Diphosphoinositol phosphates are a subclass of inositol phosphates possessing one or two high energy diphosphate groups instead of phosphoester substituents of the myo -inositol. Here we describe the enzymes responsible for their synthesis and degradation and how these may be regulated. Formation of diphosphoinositol phosphates in yeast and mammals is driven by an increase of the cellular energy charge, a lack of inorganic phosphate, and in mammals by osmotic or heat stress and in some cases by receptor mediated signaling. Known cellular actions are an improvement of the cell homeostasis by a reduction of the energy charge, increased phosphate uptake, improvement of mitochondrial performance, and an increase of insulin secretion in mammals. The underlying molecular mechanisms of action are far from being clarifi ed but an increasing body of knowledge about molecular details has highlighted their complex participation in many cellular systems and metabolic processes.

InsP6 (inositol hexakisphosphate), the most abundant inositol phosphate in metazoa, is pyrophosphorylated to InsP7 [5PP-InsP5 (diphosphoinositol pentakisphosphate)] by cytosolic and nuclear IP6Ks (InsP6 kinases) and to 1PP-InsP5 by another InsP6/InsP7 kinase family. MINPP1 (multiple inositol-polyphosphate phosphatase 1), the only known InsP6 phosphatase, is localized in the ER (endoplasmic reticulum) and lysosome lumina. A mechanism of cytosolic InsP6 dephosphorylation has remained enigmatic so far. In the present study, we demonstrated that IP6Ks change their kinase activity towards InsP6 at a decreasing ATP/ADP ratio to an ADP phosphotransferase activity and dephosphorylate InsP6. Enantio-selective analysis revealed that Ins(2,3,4,5,6)P5 is the main InsP5 product of the IP6K reaction, whereas the exclusive product of MINPP1 activity is the enantiomer Ins(1,2,4,5,6)P5. Whereas lentiviral RNAi-based depletion of MINPP1 at falling cellular ATP/ADP ratios had no significant impact on Ins(2,3,4,5,6)P5 production, the use of the selective IP6K inhibitor TNP [N2-(m-trifluorobenzyl),N6-(p-nitrobenzyl)purine] abolished the production of this enatiomer in different types of cells. Furthermore, by analysis of rat tissue and human blood samples all (main and minor) dephosphorylation products of InsP6 were detected in vivo. In summary, we identified IP6Ks as novel nuclear and cytosolic InsP6- (and InsP5-) dephosphorylating enzymes whose activity is sensitively driven by a decrease in the cellular ATP/ADP ratio, thus suggesting a role for IP6Ks as cellular adenylate energy 'sensors'.

SummaryFundamental to the life and destiny of every cell is the regulation of protein synthesis through ribosome biogenesis, which begins in the nucleolus with the production of ribosomal RNA (rRNA). Nucleolar organization is a highly dynamic and tightly regulated process; the structural factors that direct nucleolar assembly and disassembly are just as important in controlling rRNA synthesis as are the catalytic activities that synthesize rRNA. Here, we report that a signaling enzyme, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) is also a structural component in the nucleolus. We demonstrate that IP5K has functionally significant interactions with three proteins that regulate rRNA synthesis: protein kinase CK2, TCOF1 and upstream-binding-factor (UBF). Through molecular modeling and mutagenic studies, we identified an Arg-Lys-Lys tripeptide located on the surface of IP5K that mediates its association with UBF. Nucleolar IP5K spatial dynamics were sensitive to experimental procedures (serum starvation or addition of actinomycin D) that inhibited rRNA production. We show that IP5K makes stoichiometrically sensitive contributions to the architecture of the nucleoli in intact cells, thereby influencing the degree of rRNA synthesis. Our study adds significantly to the biological significance of IP5K; previously, it was the kinase activity of this protein that had attracted attention. Our demonstration that IP5K 'moonlights' as a molecular scaffold offers an unexpected new example of how the biological sophistication of higher organisms can arise from gene products acquiring multiple functions, rather than by an increase in gene number.