Adam C. Resnick, Division of Neurosurgery at the Children's Hospital of Philadelphia, Department of Neurosurgery at the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Adolfo Saiardi, Medical Research Council (MRC) Cell Biology Unit and Laboratory for Molecular Cell Biology, Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom
Inositol polyphosphates comprise a large family of water-soluble molecules derived from the combinatorial phophorylation of the six hydroxyls of myo-inositol. Second messenger roles for inositol polyphosphates in Ca2+ mobilization were first identified for what is now the best characterized family member, inositol 1,4,5-trisphosphate (InsP3). Additional anabolic and catabolic metabolism of InsP3results in the formation of a large, diverse family of higher inositol polyphosphates whose signaling roles and biologic functionality remain largely undefined. However, the recent cloning and identification of the kinases and phosphatases involved in the combinatorial modification of inositol polyphosphates has served to further define and characterize this complex metabolic network and to identify its preeminence in nearly all aspects of cell biology. Conserved from yeast to humans, inositol polyphosphates regulate a wide array of processes, including ion-channel conductance, membrane dynamics, transcription, nucleic acid metabolism, and protein phosphorylation.
The scientific study of inositol polyphosphates began in 1850 with the isolation of a crystalline “sugar” from heart muscle extracts (1). The substance was named “inosit” from the Greek root inos for muscle, fiber, or sinew. The later identification of this optically inactive inositol (the “ol” suffix added in French and English) as one of nine possible cyclohexanehexol isomers necessitated the use of prefix designations for the specific stereoisomeric configurations of the secondary hydroxyl groups about the six-carbon ring (cis-, epi-, allo-, muco-, neo-. scyllo- (+)-chiro-, (-)-chiro, and myo-) (Fig. 1). Although several, if not all, inositol isomers occur in nature, myo-inositol (myo, once again derived from the Greek word for muscle) is the biologically relevant stereoisomer in most species and functions as the structural building block for the inositol polyphosphates.
Viewed in its favored chair conformation, myo-inositol possesses five equatorial groups and one axial hydroxyl group. The modern D-numbering system for inositols is by convention counterclockwise as viewed from above and assigns the axial hydroxyl group to C2. This conformation is best illustrated by Agranoff’s turtle (2) in which the six hydroxyls are envisaged as the appendages (1-, 3-, 4-, 6-hydroxyls), head (2-hydroxyl), and tail (5-hydroxyl) of a friendly turtle. It is worth noting that among the stereoisomers of inositol, myo-inositol is unique in containing a single axial OH group (Fig. 1). This results in an achiral molecule with a plane of symmetry through C2-C5 (head to tail) and two pairs of enantiotopic hydroxyls (C1-C3 and C4-C6, the turtle’s arms and legs, respectively). Presumably, it is the chemical uniqueness of this configuration that resulted in nature’s selection of the myo isomer over others for enzymatic modification and biologic significance.
Myo-inositol’s most basic function, like other polyols (such as sorbitol), is as an osmolyte whose increased cellular concentration reflects responses to hyperosmolarity. However, inositol’s full biologic potential is only realized via chemical modification. Although inositol participates in several varied enzymatic reactions, it is the combinatorial substitution of phosphate moieties to the six hydroxyls that impart preeminent metabolic and functional significance to this deceptively simple molecule. Mathematically, 63 such combinations are possible; this number, however, is an underestimate as diphosphate (also known as pyrophosphate) moieties also exist. To date, over 30 different inositol polyphosphates have been observed across eukaryotic evolution resulting in a fairly crowded metabolic map. Complicating matters, the cellular biosynthesis of inositol polyphosphates—composed of only myo-inositol and phosphates and therefore water-soluble—is not achieved by the mere sequential phophorylation and/or dephosphorylation of the myo-inositol ring (although there are exceptions in plants and slime molds; see below). Rather, their biosynthesis is intimately connected with the metabolism of their hydrophobic, phospholipid relatives—the phosphatidylinositol (PtdIns or PI) phosphates.
Figure 1. (a) Myo-inositol and its polyphosphate derivatives. Although nine sterioisomeric configurations of inositol are possible, the turtle-like myo-inositol with its single axial hydroxyl is the most biologically relevant. Also depicted are the scyllo- and neo-inositols with zero and two axial hydroxyls, respectively. The modern D-numbering system for inositols is counterclockwise viewed from above and assigns the axial hydroxyl group to C2 of myo-inositol. (b) Inositol polyphosphates. Depicted are representatives of phophorylated derivatives of myo-inositol, including Ins(1,4,5)P3, the calcium releasing factor; InsP6, the naturally most abundant fully phosphorylated inositol polyphosphate; and InsP7, an inositol diphosphate (pyrophosphate).
Receptor-Stimulated Inositol Metabolism and the "Inositol Cycle"
The birth of receptor-stimulated inositol polyphosphate metabolism and its role in cell signaling was the observation in the early 1950s of acetylcholine-stimulated 32P incorporation into inositol lipids (termed the “PI response”) (3). However, it would take more than two decades for the mechanisms and significance of this response to come to light. Key to the eventual elucidation of the “PI response” was the realization that receptor stimulation leads to the activation of phosphoinositol-lipid-specific phospholipase C (PLC). Upon activation, PLC hydrolyzes the glycerol-phosphate bond in PtdIns(4,5)P2causing the release of water-soluble inositol 1,4,5-trisphosphate (InsP3 or Ins(1,4,5)P3) and the lipid diacyl-glycerol (DAG). After receptor-mediated catabolism, a regenerative cycle undertakes to restore PtdIns(4,5)P2 to the plasma membrane. Released DAG acts as the now familiar activator of PKC, but subsequently it reenters the inositol metabolism pathway as CMP-phosphatidic acid (CMP-PtdOH, alternatively named CDP-DAG). Synthesis of CMP-PtdOH proceeds via the phosphorylation of DAG by DAG-kinase and conjugation to a cytidine nucleotide. Inositol lipid synthesis initiates as the phosphatidic acid (PA) moiety of CMP-PtdOH is enzymatically donated to the C1-hydroxyl of myo-inositol forming PtdIns. Sequential phosphorylation of PtdIns to PtdIns(4)P and PtdIns(4,5)P2 completes the “inositol cycle” and regenerates the substrate for receptor-activated PLC hydrolysis (Fig. 2). The 32P incorporation into PtdIns initially observed in the “PI response” to acetylcholine turned out to be the result of the rapid incorporation of 32P into adenosine 5'-triphosphate (ATP) followed by the phosphorylation of DAG enroute to its attachment to myo-inositol. In addition to DAG metabolism, the completion of an “inositol cycle” is concomitantly dependent on the continuous supply of free inositol. The source of myo-inositol for PtdIns synthesis is often dephosphorylated Ins(1,4,5)P3. Alternatively, cells take up extracellular inositol or synthesize inositol de novo.
Figure 2. The ''inositol cycle.'' A simplified representation of the regenerative metabolism responsible for the synthesis of InsP3. Generation of InsP3 and DAG upon PLC-mediated hydrolysis of PtdIns(4,5)P2 is followed by sequential dephosphorylation of InsP3 and DAG's modification to CDP-DAG. The enzymatic attachment of CDP-DAG to inositol regenerates the lipid precursors that ultimately replenish PtdIns(4,5)P2.
Myo-inositol uptake and synthesis
Sodium- or proton-coupled myo-inositol transporters provide the most direct route for the initiation of inositol polyphosphate metabolism (affording the inositide researcher a simple avenue for the analysis of inositol polyphosphate dynamics using radiolabeled inositol). To date three different mammalian cotransporters have been identified (SMIT 1 and 2 are Na+-coupled, whereas HMIT uses a proton gradient). Although transporters are widely transcribed in many animal tissues, access to extracellular inositol varies among organs. In the absence or low levels of extracellular inositol, de novo synthesis of free myo-inositol is transcriptionally induced; this seems to be a universal capacity of cells conserved from bacteria to humans. Synthesis is initiated via the cyclization of glucose-6-phosphate by 1D-myo-inositol-3-phosphate synthase (MIPS) and the formation of inositol-3-phosphate (Ins(3)P). Dephosphorylation of Ins(3)P by inositol monophosphatase (IMP) yields the free inositol that can then be incorporated into inositol lipids (PtdIns). In addition to its role in de novo synthesis, IMP also functions to dephosphorylate other singly phosphorylated downstream hydrolytic products of InsP3 [Ins(1)P and Ins(4)P]. As such, IMP is situated at a metabolic intersection of de novo synthesis and the regeneration of myo-inositol, functioning as a gateway for the completion and maintenance of the “inositol cycle.”
InsP1 and InsP2, lithium, and the inositol depletion hypothesis
By and large the inositol monophosphates and bisphosphates are thought to lack messenger functions and are most often conceived as catabolic products of the regenerative portion of the inositol cycle or as components of the “off” switch of inositol polyphosphate signaling. Sequential dephosphorylation of Ins(1,4,5)P3 in most eukaryotes proceeds with the formation of Ins(1,4)P2 followed by Ins(4)P. Additional inositol bisphosphates found in animal cells are mostly composed of C3-hydroxyl phosphorylated species that are catabolic products of “higher” inositol polyphosphates resulting from the C3- and/or C6-hydroxyl phosphorylation of Ins(1,4,5)P3. Dephosphorylation of these inositol bisphosphates results in the production of either Ins(1)P or Ins(3)P. Despite their (still putative) categorization as “breakdown” products, inositol bispho- sphates, and in particular inositol monophosphates and their metabolism, have been the subject of extensive investigation because of the pharmacologic targeting of IMP by the commonly used mood stabilizer, lithium.
The use of lithium to treat manic-depressive illness dates back to 1949 (4). J.F.J. Cade, while working at a psychiatric hospital, was using guinea pigs to evaluate the effects/toxicity of injections of urine collected from psychiatric patients, testing the hypothesis that a “toxin” may be responsible for the patients’ illnesses. In the course of these and other related experiments, he began using the lithium salt of uric acid—chosen merely for its high solubility—and noted a marked depressive effect on the animals’ behavior after injection, which he later attributed to the lithium rather than to the uric acid. These experiments were followed by the successful clinical use of lithium in patients suffering from mania and bipolar disorders (4).
Studies in the early and mid-1970s evaluating the effects of lithium administration on inositol levels in rat brains identified a dramatic decrease in the free inositol levels accompanied by a concomitant increase in inositol monophosphates and suggested IMP as the plausible pharmacologic target mediating the therapeutic actions of lithium (5). Subsequent detailed biochemical studies of IMP’s inhibition by lithium demonstrated its uncompetitive inhibition by therapeutically relevant concentrations of the ion (6). Surprisingly little attention was paid to the early studies of lithium’s effects on inositide metabolism, and it was not until the formulation of the “inositol depletion hypothesis” by Berridge et al. that inositol’s mechanism of action and role in aberrant neuronal signaling came to the forefront of the psychopharmacology of lithium’s actions in the brain (7).
Stated explicitly, the proposal put forth by Berridge et al. suggests that the cells of the central nervous system (CNS) are uniquely sensitive to the inhibitory effects of lithium on IMP as a result of their limited access to extracellular inositol because of its poor penetration of the blood-brain barrier. Thus, the brain is extensively dependent on IMP’s role in de novo inositol synthesis and IMP’s functionality in the recycling of inositol monophosphates. Lithium’s inhibition of IMP results in the slowing down of the “inositol cycle” and depletes the inositol pool necessary for the production of Ins(1,4,5)P3 in response to receptor stimulation. The corollary of this hypothesis is that, in part, it is the overstimulation of inositide metabolism in the brains of manic-depressive patients that is responsible for the disease’s manifestation. In the nearly 20 years since the formulation of the “inositol depletion hypothesis,” additional substrates for lithium’s actions have been identified, including both the upstream Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase as well the serine/threonine kinase glycogen synthase kinase-3 (GSK-3), whose regulation seems to be independent of inositol metabolism, which suggests lithium’s effects on the CNS are more complicated than first envisioned. Nevertheless, it was in fact Berridge et al.’s use of lithium in examining the “PI response” that paved the way for the identification of Ins(1,4,5)P3 as the now well-known calcium-mobilizing second messenger.
Inositol Polyphosphates: Form and Function
Ins(1,4,5)P3 and Ca2+
The identification of Ins(1,4,5)P3 and its biologic function and significance in the “PI-effect” continually eluded investigators until a conceptual connection between calcium dynamics and receptor-stimulated phosphoinositide turnover was proposed by Michell (8). Working with blowfly salivary glands, Berridge and Fain demonstrated inositol’s capacity to rescue calcium responses after prolonged receptor stimulation, providing some of the first convincing evidence for inositol’s role in calcium homeostasis (9). Additional studies using lithium to monitor inositol polyphosphate accumulation allowed Berridge and Fain to hone in on Ins(1,4,5)P3 production via PLC hydrolysis of PtdIns(4,5)P2 as the likely diffusible cellular signal that coupled receptor activation to Ca2+ release from intracellular stores. Using permeabilized cells, Streb et al. conclusively demonstrated that application of exogenous InsP3 mobilized Ca2+ from the endoplasmic reticulum (10). A search for the InsP3 receptor ensued and culminated in the cloning, purification, and reconstitution of the receptor as well as the demonstration that the protein itself is an InsP3-gated Ca2+ channel (11, 12). Calcium dynamics and the regulation of the InsP3 receptor are still very active research areas. However, by the end of 1980s, a functional and elegant paradigm for soluble inositol metabolism was established.
Ins(1,3,4,5)P4: C3-hydroxyl phosphorylation
It was the unexpected identification of an alternative isomer of InsP3, Ins(1,3,4)P3, derived from the dephosphorylation of Ins(1,3,4,5)P4, that revealed that there was clearly more to inositol polyphosphate metabolism than circumscribed by the “inositol cycle” (13). Indeed within a few years’ span in the mid-to-late 1980s, the discovery of the C3-hydroxyl phosphorylation of the inositol ring ushered in an era of “proliferation” for the inositides, within both the lipid and the water-soluble arenas of their metabolism. During this time, an inositol lipid 3-kinase activity was found associated with viral Src and the middle-T antigen of the polyoma virus (14). Phophoinositide-3 kinase (PI3K) produces PtdIns(3,4,5)P3 from PtdIns(4,5)P2 upon cell-surface receptor stimulation. Structurally, PtdIns(3,4,5)P3 is in fact the lipid-bound version of Ins(1,3,4,5)P4. However, in contrast to PtdIns(4,5)P2, the D3-phosphorylated inositol lipids are not substrates for PLC, and thus they remain relegated to the “lipid domain” in their signaling role acting to recruit proteins possessing inositol lipid binding motifs to the plasma membrane (15). Like PtdIns(3,4,5)P3, the production of Ins(1,3,4,5)P4 is also mediated by a kinase.
In animals the rapid formation of Ins(1,3,4,5)P4 after the release of Ins(1,4,5)P3 in response to receptor activation is mediated by Ca2+-sensitive Ins(1,4,5)P3 3-kinases likely to have evolved soon after the emergence of InsP3-receptors. Thus, the functional roles attributed to Ins(1,3,4,5)P4 center about its potential for regulating Ca2+ responses mediated by or after InsP3-receptor activation. Conversion of Ins(1,4,5)P3 to Ins(1,3,4,5)P4 may simply serve as a means of removing Ins(1,4,5)P3 in a fashion analogous to its dephosphorylation to Ins(1,4)P2. Nonoverlapping subcellular and tissue distributions of these two metabolic activities support the hypothesis of their nonredundant role as Ins(1,4,5)P3 “off” switches. However, Ins(1,3,4,5)P4 has also been suggested to enhance the effectiveness of Ins(1,4,5)P3 by inhibiting its dephosphorylation via the 5-phosphatase (as both can serve as substrates, but with differing kinetics). Although multiple lines of evidence suggest that Ins(1,3,4,5)P4 has modulatory roles in Ca2+-homeostasis, the molecular mechanisms for these actions have remained elusive (16). Potential mediators of Ins(1,3,4,5)P4’s cellular functions have been identified as Ins(1,3,4,5)P4-binding proteins. GAP1IP4BP and GAP1m are members of the GTPase activating proteins of the Ras family displaying highly specific Ins(1,3,4,5)P4 binding (17). Binding of Ins(1,3,4,5)P4 has been mapped to the proteins’ pleck-strin homology (PH) domains, which canonically function as inositol lipid binding domains. The ability of Ins(1,3,4,5)P4 to specifically bind PH-domain-containing proteins suggests Ins(1,3,4,5)P4 may indeed function as a competitive regulator of PtdIns(3,4,5)P3/PI3K signaling. Indeed, many if not most PtdIns(3,4,5)P3 receptors also bind Ins(1,3,4,5)P4, at least in vitro. Whether an interplay between Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 is a general widespread regulatory phenomenon remains unknown.
Although its effects on Ca2+-homeostasis seem to be indirect, like its precursor (Ins(1,4,5)P3), Ins(1,3,4,5)P4 is both a messenger/cofactor molecule and a metabolite in the anabolism and catabolism of other inositol entities. C6-hydroxyl phophorylation of Ins(1,3,4,5)P4 results in the formation of Ins(1,3,4,5,6)P5, one of the most abundant inositol polyphosphates in mammalian cells, whereas the 5-phosphatase activity described above results in the formation of the alternative InsP3 isomer, Ins(1,3,4)P3. This duality of functionality—signaling molecule/transient metabolite—is likely shared with most if not all of the inositol polyphosphates and is a reflection of a more comprehensive topological theme in the evolution of the inositol polyphosphates network. Evident particularly with the advent of metazoan life are multiple and interconnected routes of synthesis for inositol polyphosphates initiated by and large with the C3-hydroxyl phosphorylation of Ins(1,4,5)P3. This complexity is likely the result of the superposition and integration of newly evolved signaling modalities with older ones, as well as the diversification of inositol polyphosphate metabolism and function in tissues and even within subcellular compartments.
The advent of InsP3-dependent Ca2+ signaling in metazoan life likely represents just such an integration and expansion of inositol polyphosphate signaling with Ins(1,3,4,5)P4 possibly acquiring a supporting role in Ca2+-homeostasis. Not surprisingly, given the preeminence of Ca2+ in cell signaling, additional Ca2+-dependent processes have been identified as targets of inositol polyphosphates with the regulation of the Ca2+-activated chloride (Cl-) channel by Ins(3,4,5,6)P4, an alternative InsP4 isomer, being one of the more well characterized.
Ins(3,4,5,6)P4: Ca2+-activated chloride (Cl-) channel regulation
Two separate biosynthetic routes have been proposed for the production of Ins(3,4,5,6)P4. The “correctness” of both pathways, whether they coexist or merely display species differences, is still a matter of debate. Regardless, both depend on the initial C3-hydroxyl phosphorylation of Ins(1,4,5)P3. The first pathway, currently only supported by experiments in avian erythrocytes, depends on several sequential dephosphorylation and phosphorylation reactions that isomerically interconvert between InsP4 and InsP3. This pathway can be summarized as follows: Ins(1,3,4,5)P4 → Ins(1,3,4)P3 →Ins(1,3,4,6)P4 → Ins(3,4,6)3 → Ins(3,4,5,6)P4 (18). A second proposed pathway, which is prevalent in mammalian cells, proceeds via the C1-hydroxyl dephophorylation of Ins(1,3,4,5,6)P5. Surprisingly, despite its use of Ins(1,3,4,5,6)P5 as a precursor for the formation of Ins(3,4,5,6)P4, this latter pathway, like the first, also seems to depend on the dephosphorylation of Ins(1,3,4,5)P4 to Ins(1,3,4)P3. In a theme of competitive protection that is likely repeated often in inositol metabolism, the generation of Ins(1,3,4)P3 after PLC activation results in the protection of the cellular pools of Ins(3,4,5,6)P4by competing as a substrate for a dual specificity kinase that phosphorylates Ins(3,4,5,6)P4 back to Ins(1,3,4,5,6)P5. Ins(1,3,4)P3 5,6-/Ins(3,4,5,6)P4 1-kinase (also named ITPK1) is one of several inositol polyphosphate kinases displaying substrate promiscuity, an enzymatically conservative yet metabolically proliferative measure on behalf of evolution that also affords the potential of competitive “crosstalk.” Startlingly, the 1- Ins(1,3,4,5,6)P5 phosphatase responsible for Ins(3,4,5,6)P4 production turns out to be the very same ITPK1. Thus, ITPK1 functions as a reversible kinase capable of the interconversion of Ins(1,3,4,5,6)P5 and Ins(3,4,5,6)P4 (19). The balance of phosphatase/kinase activities seems to be regulated by Ins(1,3,4)P3 such that Ins(1,3,4)P3 stimulates the phosphatase reaction. Reflecting the regulatory “crosstalk” involved in its production, Ins(3,4,5,6)P4 accumulation in response to persistent PLC activation occurs relatively slowly but persists long after other inositol metabolites have returned to their prestimulated levels, a feature likely important in its regulation of Ca2+-activated Cl- channels. Cl- secretion via Ca2+-activated channels serves to physiologically regulate epithelial salt and fluid secretion in the gastrointestinal tract, exocrine glands, and lungs of animals. A putative role for a PLC-dependent factor in the negative regulation of Cl- secretion was suggested by experiments in which Ca2+-mediated activation of Cl- secretion upon receptor activation of PLC were followed by a period refractory to the stimulating effects of Ca2+. Correlating channel activation and deactivation with cellular levels of downstream PLC-dependent inositol polyphosphates, Shears and associates identified Ins(3,4,5,6)P4 as the likely mediator of the inhibition of Cl-conductance and went on to show that a cell-permeant analog of Ins(3,4,5,6)P4 decreased Ca2+-dependent Cl-secretion (20). The apical distribution of ITPK1 in polarized epithelial cells localizes Ins(3,4,5,6)P4 near its presumed site of action. However, the precise mechanism or target of inhibition has remained elusive. Although much of the delineation of the physiologic importance of Ins(3,4,5,6)P4 has been derived from the study of epithelial cells, Ca2+-activated Cl- channels occur in most cell types raising the potential of Ins(3,4,5,6)P4-mediated regulation of diverse cellular process ranging from neurotransmission to smooth-muscle contraction (21).
As indicated, the regulation of Ca2+ and/or Ca2+- dependent processes by Ins(1,4,5)P3, Ins(1,3,4,5)P4, and Ins(3,4,5,6)P4 likely represents a recent addition to the repertoire of inositol polyphosphate functionality. Soon after the discovery of C3-hydroxyl phosphorylated inositol polyphosphates came the realization that more phosphorylated inositol species were indeed conserved ubiquitous inositol metabolites predating the metazoan emergence of InsP3-receptor-mediated Ca2+ signaling. These highly phosphorylated species included inositol pentakisphosphate (Ins(1,3,4,5,6)P5), known at the time to occur in avian erythrocytes, and the fully phosphorylated inositol hexakisphosphate (Ins(1,2,3,4,5,6)P6, also called phytic acid or InsP6), thought to be predominantly relegated to the plant kingdom. Although the biosynthetic pathways of highly phosphorylated inositol polyphosphates have yet to be fully elucidated, over the last decade significant strides have been made in their metabolic and functional characterization, including the identification of inositol polyphosphate kinases ancestral to the Ca2+-dependent Ins(1,4,5)P3 3-kinases. In large part these advances are the byproducts of the genetic tractability of one of the simplest model organisms, the budding yeast Saccharomyces cerevisiae.
Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5: transcriptional regulation
In the budding yeast, inositol polyphosphate synthesis proceeds via what is likely to be one of the earliest incarnations of a PLC-dependent pathway for higher inositol polyphosphate metabolism in eukaryotes. Early biochemical studies in yeast (and plants) failed to identify a calcium-sensitive Ins(1,4,5)P3 3-kinase activity analogous to that found in mammalian cells. Instead, these studies identified C6-hydroxyl phosphorylation of Ins(1,4,5)P3 and formation of Ins(1,4,5,6)P4 as the most likely first anabolic step in the production of higher inositol polyphosphates (22). Additional biochemical studies identified the sequential phosphorylation of Ins(1,4,5,6)P4 to Ins(1,3,4,5,6)P5 followed by Ins(1,2,3,4,5,6)P6 (23). These findings were interpreted as proof of the existence of disparate pathways in yeast and mammals for the metabolism and functionality of Ins(1,4,5)P3. In contrast, the eventual cloning of the yeast Ins(1,4,5)P3 kinase activity found that mammalian and yeast inositol metabolism were more closely related than initially suspected.
Biochemical and genetic characterization of the yeast Ins(1,4,5)P3 kinase revealed it to be a dual-specificity Ins(1,4, 5)P3 3/6-kinase, rapidly converting Ins(1,4,5)P3 to Ins(1,3,4,5, 6)P5 via the intermediate production of Ins(1,4,5,6)P4. Sequence comparisons of the yeast Ins(1,4,5)P3 3/6-kinase with the mammalian Ins(1,4,5)P3 3-kinases demonstrated them to be part of a closely related family and suggested the Ins(1,4,5)P3 3-kinases were merely a recent evolutionary modification and specialization of this catalytically versatile kinase family (24). The cloning and characterization of the yeast Ins(1,4,5)P3 3/6-kinase was accomplished contemporaneously by two independent groups (24, 25). Noting its catalytic versatility, the Snyder group dubbed the enzyme inositol polyphosphate multikinase (IPMK), whereas the York group chose inositol polyphosphate kinase 2 (Ipk2) as it was one of two simultaneously identified kinases necessary for the synthesis of InsP6 (26). However, yeast IPMK/Ipk2 was in fact identical with the previously characterized Arg82, which was a protein with a history of nearly two decades of research that had clearly identified it as a nuclear, transcriptional regulator.
Studies of yeast deficient for arg82 identified it as a required factor for the transcriptional regulation of genes comprising arginine anabolic and catabolic metabolism. Transcriptional regulation of these arginine-sensitive pathways permits yeast to use alternative nitrogen sources such as arginine or ornithine when the preferred sources of ammonia or glutamate are limiting. In the absence of Arg82, yeast fail to grow on media with arginine/ornithine as the sole nitrogen source (27). The identification of yeast IPMK/Ipk2 as Arg82 (henceforth called IPMK) prompted the evaluation of the role its kinase activities may play in regulating these arginine-sensitive transcriptional responses.
Monitoring growth phenotypes of various inositol polyphosphate mutants in media containing arginine or ornithine as the sole nitrogen source, York and associates identified the necessity of Plc1 and IPMK but not downstream inositol polyphosphate kinases for growth on selective media (25). These results supported the conclusion that it is the production of Ins(1,4,5,6)P4 and/or Ins(1,3,4,5,6)P5that is required for the regulation of arginine-sensitive gene expression. Although protein determinants of IPMK may play a role in its functional contribution to arginine-sensitive transcription (28), additional experiments using IPMK orthologs found in the fly and plant that lack previously identified yeast regulatory sequences suggest Ins(1,4,5,6)P4 and/or Ins(1,3,4,5,6)P5 are themselves sufficient for modulating arginine-dependent transcription; however, the molecular mechanism remains unknown (29, 30). Roles for Ins(1,4,5,6)P4 and/or Ins(1,3,4,5,6)P5 in transcriptional regulation have been supported by subsequent genetic screens in yeast that identified the requirement of Ins(1,4,5,6)P4 and/or Ins(1,3,4,5,6)Ps in phosphate-responsive regulation of chromatin remodeling and transcription (31). The ipmk null yeast are defective in the induction of the PHO5 gene in response to phosphate limitation. Remodeling of the PHO5 promoter chromatin is impaired in ipmk mutant yeast as a result of defects in the recruitment of ATP-dependent chromatin-remodeling complexes. Specific roles for either Ins(1,4,5,6)P4 or Ins(1,3,4,5,6)P5 were once again suggested as chromatin remodeling defects were also evident in plcl mutant yeast, but not in yeast deficient in downstream inositol polyphosphate kinases (31). A mechanism for Ins(1,4,5,6)P4- and/or Ins(1,3,4,5,6)P5-mediated regulation of chromatin remodeling was suggested by in vitro nucleosome mobilization assays in which Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 were found to have direct stimulatory effects on chromatin remodeling complexes (32). However, these latter in vitro studies have been faulted for the use of nonphysiologic concentrations of the inositol phosphates, which suggests additional characterization of the process is necessary.
The identification in yeast of a concise anabolic pathway for higher inositol polyphosphates in the nucleus suggests primordial roles for their metabolism consistent with earlier suppositions on the nuclear origin of lipid inositol metabolism (33). Indeed, IPMK not only functions as an inositol polyphosphate kinase, but it is also a nuclear lipid inositol PI3K kinase that predates the evolutionary emergence of the unrelated viral-Src- and middle-T-antigen associated kinases (discussed above). Like its water-soluble inositol kinase activities, IPMK-mediated synthesis of nuclear PtdIns(3,4,5)P3 has also been linked to transcriptional regulation in yeast (34). Whether transcriptional regulatory roles for Ins(1,4,5,6)P4/Ins(1,3,4,5,6)P5 (or PtdIns(3,4,5)P3) are evident in higher eukaryotes remains unknown, although roles in nuclear processes are likely, as nuclear IPMK paralogs have been identified across evolution, including mammals.
Characterization of mammalian IPMK showed it had retained its catalytic versatility and was capable of sequential phosphorylation of Ins(1,4,5)P3 to Ins(1,3,4,5,6)P5 (35); however, like the Ins(1,4,5)P3 3-kinase mammalian IPMK prefers to initiate this metabolism via C3-hydroxyl phosphorylation. Additional studies have extended IPMK’s catalytic repertoire and have suggested that in mammalian systems, IPMK serves to synthesize Ins(1,3,4,5,6)P5 via the C5-hydroxyl phosphorylation of Ins(1,3,4,6)P4, the product of Ins(1,3,4)P3 phosphorylation by ITPK1 (discussed above) (36). Thus, in mammals, two pathways may exist for the production of Ins(1,3,4,5,6)P5. One pathway is analogous to that found in yeast, mediated exclusively by IPMK, but the second follows a more circuitous route initiated by Ins(1,4,5)P3 3-kinase and ultimately is still dependent on IPMK. This latter pathway can be summarized as follows: Ins(1,4,5)P3 → Ins(1,3,4,5)P4 → Ins(1,3,4)P3 →Ins(1,3,4,6)P4 → Ins(1,3,4,5,6)P5. Whether both routes do indeed exist in mammals or whether one pathway predominates is still a matter of some debate. Perhaps the occurrence of both pathways simply reflects cytoplasmic versus nuclear signaling. Whichever the route, production of Ins(1,3,4,5,6)P5 and its more phosphorylated derivatives seems to be of preeminent importance for mammalian systems as deletion of IPMK in mice results in early embryonic lethality and severe developmental defects (37).
Ins(1,2,3,4,5,6)P6: from chelator to cofactor
Production of Ins(1,3,4,5,6)P5 is the penultimate step in the synthesis of the most abundant inositol phosphate on earth, Ins(1,2,3,4,5,6)P6. Its synthesis is achieved via the phosphorylation of Ins(1,3,4,5,6)P5’s lone remaining axial hydroxyl by an Ins(1,3,4,5,6)P5-2 kinase (IPK1) (26). InsP6’s quantitative dominance on earth is owed largely to its use by plants for phosphate storage in seeds. Although it was the first inositol polyphosphate discovered, it was not until the development and application of high-pressure liquid chromatography (HPLC) techniques in the analysis of inositol polyphosphates that its prevalence in animal cells was appreciated. With intracellular concentration in mammalian cells ranging between 10 and 100 μM, it is the most abundant inositol polyphosphate species, often exceeding most others by an order of magnitude or more. Even higher concentrations have been observed in the slime mold Dictyostelium discoideum, which possesses unique metabolic pathways for inositol polyphosphate synthesis independent of inositol lipids and their hydrolysis by PLC. In fact, in D. discoideum (and in some plants), higher inositol polyphosphate synthesis can proceed via the direct sequential phosphorylation of Ins(3)P after cyclization of glucose-6-phosphate. In these unusual organisms InsP6 concentrations can reach as high as 700 μM. Because of its high charge density, InsP6 is a strong chelator that readily forms insoluble salts with polyvalent cations resulting in its precipitation at higher concentrations. Under cellular ionic condition found in animal cells, soluble InsP6 concentrations are limited to <50 μM (likely in the form a neutral, stable pentam-agnisium salt). Thus, it is likely that much of cellular InsP6 is actually found in a “bound state.”
Several proteins have been identified to bind InsP6. As InsP6 interacts strongly with positively charged residues, caution has been urged in interpreting in vitro binding experiments. Nevertheless, physiologic roles for InsP6 have been suggested in several processes throughout the cell. In the cytoplasm, InsP6 binds tightly to clathrin assembly proteins negatively regulating the assembly of clathrin-coated vesicles and receptor-mediated endocytosis at the plasma membrane (38). Nuclear roles for InsP6 have also been suggested. In mammals, DNA double-strand breaks can be repaired by nonhomologous end-joining (NHEJ) requiring the activity of DNA-dependent protein kinase (DNA-PK). In vitro studies demonstrated the stimulation of DNA-PK-dependent NHEJ activity by InsP6 and identified the Ku70/80 subunits of DNA-PK as direct InsP6 targets (39). Recently, a crystallographic study unexpectedly found InsP6 bound within the enzymatic core of an RNA editing enzyme (ADAR2) belonging to a class of adenosine deaminases and further demonstrated its requirement as a cofactor for this class of enzymes (40). Consistent with a conserved role in processes involving nucleic acids, a yeast genetic screen for required factors involved in mRNA export unambiguously identified Plc1, IPMK, and Ipk1 and their sequential enzymatic roles in InsP6 synthesis (26). Finally, like the IPMK mouse knockout, IPK1 knockout mice also display early embryonic lethality demonstrating a critical role for higher inositol polyphosphate synthesis in mammalian development (41).
In contrast to the rapid changes upon receptor activation in intracellular concentrations of lower inositol polyphosphates (e.g., Ins(1,4,5)P3), InsP6 concentrations seem immutable. This finding, along with observations of the sluggish incorporation of radiolabeled inositol into InsP6, prompted interpretations of slow turnover rates for InsP6 in vivo. However, the identification of InsP6 as a precursor to even more phosphorylated inositol polyphosphates overturned these misconceptions and revealed the true dynamic nature of cellular InsP6.
PP-InsP5 and PP2-InsP4: signaling and protein phosphorylation
That more than six phosphates can fit onto an inositol ring was first discovered in D. discoideum (42) and fluoride-treated pancreatoma cells (43). Subsequent studies confirmed the widespread evolutionary conservation of the diphosphate-containing inositols. These “high energy” molecules contain the fully phosphorylated inositol ring of InsP6 with additional pyrophosphate moieties on either one or two a phosphates. The best characterized diphosphorylated inositol polyphosphates are the diphosphoinositol pentakispho- sphate (InsP7, PP-InsP5) and bis-diphosphoinositol tetrakisphosphate (InsP8, PP2-InsP4) species. In most cell types, these species are present in submicromolar concentrations, representing 1-5% of total InsP6 levels. Remarkably, in mammalian cells, up to 50% of InsP6cycles through the more phosphorylated diphosphates every hour (43). Once again the slime mold represents the extreme of higher inositol polyphosphates metabolism with diphosphates species reaching concentrations of 200 μM. Nuclear magnetic resonance spectroscopy analysis of inositol diphosphates in D. discoideum showed a single InsP8 isomer possessing pyrophosphates on C5 and C6 (5,6-(PP)2-InsP4) and the two respective InsP7 isomers, 5-PP-InsP5 and 6-PP-InsP5. So far, in mammalian cells, only a single InsP7 isomer has been detected and demonstrated to be the C5-pyrophosphorylated species. The standard free energy of hydrolysis of the pyrophosphate bond in InsP7 (the nonphysiologic 1-PP-InsPs) has been estimated at 6.6 kcal/mol, higher than that of adenosine5'-diphosphate (ADP) (6.4kcal/mol) and only slightly lower than that of ATP (7.3 kcal/mol). These values are likely to be quite higher for InsP8 isomers with vicinal pyrophosphates as a result of steric constraints and strong electrostatic repulsion. In addition to InsP6-derived diphosphates, inositol species derived from the pyrophophorylation of InsP4 and InsP5 have also been observed while triphosphate-containing InsP8 species have not been ruled out, implying an overall more diverse ensemble of high energy inositols than first suspected (44).
The enzymatic capacity for the synthesis of diphosphorylated inositides has so far been attributed to a single class of InsP6-kinases conserved across the evolutionary spectrum. Indeed the InsP6-kinases, along with IPMK and Ins(1,4,5)P3 3-kinases, define a single, highly related, conserved family with representative members identified in every eukaryotic genome studied thus far. Surprisingly, studies examining the occurrence of this kinase family in some of the “earliest” eukaryotes suggest InsP6-kinases may be founding representatives as exemplified in the Giardia genome, which contains a single InsP6-kinase (45). Along with IPMK and Ipkl, the IP6-kinase (denoted Kcsl in yeast) completes the list of identified inositol polyphosphate kinases in the yeast genome. InsP6-kinase can mediate the production of both InsP7 and InsP8 as well as diphosphate derivatives of InsP4 and InsP5 (46, 47). However deletion studies in yeast (as well as the absence of a clear paralog in plants, although they contain IPMK) suggest additional enzymes—including an InsP7-kinase—capable of inositol pyrophosphorylation may exist (48, 49). It is worth noting that at least in vitro mammalian and yeast IPMKs have also been shown to generate inositol diphosphates (35).
As yeast contains only a single InsP6-kinase (mammals contain three isoforms), much of what we know regarding the functional roles of inositol diphosphates is the result of loss-of-function studies in the kcsl yeast mutant. Expanding on roles suggested for InsP6 in vesicle formation and endocytosis, in yeast, InsP7/InsP8 seem to regulate vesicular trafficking. Loss of Kcsl results in altered vacuolar morphology with the appearance of smaller fragmented vacuoles and the accumulation of membranous, vesicular structures as a result of aberrant endosomal processing (50). Inositol diphosphates also likely complement the transcriptional regulatory roles of InsP4/InsP5 in phosphate metabolism. InsP6-kinase was in fact identified as a cDNA stimulating inorganic phosphate uptake into Xenopus oocytes (51). Furthermore, yeast deficient in inositol diphosphate synthesis lack polyphosphate synthesis/storage (52). Recently, a remarkable role for inositol diphosphates in telomere length maintenance was revealed by studying ipkl-deficient yeast (49, 53). Telomeres consist of chromosomal caps of long, repetitive DNA sequences that prevent nucleolytic degradation and protect more internal coding sequences from chromosomal shortening associated with cell division. In the absence of InsP6, InsP6-kinase can use InsP5, which accumulates in yeast in the absence of Ipkl. As a result, in ipkl-deficient yeast, the products of InsP5 pyrophosphorylation, PP-InsP4, and (PP)2-InsP3, also accumulate to levels not observed in wild-type yeast and in fact exceed concentrations normally observed for InsP7 (PP-InsP5) and InsP8 [(PP)2-InsP4]. When compared with wild-type yeast, ipkl-deficient yeast display shortened telomere length, whereas yeast deficient in InsP6-kinase activity display increased telomere length, which suggests inositol diphosphates (increased in ipkl-deficient yeast and absent in kcsl-deficient yeast) negatively regulate telomere extension. Although the mechanism for these effects remains unknown, inositol diphosphates may target the regulatory actions of phosphatidylinositol 3-kinase-related kinases (protein kinases of evolutionary relation to PI3Ks) known to be involved in telomere-length maintenance (44). While the functional significance for inositol diphosphates observed in yeast is likely to be conserved in mammalian cells, the roles for InsP7/InsP8 in mammalian physiology are more difficult to study because of the presence of three isoforms of InsP6-kinase with distinct subcellular distributions. In mammals, several lines of evidence suggest inositol diphosphates regulate cell death/apoptosis (54) and are dynamically regulated in response to environmental stress (48).
As is the case for other inositol polyphosphates, binding/ allosteric mechanisms for inositol diphosphates’ functionality have also been proposed, although it must be noted that binding partners must demonstrate significant specificity to allow for meaningful regulation by InsP7/InsP8 within the cellular context of the much more abundant InsP6. In D. discoideum, where the levels of InsP7/InsP8 rival those of InsP6, evidence for the specific regulation by inositol diphosphates of PH-domain lipid binding (analogous to that suggested for Ins(1,3,4,5)P3 discussed above) has revealed a role in PtdIns(3,4,5)P3-dependent chemotaxis (55). However, the high energy potential of the β-phosphates in inositol diphosphates suggests unique functionality for this class of inositol polyphosphates beyond allostery/competition. Indeed, early characterization of InsP6-kinase demonstrated its capacity for ATP formation in the reverse by transferring a β-phosphate from InsP7 to ADP (56). More recently, Snyder and associates have demonstrated the occurrence of likely more functionally significant acceptors of the high energy β-phosphates than ADP: proteins (57).
InsP7 seems to physiologically phosphorylate a variety of protein targets, although its activity seems restricted to eukaryotes. Identification of specific targets in yeast suggested a phosphorylation consensus sequence consisting of an acidic, polyserine stretch interspersed with aspartate or glutamate residues. Such stretches proved to be excellent CK2 (formerly casein kinase-2) substrates. However, careful characterization of the process has demonstrated InsP7-mediated phosphorylation to be quite distinct from ATP/kinase-mediated phosphorylation. In contrast to ATP, InsP7 does not require separate protein kinases but directly phosphorylates its targets in a nonenzymatic and temperature-dependent reaction. Nevertheless, protein kinases such as CK2 are indeed critical for the process but function only to “prime” targets. The significance of kinase-mediated pre-phophorylation/priming and its requirement for InsP7-mediated nonenzymatic phosphorylation has only recently been elucidated. Biochemical characterization of phophorylated substrates suggests the chemical nature of InsP7-phosphorylated serines is quite distinct from canonical phospho-serines. In a dramatic twist to protein phosphorylation, InsP7-mediated phosphorylation is in fact serine pyrophosphorylation (R. Bhandari and S.H. Snyder, personal communication).
Although the physiologic roles of this novel posttranslational modification need to be evaluated, serine pyrophosphorylation likely expands on the already well-defined roles of canonical protein phosphorylation in modifying protein conformation, regulating catalytic activity, determining protein localization, or altering protein-protein interactions. Experiments assessing these possibilities are currently underway.
As the most recently identified members of the inositol polyphosphate family, inositol diphosphates expand an already complex and universal signaling network. Although it has been more than 20 years since the discovery of Ins(1,4,5)P3 as a second messenger, much of the functional and cell signaling roles of most inositol polyphosphates remains poorly understood. Nevertheless it is clear that inositol polyphosphates serve to dynamically organize, regulate, and orchestrate nearly all aspects of cell biology (Fig. 3). Full elucidation of their complex metabolism and roles will likely necessitate the development of better and less laborious analytic methods for the evaluation/identification of their dynamic regulation as well as methods to track their subcellular localization. Although such tools (as well as pharmacologic inhibitors) exist for their lipid counterparts, the inositol polyphosphates have lagged behind, in part because of their greater complexity. Nevertheless, the molecular identification and cloning of the inositol polyphosphate kinases (and phosphatases) achieved in the last decade has allowed for the generation of new experimental/genetic models. Although preliminary characterizations of these have confirmed inositol polyphosphates’ preeminent importance in cell biology and physiology, they have also revealed how much more we have yet to learn.
Although every effort was made to cite relevant original research, unavoidable omissions because of space constraints exist, for this, the authors apologize. More complete descriptions and referencing of original sources can be found under “Further Reading.”
Figure 3. Inositol polyphosphates and their cellular functions. Inositol polyphosphates serve to dynamically organize, regulate, and orchestrate nearly all aspects of cell biology. Depicted are the various roles discussed in the text for Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4, Ins(1,4,5,6)P4/Ins(1,3,4,5,6)P5, InsP6, and InsP7, only seven of the more than 30 inositol polyphosphates identified to date.
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