VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis

Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP₈ by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP₈ in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP₈ by COI1-JAZ is a critical component in jasmonate-regulated defenses.     

Roles for inositol polyphosphate kinases in the regulation of nuclear processes and developmental biology

Our laboratory studies the biology and enzyme regulation of inositol signal transduction pathways, which are activated in response to a wide range of stimuli. As a six-carbon cyclitol, inositol and its numerous phosphorylated derivatives efficiently generate combinatorial ensembles of signaling molecules. Through the cloning and characterization of inositol polyphosphate kinases (IPK), novel roles for inositol tetrakisphosphate (IP₄), inositol pentakisphosphate (IP₅), and inositol hexakisphosphate (IP₆) and inositol pyrophosphates (PP-IPs), have been identified. Studies have linked the IPKs and their inositide products to the regulation of nuclear processes including gene expression, chromatin remodeling, mRNA export, DNA repair and telomere maintenance. Analysis of IPK knockout animals has revealed a role for production of IPs in regulation of embryogenesis and organism development.The discoveries of the IPK proteins and their connection to nuclear signaling have generated significant interest in the field. Furthermore, they have provided interesting clues into the evolution of inositide-signaling pathways. Ipk2/IPMK and IPS/IP6K family members are conserved from yeast to man. In contrast, the IP₃ 3-kinase (ITPK) branch is observed in selected metazoans and not in plant or fungi. This may imply that Ipk2 and IPS activities evolved first among the group. The promiscuity of the Ipk2 protein further supports this notion and may provide the cell with a means to generate many IP species in a genetically economical fashion. Studies of yeast inositide signaling reveal that these simple eukaryotes do not have an IP₃ receptor in their genome and do not utilize diacylglycerol to activate protein kinase C. Thus, it appears that the canonical “text book” aspects of inositide-signaling pathways are not conserved throughout eukaryotic evolution. In light of the conservation of Ipk2/IPMK, Ipk1 and IPS/IP6K pathways from yeast to man it is interesting to speculate that a primordial role of phospholipase C-induced, IPK-dependent inositide signaling was to regulate nuclear processes. As calcium and PKC signaling evolved in metazoans, these may have greatly enhanced signaling capabilities. Recent studies demonstrating an essential role for IP₅, IP₆ and possibly PP-IP production in metazoan development highlight the importance of IPK signaling in cellular responses in metazoans. With these thoughts in mind, we eagerly await future studies aimed at further elucidating how these signaling codes participate in developmental processes and the control of gene expression, mRNA export, and DNA metabolism.     

Inositol hexakisphosphate kinase 1 is a metabolic sensor in pancreatic β-cells

Diphosphoinositol pentakisphosphate (IP₇) is critical for the exocytotic capacity of the pancreatic β-cell, but its regulation by the primary instigator of β-cell exocytosis, glucose, is unknown. The high K_m for ATP of the IP₇-generating enzymes, the inositol hexakisphosphate kinases (IP6K1 and 2) suggests that these enzymes might serve as metabolic sensors in insulin secreting β-cells and act as translators of disrupted metabolism in diabetes. We investigated this hypothesis and now show that glucose stimulation, which increases the ATP/ADP ratio, leads to an early rise in IP₇ concentration in β-cells. RNAi mediated knock down of the IP6K1 isoform inhibits both glucose-mediated increase in IP₇ and first phase insulin secretion, demonstrating that IP6K1 integrates glucose metabolism and insulin exocytosis. In diabetic mouse islets the deranged ATP/ADP levels under both basal and glucose-stimulated conditions are mirrored in both disrupted IP₇ generation and insulin release. Thus the unique metabolic sensing properties of IP6K1 guarantees appropriate concentrations of IP₇ and thereby both correct basal insulin secretion and intact first phase insulin release. In addition, our data suggest that a specific cell signaling defect, namely, inappropriate IP₇ generation may be an essential convergence point integrating multiple metabolic defects into the commonly observed phenotype in diabetes.     

The inositol pyrophosphate pathway in health and diseases

Inositol pyrophosphates (IPPs) are present in organisms ranging from plants, slime moulds and fungi to mammals. Distinct classes of kinases generate different forms of energetic diphosphate‐containing IPPs from inositol phosphates (IPs). Conversely, polyphosphate phosphohydrolase enzymes dephosphorylate IPPs to regenerate the respective IPs. IPPs and/or their metabolizing enzymes regulate various cell biological processes by modulating many proteins via diverse mechanisms. In the last decade, extensive research has been conducted in mammalian systems, particularly in knockout mouse models of relevant enzymes. Results obtained from these studies suggest impacts of the IPP pathway on organ development, especially of brain and testis. Conversely, deletion of specific enzymes in the pathway protects mice from various diseases such as diet‐induced obesity (DIO), type‐2 diabetes (T2D), fatty liver, bacterial infection, thromboembolism, cancer metastasis and aging. Furthermore, pharmacological inhibition of the same class of enzymes in mice validates the therapeutic importance of this pathway in cardio‐metabolic diseases. This review critically analyses these findings and summarizes the significance of the IPP pathway in mammalian health and diseases. It also evaluates benefits and risks of targeting this pathway in disease therapies. Finally, future directions of mammalian IPP research are discussed.     

Regulation of nuclear processes by inositol polyphosphates

Inositide signaling pathways represent a multifaceted ensemble of cellular switches capable of regulating a number of processes, for example, intracellular calcium release, membrane trafficking, chemotaxis, ion channel activity and several nuclear functions. Over 30 inositide messengers are found in eukaryotic cells that may be grouped into two classes: (1) inositol lipids, phosphatidylinositols or phosphoinositides (PIPs) and (2) water-soluble inositol polyphosphates (IPs). This review will focus on inositol polyphosphate kinases (IPK) and inositol pyrophosphate synthases (IPS) responsible for the cellular production of IP(4), IP(5) IP(6) and PP-IPs. Of interest, IPK and IPS proteins localize, in part, within the nucleus and their activities are necessary for proper regulation of gene expression, mRNA export, DNA repair and telomere maintenance. The breadth of nuclear processes regulated and the evolutionary conservation of the genes involved in their synthesis have sparked renewed interest in inositide messengers derived from sequential phosphorylation of inositol 1,4,5-trisphosphate.     

The families of kinases removing the Ca2+ releasing second messenger Ins(1,4,5)P3

The formation and degradation of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] are of great metabolic importance, because of its role in the mediation of calcium release from intracellular stores. The concentration of Ins(1,4,5)P3 in the cell is regulated by three signaling enzymes: phospholipase C isoforms release Ins(1,4,5)P3 from the plasma membrane by hydrolysis of phosphatidyl inositol 4,5-bisphosphate, whereas inositol phosphate 5-phosphatases remove it by dephosphorylation and a group of inositol phosphate kinases eliminate it by further phosphorylation at its 3- or 6-hydroxy group. The latter group is formed by the three isoforms of Ins(1,4,5)P3 3-kinase (IP3K) and inositol phosphate multikinase. In this article the tissue specific gene expression, molecular structure, role in calcium oscillations, regulation by calcium calmodulin, by phosphorylation and by intracellular localization of the IP3K isoforms are discussed. Another important aspect is the evolution of diverse inositol phosphate metabolizing enzymes from a eukaryotic founder by different mechanisms of gene diversification. Finally the role of IPMK in calcium signaling will be elucidated in more detail.     

Molecular definition of a novel inositol polyphosphate metabolic pathway initiated by inositol 1,4,5-trisphosphate 3-kinase activity in Saccharomyces cerevisiae

The production of inositol polyphosphate (IPs) and pyrophosphates (PP-IPs) from inositol 1,4,5-trisphosphate (I(1,4,5)P3) requires the 6-/3-/5-kinase activity of Ipk2 (also known as Arg82 and inositol polyphosphate multikinase). Here, we probed the distinct roles for I(1,4,5)P3 6- versus 3-kinase activities in IP metabolism and cellular functions reported for Ipk2. Expression of either I(1,4,5)P3 6- or 3-kinase activity rescued growth of ipk2-deficient yeast at high temperatures, whereas only 6-kinase activity enabled growth on ornithine as the sole nitrogen source. Analysis of IP metabolism revealed that the 3-kinase initiated the synthesis of novel pathway consisting of over eleven IPs and PP-IPs. This pathway was present in wild-type and ipk2 null cells, albeit at low levels as compared with inositol hexakisphosphate synthesis. The primary route of synthesis was: I(1,4,5)P3 --> I(1,3,4,5)P4 --> I(1,2,3,4,5)P5 --> PP-IP4 --> PP2-IP3 and required Kcs1 (or possibly Ipk2), Ipk1, a novel inositol pyrophosphate synthase, and then Kcs1 again, respectively. Mutation of kcs1 ablated this pathway in ipk2 null cells and overexpression of Kcs1 in ipk2 mutant cells phenocopied IP3K expression, confirming it harbors a novel 3-kinase activity. Our work provides a revised genetic map of IP metabolism in yeast and evidence for dosage compensation between IPs and PP-IPs downstream of I(1,4,5)P3 in the regulation of nucleocytoplasmic processes.     

Emerging cell biological functions of phosphatidylinositol 5 phosphate 4 kinase

The generation of phosphoinositides (PIs) with spatial and temporal control is a key mechanism in cellular organization and signaling. The synthesis of PIs is mediated by PI kinases, proteins that are able to phosphorylate unique substrates at specific positions on the inositol headgroup to generate signaling molecules. Phosphatidylinositol 5 phosphate 4 kinase (PIP4K) is one such lipid kinase that is able to specifically phosphorylate phosphatidylinositol 5 phosphate, the most recently discovered PI to generate the well-known and abundant PI, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P₂]. PIP4K appears to be encoded only in metazoan genomes, and several genetic studies indicate important physiological functions for these enzymes in metabolism, immune function, and growth control. PIP4K has recently been reported to localize to multiple cellular compartments, including the nucleus, plasma membrane, endosomal systems, and autophagosome. However, the biochemical activity of these enzymes that is relevant to these physiological functions remains elusive. We review recent developments in this area and highlight emerging roles for these enzymes in cellular organization.     

Inositol polyphosphates contribute to cellular circadian rhythms: Implications for understanding lithium's molecular mechanism

Most living organisms maintain cell autonomous circadian clocks that synchronize critical biological functions with daily environmental cycles. In mammals, the circadian clock is regulated by inputs from signaling pathways including glycogen synthase kinase 3 (GSK3). The drug lithium has actions on GSK3, and also on inositol metabolism. While it is suspected that lithium's inhibition of GSK3 causes rhythm changes, it is not known if inositol polyphosphates can also affect the circadian clock. We examined whether the signaling molecule inositol hexaphosphate (IP₆) has effects on circadian rhythms. Using a bioluminescent reporter (Per2::luc) to measure circadian rhythms, we determined that IP₆ increased rhythm amplitude and shortened period in NIH3T3 cells. The IP₆ effect on amplitude was attenuated by selective siRNA knockdown of GSK3B and pharmacological blockade of AKT kinase. However, unlike lithium, IP₆ did not induce serine-9 phosphorylation of GSK3B. The synthesis of IP₆ involves the enzymes inositol polyphosphate multikinase (IPMK) and inositol pentakisphosphate 2-kinase (IPPK). Knockdown of Ippk had effects opposite to those of IP₆, decreasing rhythm amplitude and lengthening period. Ipmk knockdown had few effects on rhythm alone, but attenuated the effects of lithium on rhythms. However, lithium did not change the intracellular content of IP₆ in NIH3T3 cells or neurons. Pharmacological inhibition of the IP₆ kinases (IP6K) increased rhythm amplitude and shortened period, suggesting secondary effects of inositol pyrophosphates may underlie the period shortening effect, but not the amplitude increasing effect of IP₆. Overall, we conclude that inositol phosphates, in particular IP₆ have effects on circadian rhythms. Manipulations affecting IP₆ and related inositol phosphates may offer a novel means through which circadian rhythms can be regulated.     

Preparation of quality inositol pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP(5;)) and inositol hexakisphosphate (phytic acid or IP(6;)). IP(5;) and IP(6;) are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds(1). Phosphorylation of IP(6;) generates diphoshoinositolpentakisphosphate (IP(7;) or PP-IP(5;)) and bisdiphoshoinositoltetrakisphosphate (IP(8;) or (PP)(2;)-IP(4;)). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution(2-4). The IP(6;) kinases (IP(6;)Ks) posses an enormous catalytic flexibility, converting IP(5;) and IP(6;) to PP-IP(4;) and IP(7;) respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules(5,6). Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP(1;) (also referred as PP-IP(5;)K), which is able to convert IP(6;) to IP(7;) and IP(8;)(7,8). Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion(9), telomere length(10,11), chemotaxis(12), vesicular trafficking(13), phosphate homeostasis(14) and HIV-1 gag release(15). Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT(16). Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins(17). The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus(18,19). These procedures use acidic conditions that might lead to inositol pyrophosphate degradation(20) and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories. In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP(6;)-kinase and PP-IP(5;)-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates(20). Following IP(6;)K1 and PP-IP(5;)K enzymatic reactions using IP(6;) as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.     

Integration of inositol phosphate signaling pathways via human ITPK1

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role.     

Expression of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) and its role in neural tube defects

ITPK1 is the rate-limiting enzyme in the pathway leading to formation of the highly phosphorylated inositol phosphates including IP₆ and the inositol pyrophosphates. One or more of these metabolites are essential for life as deletion of either of the kinases that form IP₅ or IP₆ in mice results in embryonic lethality. We have produced mice harboring a hypomorphic allele for Itpk1, and mice homozygous for this gene trap allele produce low but detectable levels of active enzyme. We have studied the expression of Itpk1 in various tissues and found that the enzyme is highly expressed in smooth muscle of vessels and other tissues. In addition, these mice have neural tube defects in 12% of homozygous embryos. Since the levels of enzyme expression vary greatly in homozygous animals, we speculate that relative deficiency of one or more inositol phosphates accounts for these defects. We plan to feed an inositol deficient diet or one with supplemental inositol to animals to demonstrate altered prevalence of neural tube defects.     

Analysis of Dictyostelium discoideum Inositol Pyrophosphate Metabolism by Gel Electrophoresis

The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP₆ or Phytic acid) and its derivative inositol pyrophosphates, IP₇ and IP₈. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP₉ in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP₅) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP₈ was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP₅K, doesn’t’ play a major role in the IP₈ developmental increase.     

Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP₇, is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP₇ regulation in β-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP₇. Although the inhibitors have diverse targets, they all perturbed IP₇ levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP₇ levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high K_m for ATP makes IP₇ synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP₇ concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP₇ indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic β-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP₇’s role in cellular signal transduction is likely to have been underestimated.     

A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine

Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO₂) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP₆ (phytate), its pyrophosphate derivatives InsP₇ and InsP₈, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP₆, InsP₇ and InsP₈ were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO₂ bead purification also enabled us to quantify InsP₆ in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP₆ phosphatase in human plasma, and secondly, InsP₆ is undetectable in either fluid. These observations seriously question reports that InsP₆ is present in human biofluids and the advisability of using InsP₆ as a dietary supplement.     

Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants

Inositol pyrophosphates are unique cellular signaling molecules with recently discovered roles in energy sensing and metabolism. Studies in eukaryotes have revealed that these compounds have a rapid turnover, and thus only small amounts accumulate. Inositol pyrophosphates have not been the subject of investigation in plants even though seeds produce large amounts of their precursor, myo‐inositol hexakisphosphate (InsP₆). Here, we report that Arabidopsis and maize InsP₆ transporter mutants have elevated levels of inositol pyrophosphates in their seed, providing unequivocal identification of their presence in plant tissues. We also show that plant seeds store a little over 1% of their inositol phosphate pool as InsP₇ and InsP₈. Many tissues, including, seed, seedlings, roots and leaves accumulate InsP₇ and InsP₈, thus synthesis is not confined to tissues with high InsP₆. We have identified two highly similar Arabidopsis genes, AtVip1 and AtVip2, which are orthologous to the yeast and mammalian VIP kinases. Both AtVip1 and AtVip2 encode proteins capable of restoring InsP₇ synthesis in yeast mutants, thus AtVip1 and AtVip2 can function as bonafide InsP₆ kinases. AtVip1 and AtVip2 are differentially expressed in plant tissues, suggesting non‐redundant or non‐overlapping functions in plants. These results contribute to our knowledge of inositol phosphate metabolism and will lay a foundation for understanding the role of InsP₇ and InsP₈ in plants.     

Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Inositol phosphates (IPs) and their turnover products have been implicated to play important roles in stress signaling in eukaryotic cells. In higher plants genes encoding inositol polyphosphate kinases have been identified previously, but their physiological functions have not been fully resolved. Here we expressed Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2β) in two heterologous systems, i.e. the yeast Saccharomyces cerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance. Expression of AtIpk2β rescued the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82Δ) that lacks inositol polyphosphate multikinase activity encoded by the ARG82/IPK2 gene. Transgenic tobacco plants constitutively expressing AtIpk2β under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants. Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2β in plant stress responses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Jasmonic acid perception by COI1 involves inositol polyphosphates in Arabidopsis thaliana

Plant responses to wounding are part of their defense responses against insects, and are tightly regulated. The isoleucin conjugate of jasmonic acid (JA‐Ile) is a major regulatory molecule. We have previously shown that inositol polyphosphate signals are required for defense responses in Arabidopsis; however, the way in which inositol polyphosphates contribute to plant responses to wounding has so far remained unclear. Arabidopsis F‐box proteins involved in the perception of JA‐Ile (COI1) and auxin (TIR1) are structurally similar. Because TIR1 has recently been shown to contain inositol hexakisphosphate (InsP₆) as a co‐factor of unknown function, here we explored the possibility that InsP₆ or another inositol polyphosphate is required for COI1 function. In support of this hypothesis, COI1 variants with changes in putative inositol polyphosphate coordinating residues exhibited a reduced interaction with the COI1 target, JAZ9, in yeast two‐hybrid tests. The equivalent COI1 variants displayed a reduced capability to rescue jasmonate‐mediated root growth inhibition or silique development in Arabidopsis coi1 mutants. Yeast two‐hybrid tests using wild‐type COI1 in an ipk1Δ yeast strain exhibiting increased levels of inositol pentakisphosphate (InsP₅) and reduced levels of InsP₆ indicate an enhanced COI1/JAZ9 interaction. Consistent with these findings, Arabidopsis ipk1‐1 mutants, also with increased InsP₅ and reduced InsP₆ levels, showed increased defensive capabilities via COI1‐mediated processes, including wound‐induced gene expression, defense against caterpillars or root growth inhibition by jasmonate. The combined data from experiments using mutated COI1 variants, as well as yeast and Arabidopsis backgrounds altered in inositol polyphosphate metabolism, indicate that an inositol polyphosphate, and probably InsP₅, contributes to COI1 function.     

Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases

Eukaryotes possess numerous inositol phosphate (IP) and diphosphoinositol phosphate (PP-IPs or inositol pyrophosphates) species that act as chemical codes important for intracellular signaling pathways. Production of IP and PP-IP molecules occurs through several classes of evolutionarily conserved inositol phosphate kinases. Here we report the characterization of a human inositol hexakisphosphate (IP6) and diphosphoinositol pentakisphosphate (PP-IP5 or IP7) kinase with similarity to the yeast enzyme Vip1, a recently identified IP6/IP7 kinase (Mulugu, S., Bai, W., Fridy, P. C., Bastidas, R. J., Otto, J. C., Dollins, D. E., Haystead, T. A., Ribeiro, A. A., and York, J. D. (2007) Science 316, 106-109). Recombinant human VIP1 exhibits in vitro IP6 and IP7 kinase activities and restores IP7 synthesis when expressed in mutant yeast. Expression of human VIP1 in HEK293T cells engineered to produce high levels of IP7 results in dramatic increases in bisdiphosphoinositol tetrakisphosphate (PP2-IP4 or IP8). Northern blot analysis indicates that human VIP1 is expressed in a variety of tissues and is enriched in skeletal muscle, heart, and brain. The subcellular distribution of tagged human VIP1 is indicative of a cytoplasmic non-membrane localization pattern. We also characterized human and mouse VIP2, an additional gene product with nearly 90% similarity to VIP1 in the kinase domain, and observed both IP6 and IP7 kinase activities. Our data demonstrate that human VIP1 and VIP2 function as IP6 and IP7 kinases that act along with the IP6K/Kcs1-class of kinases to convert IP6 to IP8 in mammalian cells, a process that has been found to occur in response to various stimuli and signaling events.