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Phosphate (Pi) serves countless metabolic pathways and is involved in macromolecule synthesis, energy storage, cellular signaling, and bone maintenance. Herein, we describe the coordination of Pi uptake and efflux pathways to maintain mammalian cell Pi homeostasis. We discover that XPR1, the presumed Pi efflux transporter, separately supervises rates of Pi uptake. This direct, regulatory interplay arises from XPR1 being a binding partner for the Pi uptake transporter PiT1, involving a predicted transmembrane helix/extramembrane loop in XPR1, and its hitherto unknown localization in a subset of intracellular LAMP1-positive puncta (named "XLPVs"). A pharmacological mimic of Pi homeostatic challenge is sensed by the inositol pyrophosphate IP 8 , which functionalizes XPR1 to respond in a temporally hierarchal manner, initially adjusting the rate of Pi efflux, followed subsequently by independent modulation of PiT1 turnover to reset the rate of Pi uptake. These observations generate a unifying model of mammalian cellular Pi homeostasis, expanding opportunities for therapeutic intervention. [Display omitted] • Organellar XPR1 regulates cellular Pi fluxes • XPR1 physically interacts with PiT1 to temporally coordinate Pi uptake and Pi efflux • The IP 8 /XPR1 signaling axis determines PiT1 protein expression and hence Pi uptake Li et al. demonstrate that organellar XPR1 regulates inorganic phosphate fluxes in mammalian cells. Control of phosphate influx by XPR1 is mediated through its interaction with the phosphate transporter PiT1 that prevents PiT1 degradation. The signaling molecule IP 8 functionalizes XPR1-dependent regulation of PiT1 expression to supervise cellular phosphate homeostasis. [ABSTRACT FROM AUTHOR]

The role of phosphinositide metabolism, an essential role, in synaptic vesicle recycling is discussed relative to results of a study. Synaptojanin 1-deficient mice were found to show neurological defects and die soon after birth. In neurons of mutants, clathrin-coated vesicles accumulate int he cytomatrix-rich area surrounding the synaptic vesicle cluster in nerve endings.

Abstract The protein kinase family is characterized by substantial conservation of architectural elements that are required for both ATP binding and phosphotransferase activity. Many of these structural features have also been identified in homologous enzymes that phosphorylate a variety of alternative, non-protein substrates. A comparative structural analysis of these different kinase sub-classes is a portal to a greater understanding of reaction mechanisms, enzyme regulation, inhibitor-development strategies, and superfamily-level evolutionary relationships. To serve such advances, we review structural elements of the protein kinase fold that are conserved in the subfamily of inositol phosphate kinases (InsPKs) that share a PxxxDxKxG catalytic signature: inositol 1,4,5-trisphosphate kinase (IP3K), inositol hexakisphosphate kinase (IP6K), and inositol polyphosphate multikinase (IPMK). We describe conservation of the fundamental two-lobe kinase architecture: an N-lobe constructed upon an anti-parallel β-strand scaffold, which is coupled to a largely helical C-lobe by a single, adenine-binding hinge. This equivalency also includes a G-loop that embraces the β/γ-phosphates of ATP, a transition-state stabilizing residue (Lys/His), and a Mg-positioning aspartate residue within a catalytic triad. Furthermore, we expand this list of conserved structural features to include some not previously identified in InsPKs: a 'gatekeeper' residue in the N-lobe, and an 'αF'-like helix in the C-lobe that anchors two structurally-stabilizing, hydrophobic spines, formed from non-consecutive residues that span the two lobes. We describe how this wide-ranging structural homology can be exploited to develop lead inhibitors of IP6K and IPMK, by using strategies similar to those that have generated ATP-competing inhibitors of protein-kinases. We provide several examples to illustrate how such an approach could benefit human health. [ABSTRACT FROM AUTHOR]

Human inositol phosphate multikinase (HsIPMK) critically contributes to intracellular signaling through its inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 3-kinase activities. This catalytic profile is not conserved; orthologs from Arabidopsis thaliana and Saccharomyces cerevisiae are predominantly Ins(1,4,5)P3 6-kinases, and the plant enzyme cannot phosphorylate PtdIns(4,5)P2. Therefore, crystallographic analysis of the yeast and plant enzymes, without bound inositol phosphates, do not structurally rationalize HsIPMK activities. Here, we present 1.6-Å resolution crystal structures of HsIPMK in complex with either Ins(1,4,5)P3 or PtdIns(4,5)P2. The Ins(1,4,5)P3 headgroup of PtdIns(4,5)P2 binds in precisely the same orientation as free Ins(1,4,5)P3 itself, indicative of evolutionary optimization of 3-kinase activities against both substrates. We report on nucleotide binding between the separate N- and C-lobes of HsIPMK. The N-lobe exhibits a remarkable degree of conservation with protein kinase A (root mean square deviation = 1.8 Å), indicating common ancestry. We also describe structural features unique to HsIPMK. First, we observed a constrained, horseshoeshaped substrate pocket, formed from an α-helix, a 310 helix, and a recently evolved tri-proline loop. We further found HsIPMK activities rely on a preponderance of Gln residues, in contrast to the larger Lys and Arg residues in yeast and plant orthologs. These conclusions are supported by analyzing 14 single-site HsIPMK mutants, some of which differentially affect 3-kinase and 6-kinase activities. Overall, we structurally rationalize phosphorylation of Ins(1,4,5)P3 and PtdIns(4,5)P2 by HsIPMK. [ABSTRACT FROM AUTHOR]

The inositol pyrophosphates (diphosphoinositol polyphosphates), which include 1-InsP 7 , 5-InsP 7 , and InsP 8 , are highly ‘energetic’ signaling molecules that play important roles in many cellular processes, particularly with regards to phosphate and bioenergetic homeostasis. Two classes of kinases synthesize the PP-InsPs: IP6Ks and PPIP5Ks. The significance of the IP6Ks - and their 5-InsP 7 product - has been widely reported. However, relatively little is known about the biological significance of the PPIP5Ks. The purpose of this review is to provide an update on developments in our understanding of key features of the PPIP5Ks, which we believe strengthens the hypothesis that their catalytic activities serve important cellular functions. Central to this discussion is the recent discovery that the PPIP5K is a rare example of a single protein that catalyzes a kinase/phosphatase futile cycle. [ABSTRACT FROM AUTHOR]

The inositol pyrophosphates (PP-InsPs) are a specialized group of “energetic” signaling molecules found in yeasts, plants and animals. PP-InsPs boast the most crowded three dimensional phosphate arrays found in Nature; multiple phosphates and diphosphates are crammed around the six-carbon, inositol ring. Yet, phosphate esters are also a major energy currency in cells. So the synthesis of PP-InsPs, and the maintenance of their levels in the face of a high rate of ongoing turnover, all requires significant bioenergetic input. What are the particular properties of PP-InsPs that repay this investment of cellular energy? Potential answers to that question are discussed here, against the backdrop of a recent hypothesis that signaling by PP-InsPs is evolutionarily ancient. The latter idea is extended herein, with the proposal that the primordial origins of PP-InsPs is reflected in the apparent lack of isomeric specificity of certain of their actions. Nevertheless, there are other aspects of signaling by these polyphosphates that are more selective for a particular PP-InsP isomer. Consideration of the nature of both specific and non-specific effects of PP-InsPs can help rationalize why such molecules possess so many phosphates. [ABSTRACT FROM AUTHOR]

Summary: Dietary InsP6 can modulate eukaryotic cell proliferation and has complex nutritive consequences, but its metabolism in the mammalian gastrointestinal tract is poorly understood. Therefore, we performed phylogenetic analyses of the gastrointestinal microbiome in order to search for candidate InsP6 phosphatases. We determined that prominent gut bacteria express homologs of the mammalian InsP6 phosphatase (MINPP) and characterized the enzyme from Bacteroides thetaiotaomicron (BtMinpp). We show that BtMinpp has exceptionally high catalytic activity, which we rationalize on the basis of mutagenesis studies and by determining its crystal structure at 1.9 Å resolution. We demonstrate that BtMinpp is packaged inside outer membrane vesicles (OMVs) protecting the enzyme from degradation by gastrointestinal proteases. Moreover, we uncover an example of cross-kingdom cell-to-cell signaling, showing that the BtMinpp-OMVs interact with intestinal epithelial cells to promote intracellular Ca2+ signaling. Our characterization of BtMinpp offers several directions for understanding how the microbiome serves human gastrointestinal physiology. [Copyright &y& Elsevier]

Abstract: The diphosphoinositol polyphosphates (“inositol pyrophosphates”; PP-InsPs) regulate many cellular processes in eukaryotes, including stress responses, apoptosis, vesicle trafficking, cytoskeletal dynamics, exocytosis, telomere maintenance, insulin signaling and neutrophil activation. Thus, the enzymes that control the metabolism of the PP-InsPs serve important cell signaling roles. In order to fully characterize how these enzymes are regulated, we need to determine the atomic-level architecture of their active sites. Only then can we fully appreciate reaction mechanisms and their modes of regulation. In this review, we summarize published information obtained from the structural analysis of a human diphosphoinositol polyphosphate phosphohydrolase (DIPP), and a human diphosphoinositol polyphosphate kinase (PPIP5K). This work includes the analysis of crystal complexes with substrates, products, transition state analogs, and a novel phosphonoacetate substrate analog. [Copyright &y& Elsevier]

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, IP 7 , is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP 7 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 IP 7 . Although the inhibitors have diverse targets, they all perturbed IP 7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP 7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high K m for ATP makes IP 7 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 IP 7 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 IP 7 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, IP 7 ’s role in cellular signal transduction is likely to have been underestimated. [ABSTRACT FROM AUTHOR]

Growth-blocking peptide (GBP), an insect cytokine, was first found in armyworm Mythimna separata. A functional analogue of GBP, stress-responsive peptide (SRP), was also identified in the same species. SRP gene expression has been demonstrated to be enhanced by GBP, indicating that both cytokines are organized within a hierarchical regulatory network. Although GBP1 (CG15917) and GBP2 (CG11395) have been identified in Drosophila melanogaster , immunological functions have only been characterized for GBP1. It is expected that the biological responses of two structurally similar peptides should be coordinated, but there is little information on this topic. Here, we demonstrate that GBP2 replicates the GBP1-mediated cellular immune response from Drosophila S2 cells. Moreover, the GBP2-induced response was silenced by pre-treatment with dsRNA targeting the GBP receptor gene, Mthl10. Furthermore, treatment of S2 cells with GBP2 enhanced GBP1 expression levels, but GBP1 did not affect GBP2 expression. GBP2 derived enhancement of GBP1 expression was not observed in the presence of GBP1, indicating that GBP2 is an upstream expressional regulator of a GBP1/GBP2 cytokine network. GBP2-induced enhancement of GBP1 expression was not observed in Mthl10 knockdown cells. Enhancement of GBP2 expression was observed in both Drosophila larvae and S2 cells under heat stress conditions; expressional enhancement of both GBP1 and GBP2 was eliminated in Mthl10 knockdown cells and larvae. Finally, Ca2+ mobilization assay in GCaMP3-expressing S2 cells demonstrated that GBP2 mobilizes Ca2+ upstream of Mthl10. Our finding revealed that Drosophila GBP1 and GBP2 control immune responses as well as their own expression levels through a hierarchical cytokine network, indicating that Drosophila GBP1/GBP2 system can be a simple model that is useful to investigate the detailed regulatory mechanism of related cytokine complexes. [Display omitted] • Drosophila GBP2 induces the GBP1-like immune response through the GBP1 receptor. • GBP2 enhanced GBP1 mRNA levels, but GBP1 did not affect GBP2 mRNA levels. • GBP2 is an upstream regulator of a GBP1/GBP2 cytokine network. • Drosophila GBP2 and GBP1 are orthologs of armyworm GBP and SRP, respectively. [ABSTRACT FROM AUTHOR]

Abstract: Genetic manipulation of diphosphoinositol polyphosphate synthesis impacts many biological processes (reviewed in S.B. Shears, Biochem. J. 377, 2004, 265–280). These observations lacked a cell-signalling context, until the recent discovery that bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4 or “InsP8”) accumulates rapidly in mammalian cells in response to hyperosmotic stress (X. Pesesse, K. Choi, T. Zhang, and S. B. Shears J. Biol. Chem. 279, 2004, 43378–43381). We now investigate how widely applicable is this new stress-response. [PP]2-InsP4 did not respond to mechanical strain or oxidative stress in mammalian cells. Furthermore, despite tight conservation of many molecular stress responses across the phylogenetic spectrum, we show that cellular [PP]2-InsP4 levels do not respond significantly to osmotic imbalance, heat stress and salt toxicity in Saccharomyces cerevisiae. In contrast, we show that [PP]2-InsP4 is a novel sensor of mild thermal stress in mammalian cells: [PP]2-InsP4 levels increased 3–4 fold when cells were cooled from 37 to 33 °C, or heated to 42 °C. Increases in [PP]2-InsP4 levels following heat-shock were evident <5 min, and reversible (t 1/2 =7 min) once cells were returned to 37 °C. These responses were blocked by pharmacological inhibition of the ERK/MEK pathway. Additional control processes may lie upstream of [PP]2-InsP4 synthesis, which was synergistically activated when heat stress and osmotic stress were combined. Our data add to the repertoire of signaling responses following thermal challenges, a topic of current interest for its possible therapeutic value. [Copyright &y& Elsevier]

The inositol pyrophosphates (PP-IPs) are specialized members of the wider inositol phosphate signaling family that possess functionally significant diphosphate groups. The PP-IPs exhibit remarkable functionally versatility throughout the eukaryotic kingdoms. However, a quantitatively minor PP-IP – 1,5 bisdiphosphoinositol tetrakisphosphate (1,5-IP 8) – has received considerably less attention from the cell signalling community. The main purpose of this review is to summarize recently-published data which have now brought 1,5-IP 8 into the spotlight, by expanding insight into the molecular mechanisms by which this polyphosphate regulates many fundamental biological processes. [ABSTRACT FROM AUTHOR]

Diphosphoinositol pentakisphosphate (IP 7 ) 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 IP 7 -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 IP 7 concentration in β-cells. RNAi mediated knock down of the IP6K1 isoform inhibits both glucose-mediated increase in IP 7 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 IP 7 generation and insulin release. Thus the unique metabolic sensing properties of IP6K1 guarantees appropriate concentrations of IP 7 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 IP 7 generation may be an essential convergence point integrating multiple metabolic defects into the commonly observed phenotype in diabetes. [ABSTRACT FROM AUTHOR]

Inositol pyrophosphates (PP-InsPs) are "energetic" intracellular signals that are ubiquitous in animals, plants, and fungi; structural and biochemical characterization of PP-InsP metabolic enzymes provides insight into their evolution, reaction mechanisms, and regulation. Here, we describe the 2.35-Å-resolution structure of the catalytic core of Siw14, a 5-PP-InsP phosphatase from Saccharomyces cerevisiae and a member of the protein tyrosine-phosphatase (PTP) superfamily. Conclusions that we derive from structural data are supported by extensive site-directed mutagenesis and kinetic analyses, thereby attributing new functional significance to several key residues. We demonstrate the high activity and exquisite specificity of Siw14 for the 5-diphosphate group of PP-InsPs. The three structural elements that demarcate a 9.2-Å-deep substrate-binding pocket each have spatial equivalents in PTPs, but we identify how these are specialized for Siw14 to bind and hydrolyze the intensely negatively charged PP-InsPs. (a) The catalytic P-loop with the CX5R(S/T) PTP motif contains additional, positively charged residues. (b) A loop between the β5 and α6 helices, corresponding to the Q-loop in PTPs, contains a lysine and an arginine that extend into the catalytic pocket due to displacement of the-5 helix orientation through intramolecular crowding caused by three bulky, hydrophobic residues. (c) The general-acid loop in PTPs is replaced in Siw14 with a flexible loop that does not use an aspartate or glutamate as a general acid. We propose that an acidic residue is not required for phosphoanhydride hydrolysis. [ABSTRACT FROM AUTHOR]

Proteins responsible for Pi homeostasis are critical for all life. In Saccharomyces cerevisiae, extracellular [Pi] is "sensed" by the inositol-hexakisphosphate kinase (IP6K) that synthesizes the intracellular inositol pyrophosphate 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) as follows: during a period of Pi starvation, there is a decline in cellular [ATP]; the unusually low affinity of IP6Ks for ATP compels 5-InsP7 levels to fall in parallel (Azevedo, C., and Saiardi, A. (2017) Trends. Biochem. Sci. 42, 219-231. Hitherto, such Pi sensing has not been documented in metazoans. Here, using a human intestinal epithelial cell line (HCT116), we show that levels of both 5-InsP7 and ATP decrease upon [Pi] starvation and subsequently recover during Pi replenishment. However, a separate inositol pyrophosphate, 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8), reacts more dramatically (i.e. with a wider dynamic range and greater sensitivity). To understand this novel InsP8 response, we characterized kinetic properties of the bifunctional 5-InsP7 kinase/InsP8 phosphatase activities of full-length diphosphoinositol pentakisphosphate kinases (PPIP5Ks). These data fulfil previously published criteria for any bifunctional kinase/phosphatase to exhibit concentration robustness, permitting levels of the kinase product (InsP8 in this case) to fluctuate independently of varying precursor (i.e. 5-InsP7) pool size. Moreover, we report that InsP8 phosphatase activities of PPIP5Ks are strongly inhibited by Pi (40-90% within the 0-1 mM range). For PPIP5K2, Pi sensing by InsP8 is amplified by a 2-fold activation of 5-InsP7 kinase activity by Pi within the 0-5 mM range. Overall, our data reveal mechanisms that can contribute to specificity in inositol pyrophosphate signaling regulating InsP8 turnover independently of 5-InsP7, in response to fluctuations in extracellular supply of a key nutrient. [ABSTRACT FROM AUTHOR]

We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional [sup1]H-[sup1]H and [sup1]H-[sup31]P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)[sub2]-IP[sub4] is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)[sub2]-IP[sub4] (Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates. [ABSTRACT FROM AUTHOR]

We demonstrate that physiologically relevant perturbations in the osmotic environment rheostatically regulate a gatekeeping function for the nucleolus that controls the spatial dynamics and functions of nucleolin. HeLa cells and U2-OS osteosarcoma cells were osmotically challenged with 100-200 mr~i sorbitol, and the intranuclear distribution of nucleolin was monitored by confocal microscopy. Nucleolin that normally resides in the innermost fibrillar core of the nucleolus, where it assists rDNA transcription and replication, was expelled within 30 mm of sorbitol addition, The nucleolin was transferred into the nucleo- plasm, but it distributed there non-uniformly; locally high levels accumulated in 4',6-diamidino-2-phenylindole-negative zones containing euchromatic (transcriptionally active) DNA. Inositol pyrophosphates also responded within 30 mm of hyperosmotic stress: levels of bisdiphosphoinositol tetrakisphosphate in- creased 6-fold, and this was matched by decreased levels of its precursor, diphosphoinositol pentakisphosphate. Such fluctuations in inositol pyrophosphate levels are of considerable interest, because, according to previously published in vitro data, they reg- ulate the degree of phosphorylation of nucleolin through a novel kinase-independent phosphotransferase reaction (Saiardi, A., Bhandari, A., Resnick, R., Cain, A., Snowman, A. M., and Snyder, 5. H. (2004) Science 306, 2101-2105). However, by pharmacologically intervening in inositol pyrophosphate metabolism, we found that it did not supervise the osmotically driven switch in the biological activities of nucleolin in vivo. [ABSTRACT FROM AUTHOR]

Mammalian cells utilize multiple signaling mechanisms to protect against the osmotic stress that accompanies plasma membrane ion transport, solute uptake, and turnover of protein and carbohydrates (Schliess, F., and Haussinger, D. (2002) Biol. Chem. 383, 577–583). Recently, osmotic stress was found to increase synthesis of bisdiphosphoinositol tetrakisphosphate ((PP)2-InsP4), a high energy inositol pyrophosphate (Pesesse, X., Choi, K., Zhang, T., and Shears, S. B. (2004)]. Biol. Chem. 279, 43378-43381). Here, we describe the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIPSK) that synthesizes (PP)2-InsP4. Partial amino acid sequence, obtained by mass spectrometry, matched the sequence of a 160-kDa rat protein containing a putative ATPgrasp kinase domain. BLAST searches uncovered two human isoforms (PPIP5K1 (160 kDa) and PPIPSK2 (138 kDa)). Recombinant human PPIPSK1, expressed in Escherichia coli, was found to phosphorylate diphosphoinositol pentakisphosphate (PP-lnsP5) to (PP)2-InsP4 (Vmax = 8.3 nmol/mg of protein/min; Km = 0.34 μM). Overexpression in human embryonic kidney cells of either PPIPSK1 or PPIPSK2 substantially increased levels of (PP)2-InsP4, whereas overexpression of a catalytically dead PPIPSK1D332A mutant had no effect. PPIPSK1 and PPIP5K2 were more active against PP-InsP5 than InsP6, both in vitro and in vivo. Analysis by confocal immunofluorescence showed PPIPSK1 to be distributed throughout the cytoplasm but excluded from the nucleus. Immunopurification of overexpressed PPIPSK1 from osmotically stressed HEK cells (0.2 M sorbitol; 30 min) revealed a persistent, 3.9 ± 0.4-fold activation when compared with control cells. PPlPSKs are likely to be important signaling enzymes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: To understand how a signaling molecule''s activities are regulated, we need insight into the processes controlling the dynamic balance between its synthesis and degradation. For the Ins(1,3,4,5,6)P5 signal, this information is woefully inadequate. For example, the only known cytosolic enzyme with the capacity to degrade Ins(1,3,4,5,6)P5 is the tumour-suppressor PTEN [J.J. Caffrey, T. Darden, M.R. Wenk, S.B. Shears, FEBS Lett. 499 (2001) 6 ], but the biological relevance has been questioned by others [E.A. Orchiston, D. Bennett, N.R. Leslie, R.G. Clarke, L. Winward, C.P. Downes, S.T. Safrany, J. Biol. Chem. 279 (2004) 1116 ]. The current study emphasizes the role of physiological levels of PTEN in Ins(1,3,4,5,6)P5 homeostasis. We employed two cell models. First, we used a human U87MG glioblastoma PTEN-null cell line that hosts an ecdysone-inducible PTEN expression system. Second, the human H1299 bronchial cell line, in which PTEN is hypomorphic due to promoter methylation, has been stably transfected with physiologically relevant levels of PTEN. In both models, a novel consequence of PTEN expression was to increase Ins(1,3,4,5,6)P5 pool size by 30–40% (p <0.01); this response was wortmannin-insensitive and, therefore, independent of the PtdIns 3-kinase pathway. In U87MG cells, induction of the G129R catalytically inactive PTEN mutant did not affect Ins(1,3,4,5,6)P5 levels. PTEN induction did not alter the expression of enzymes participating in Ins(1,3,4,5,6)P5 synthesis. Another effect of PTEN expression in U87MG cells was to decrease InsP6 levels by 13% (p <0.02). The InsP6-phosphatase, MIPP, may be responsible for the latter effect; we show that recombinant human MIPP dephosphorylates InsP6 to d/l-Ins(1,2,4,5,6)P5, levels of which increased 60% (p <0.05) following PTEN expression in U87MG cells. Overall, our data add higher inositol phosphates to the list of important cellular regulators [Y. Huang, R.P. Wernyj, D.D. Norton, P. Precht, M.C. Seminario, R.L. Wange, Oncogene, 24 (2005) 3819 ] the levels of which are modulated by expression of the highly pleiotropic PTEN protein. [Copyright &y& Elsevier]

In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca2+i) mobilization. We have shown that Ca2+i signals resulting from apical purinoceptor (P2Y2-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y2-R-dependent Ca2+i signals in CF human airway epithelia. We show that the increased Ca2+i mobilization in CF was not specific to P2Y2-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y2-R or a more efficient coupling between P2Y2-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y2-R activation-promoted Ca2+i signals in CF epithelia resulted from an increased density and Ca2+i storage capacity of apically confined endoplasmic reticulum (ER) Ca2+i stores. To address whether the ER up-regulation resulted from ER retention of misfolded ΔF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of ΔF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, ΔF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y2-R activation-mobilized Ca2+i, which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether luminal infection/inflammation triggers an up-regulation of the apically confined ER 2-f . . Ca stores, normal airway epithelia were chronically exposed to supernatant from mucopurulent material from CF airways. Supernatant treatment expanded the apically confined ER, resulting in larger apical P2Y2-R 2+ activation-dependent Ca2+i responses, which reproduced the increased Ca2+i signals observed in CF epithelia. In conclusion, the mechanism for the larger Ca2+i signals elicited by apical P2Y2-R activation in CF airway epithelia is an expansion of the apical ER Ca2+i stores triggered by chronic luminal airway infection/inflammation. Greater ER-derived Ca2+i, signals may provide a compensatory mechanism to restore, at least acutely, mucus clearance in CF airways. [ABSTRACT FROM AUTHOR]

Evidence has accumulated that inositol pyrophosphates (diphosphoinositol pentakisphosphate (PP-InsP5) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4)) are intracellular signals that regulate many cellular processes including endocytosis, vesicle trafficking, apoptosis, and DNA repair. Yet, in contrast to the situation with all other second messengers, no one studying multicellular organisms has previously described a stimulus that acutely and specifically elevates cellular levels of PP-InsP5 or [PP]2-InsP4. We now show up to 25-fold elevations in [PP]2-InsP4 levels in animal cells. Importantly, this does not involve classical agonists. Instead, we show that this [PP]2-InsP4 response is a novel consequence of the activation of ERK1/2 and p38MAPα/β kinases by hyperosmotic stress. JNK did not participate in regulating [PP]2-InsP4 levels. Identification of [PP]2-InsP4 as a sensor of hyperosmotic stress opens up a new area of research for studies into the cellular activities of higher inositol phosphates. [ABSTRACT FROM AUTHOR]

Multiple inositol polyphosphate phosphatase (MIPP) is an enzyme that, in vitro, has the interesting property of degrading higher inositol polyphosphates to the Ca[sup 2+] second messenger, inositol 1,4,5-trisphosphate (Ins(1,4,5)P[sub 3]), independently of inositol lipid breakdown. We hypothesized that a truncated cytosolic form of the largely endoplasmic reticulum-confined MIPP (cyt-MIPP) could represent an important new tool in the investigation of Ins(1,4,5) P[sub 3]dependent intracellular Ca[sup 2+] homeostasis. To optimize our ability to judge the impact of cyt-MIPP on intracellular Ca[sup 2+] concentration ([Ca[sup 2+]][sub i]) we chose a poorly responsive β-cell line (HIT M222.2) with an abnormally low [Ca[sup 2+]][sub i]. Our results show for the first time in an intact mammalian cell that cyt-MIPP expression leads to a significant enhancement of Ins(1,4,5)P[sub 3] concentration. This is achieved without a significant interference from other cyt-MIPP-derived inositol phosphates. Furthermore, the low basal [Ca[sup 2+]][sub i] of these cells was raised to normal levels (35 to 115 nM) when they expressed cyt-MIPP. Noteworthy is that the normal feeble glucose-induced Ca[sup 2+] response of HIT M2.2.2 cells was enhanced dramatically by mechanisms related to this increase in basal [Ca[sup 2+]][sub i]. These data support the use of cyt-MIPP as an important tool in investigating Ins(1,4,5)P[sub 3]dependent Ca[sup 2+] homeostasis and suggest a close link between Ins(1,4,5)P[sub 3] concentration and basal [Ca[sup 2+]][sub i], the latter being an important modulator of Ca[sup 2+] signaling in the pancreatic β-cell. [ABSTRACT FROM AUTHOR]

The inositol pyrophosphates (PP-InsPs) are a unique subgroup of intracellular signals with diverse functions, many of which can be viewed as reflecting an overarching role in metabolic homeostasis. Thus, considerable attention is paid to the enzymes that synthesize and metabolize the PP-InsPs. One of these enzyme families - the diphosphoinositol pentakisphosphate kinases (PPIP5Ks) - provides an extremely rare example of separate kinase and phosphatase activities being present within the same protein. Herein, we review the current state of structure/function insight into the PPIP5Ks, the separate specialized activities of the two metazoan PPIP5K genes, and we describe a phylogenetic analysis that places PPIP5K evolutionary origin within the Excavata, the very earliest of eukaryotes. These different aspects of PPIP5K biology are placed in the context of a single, overriding question. Why are they bifunctional: i.e., what is the particular significance of the ability to turn PP-InsP signaling on or off from two separate 'switches' in a single protein? [ABSTRACT FROM AUTHOR]

Processing of substrates by enzymes can only be fully understood through their conformational dynamics; this is particularly true for the diphosphoinositol pentakisphosphate kinase PPIP5K2, an enzyme with critical roles in cell signaling and bioenergetic homeostasis. PPIP5K2 is remarkable for the reversible nature of its kinase activity, its unique ligand-stimulated ATPase activity, and the substrate traveling between two ligand-binding sites. Here we use molecular dynamics and data analysis techniques to rationalize these PPIP5K2 activities, thereby increasing our understanding of complex enzymatic mechanisms. In particular, we demonstrate how the enzyme's distinctive, ratchet-like mechanism harnesses the energy of random fluctuations to significantly reduce the entropy toll for intramolecular substrate transfer. We show that pre-reaction pulling forces along the reaction coordinate are predictive of the various PPIP5K2 catalytic activities. An unexpected possibility, raised by these computational studies, that 3,5-IP8 might be a substrate for dephosphorylation was experimentally interrogated and confirmed in a luciferase assay. • Molecular dynamics was used to rationalize the catalytic versatility of PPIP5K2 • A distinctive ratchet mechanism is involved in intramolecular substrate transfer • Enzyme-substrate forces are predictive of the various PPIP5K2 catalytic activities • A new substrate was computationally predicted and experimentally confirmed Remarkable catalytic versatility of the pyrophosphate kinase PPIP5K2 can only be fully understood through its conformational plasticity. An et al. used molecular dynamics to study its enzymatic mechanisms. They revealed a distinctive ratchet-like mechanism of substrate transfer and showed how enzyme-substrate forces explain the enzyme's catalytic activities. [ABSTRACT FROM AUTHOR]