mean ± S.E. Statistical significance comparing transcription levels to KO + IP6K1 control was determined by unpaired t test.

FIGURE 7.

Model of regulation of mIno1 transcription by IP6K1. PA binding facilitates localization of IP6K1 to the nucleus, where it associates with chromatin and synthesizes IP₇/PP-IP₄. IP₇/PP-IP₄ represses mIno1 expression by promoting methylation of mIno1 DNA and/or inhibiting the transcription apparatus.