Search

Background: Bone hardness and strength depends on mineralization, which involves a complex process in which calcium phosphate, produced by bone-forming cells, was shed around the fibrous matrix. This process is strictly regulated, and a number of signal transduction systems were interested in calcium metabolism, such as the phosphoinositide (PI) pathway and related phospholipase C (PLC) enzymes. Objectives: Our aim was to search for common patterns of expression in osteoblasts, as well as in ES and SS. Methods: We analysed the PLC enzymes in human osteoblasts and osteosarcoma cell lines MG-63 and SaOS-2. We compared the obtained results to the expression of PLCs in samples of patients affected with Ewing sarcoma (ES) and synovial sarcoma (SS). Results: In osteoblasts, MG-63 cells and SaOS-2 significant differences were identified in the expression of PLC δ4 and PLC η subfamily isoforms. Differences were also identified regarding the expression of PLCs in ES and SS. Most ES and SS did not express PLCB1, which was expressed in most osteoblasts, MG-63 and SaOS-2 cells. Conversely, PLCB2, unexpressed in the cell lines, was expressed in some ES and SS. However, PLCH1 was expressed in SaOS-2 and inconstantly expressed in osteoblasts, while it was expressed in ES and unexpressed in SS. The most relevant difference observed in ES compared to SS regarded PLC ε and PLC η isoforms. Conclusion: MG-63 and SaOS-2 osteosarcoma cell lines might represent an inappropriate experimental model for studies about the analysis of signal transduction in osteoblasts

Four phospholipase C β (PLCB) isoforms, PLCB1, PLCB2, PLCB3 and PLCB4, have been previously investigated regarding their roles in the metabolism of inositol lipids and cancer. The present study aimed to explore the association between PLCB1-4 and hepatocellular carcinoma (HCC). Data from 212 patients with hepatitis B virus-associated HCC were used to analyze the diagnostic and prognostic significance of PLCB genes in. A nomogram predicted the survival probability. Gene set enrichment analysis explored gene ontology terms and the metabolic pathways associated with PLCB genes. Validation of the prognostic values of PLCB genes was performed using the Gene Expression Profiling Interactive Analysis website. PLCB1 and PLCB2 were revealed to have diagnostic value for HCC (0.869 and 0.836 area under the curve, respectively; both P≤0.05). The combination analysis of these genes had an advantage over each alone (0.905 PLCB1 and PLCB2, and 0.877 PLCB1 and PLCB3 area under the curve; P≤0.05). PLCB1 was associated with overall survival (OS) and recurrence-free survival (RFS; adjusted P=0.002 and P=0.001, respectively). A nomogram predicted survival probability of patients with HCC at 1, 3- and 5-years. Gene set enrichment analysis indicated that PLCB1 and PLCB2 are involved in the cell cycle, cell division and the PPAR signaling pathway, among other functions. Validation using GEPIA revealed that PLCB1 and PLCB2 were associated with OS and PLCB1 and PLCB4 were associated with RFS. PLCB1 and PLCB2 exhibited diagnostic value for HCC and their combination had an advantage over each individually. PLCB1 has OS and RFS prognostic value for patients with HCC.

SummaryPhospholipase C (PLC)-β2 (gene PLCB2) is a critical regulator of platelet responses upon activation. Mechanisms regulating of PLC-β2 expression in platelets/MKs are unknown. Our studies in a patient with platelet PLC-β2 deficiency revealed the PLCB2 coding sequence to be normal and decreased platelet PLC-β2 mRNA, suggesting a defect in transcriptional regulation. PLCB2 5’- upstream region of the patient revealed a heterozygous 13 bp deletion (-1645/-1633 bp) encompassing a consensus sequence for nuclear factor-κB (NF-κB). This was subsequently detected in three of 50 healthy subjects. To understand the mechanisms regulating PLC-β2, we studied the effect of this variation in the PLCB2. Gel-shift studies using nuclear extracts from human erythroleukaemia (HEL) cells or recombinant p65 showed NF-κB binding to oligonucleotide with NF-κB site; in luciferase reporter studies its deletion reduced PLCB2 promoter activity. PLCB2 expression was decreased by siRNA knockdown of NF-κB p65 subunit and increased by p65 overexpression. By immunoblotting platelet PLC-β2 in 17 healthy subjects correlated with p65 (r=0.76, p=0.0005). These studies provide the first evidence that NF-kB regulates MK/platelet PLC-β2 expression. This interaction is important because of the major role of PLC-β2 in platelet activation and of NF-κB in processes, including inflammation and atherosclerosis, where both are intimately involved.Supplementary Material to this article is available online at www.thrombosis-online.com.

The epsilon isoform of mammalian diacylglycerol kinase (DGKϵ) is an enzyme that associates strongly with membranes and acts on a lipid substrate, diacylglycerol. The protein has one segment that is predicted to be a transmembrane helix, but appears to interconvert between a transmembrane helix and a re-entrant helix. Despite the hydrophobicity of this segment and the fact that the lipid substrate is also hydrophobic, removal of this hydrophobic segment by truncating the protein at the amino terminus has no effect on its enzymatic activity. The amino acid sequence of the catalytic segment of DGKϵ is highly homologous to that of a bacterial DGK, DgkB. This has allowed us to predict a conformation of DGKϵ based on the known crystal structure of DgkB. An important property of DGKϵ is that it is specific for diacylglycerol species containing an arachidonoyl group. The region of DGKϵ that interacts with this group is found within the accessory domain of the protein and not in the active site nor in the hydrophobic amino terminus. The nature of the acyl chain specificity of the enzyme indicates that DGKϵ is associated with the synthesis of phosphatidylinositol. Defects or deletion of the enzyme give rise to several disease states.

Endothelial cells (EC) are the first elements exposed to mediators circulating in the bloodstream, and react to stimulation with finely tuned responses mediated by different signal transduction pathways, leading the endothelium to adapt. Neuropeptide Y (NPY), the most abundant peptide in heart and brain, is mainly involved in the neuroendocrine regulation of the stress response. The regulatory roles of NPY depend on many factors, including its enzymatic processing, receptor subtypes and related signal transduction systems, including the phosphoinositide (PI) pathway and related phospholipase C (PI-PLC) family of enzymes. The panel of expression of PI-PLC enzymes differs comparing quiescent versus differently stimulated human EC. Growing evidences indicate that the regulation of the expression of PLC genes, which codify for PI-PLC enzymes, might act as an additional mechanism of control of the PI signal transduction pathway. NPY was described to potentiate the activation of PI-PLC enzymes in different cell types, including EC. In the present experiments, we stimulated human umbilical vein EC using different doses of NPY in order to investigate a possible role upon the expression PLC genes. NPY reduced the overall transcription of PLC genes, excepting for PLCE. The most significant effects were observed for PLCB2 and PLCD1, both isoforms recruited by means of G-proteins and G-protein-coupled receptors. NPY behavior was comparable with other PI-PLC interacting molecules that, beside the stimulation of phospholipase activity, also affect the upcoming enzymes' production acting upon gene expression. That might represent a mode to regulate the activity of PI-PLC enzymes after activation.

Phosphatidylinositol 4-kinase type IIIα (PI4KA) is a host factor essential for hepatitis C virus replication and hence is a target for drug development. PI4KA has also been linked to endoplasmic reticulum exit sites and generation of plasma membrane phosphoinositides. Here, we developed highly specific and potent inhibitors of PI4KA and conditional knock-out mice to study the importance of this enzyme in vitro and in vivo. Our studies showed that PI4KA is essential for the maintenance of plasma membrane phosphatidylinositol 4,5-bisphosphate pools but only during strong stimulation of receptors coupled to phospholipase C activation. Pharmacological blockade of PI4KA in adult animals leads to sudden death closely correlating with the drug's ability to induce phosphatidylinositol 4,5-bisphosphate depletion after agonist stimulation. Genetic inactivation of PI4KA also leads to death; however, the cause in this case is due to severe intestinal necrosis. These studies highlight the risks of targeting PI4KA as an anti-hepatitis C virus strategy and also point to important distinctions between genetic and pharmacological studies when selecting host factors as putative therapeutic targets.

Although inositol pyrophosphates have diverse roles in phosphate signaling and other important cellular processes, little is known about their functions in the biosynthesis of inositol and phospholipids. Here, we show that KCS1, which encodes an inositol pyrophosphate kinase, is a regulator of inositol metabolism. Deletion of KCS1, which blocks synthesis of inositol pyrophosphates on the 5-hydroxyl of the inositol ring, causes inositol auxotrophy and decreased intracellular inositol and phosphatidylinositol. These defects are caused by a profound decrease in transcription of INO1, which encodes myo-inositol-3-phosphate synthase. Expression of genes that function in glycolysis, transcription, and protein processing is not affected in kcs1Δ. Deletion of OPI1, the INO1 transcription repressor, does not fully rescue INO1 expression in kcs1Δ. Both the inositol pyrophosphate kinase and the basic leucine zipper domains of KCS1 are required for INO1 expression. Kcs1 is regulated in response to inositol, as Kcs1 protein levels are increased in response to inositol depletion. The Kcs1-catalyzed production of inositol pyrophosphates from inositol pentakisphosphate but not inositol hexakisphosphate is indispensable for optimal INO1 transcription. We conclude that INO1 transcription is fine-tuned by the synthesis of inositol pyrophosphates, and we propose a model in which modulation of Kcs1 controls INO1 transcription by regulating synthesis of inositol pyrophosphates. Background: Regulation of inositol metabolism is crucial for cellular functions. Results: Inositol pyrophosphate-deficient cells exhibit defective inositol biosynthesis. Protein levels of the inositol pyrophosphate biosynthetic enzyme Kcs1 are dynamically altered in response to inositol. Conclusion: INO1 transcription and inositol biosynthesis are regulated by modulation of inositol pyrophosphate synthesis. Significance: Inositol pyrophosphates are novel regulators of biosynthesis of inositol and inositol phospholipids.

The molecular mechanisms by which receptors regulate the Ras Binding Domains of the PIP3-generating, class I PI3Ks remain poorly understood, despite their importance in a range of biological settings, including tumorigenesis, activation of neutrophils by pro-inflammatory mediators, chemotaxis of Dictyostelium and cell growth in Drosophila. We provide evidence that G protein-coupled receptors (GPCRs) can stimulate PLCb2/b3 and diacylglycerol- dependent activation of the RasGEF, RasGRP4 in neutrophils. The genetic loss of RasGRP4 phenocopies knock-in of a Ras-insensitive version of PI3Kc in its effects on PI3Kc-dependent PIP3 accumulation, PKB activation, chemokinesis and reactive oxygen species (ROS) formation. These results establish a new mechanism by which GPCRs can stimulate Ras, and the broadly important principle that PLCs can control activation of class I PI3Ks.

Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], produce the Ca 2+ -mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca 2+ . One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca 2+ , modestly activated by GTPγS in vitro , and inhibited by the compound U73122 [1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P 2 levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena . By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P 3 -Ca 2+ regulator y axis in ciliates.

Phosphatidylinositol is an important membrane lipid in Saccharomyces cerevisiae and other eukaryotes. Phosphatidylinositol and its metabolites (phosphoinositides, inositol polyphosphates, etc.) affect many cellular processes with implications in human diseases. Phosphatidylinositol synthesis in S. cerevisiae requires the essential PIS1 gene. Recent studies reveal that PIS1 expression is regulated at the level of transcription in response to carbon source, oxygen, and zinc. However, the consequence of this regulation on phosphatidylinositol levels and functions has not been thoroughly studied. To investigate this, we created a strain with a galactose-inducible GAL1-PIS1 gene. In this strain, the amount of phosphatidylinositol correlated with PIS1 expression but did not exceed c. 25% of the total phospholipid composition. Interestingly, we found that 4% phosphatidylinositol was sufficient for cell growth. We also found that reduced PIS1 expression yielded derepression of two phospholipid biosynthetic genes (INO1 and CHO1) and the INO2 regulatory gene. Consistent with this derepression, reduced PIS1 expression also yielded an overproduction of inositol (Opi(-)) phenotype. The effect on transcription of the INO1, CHO1, and INO2 genes is consistent with the accepted model that phosphatidic acid (PA) is the signal for regulation of these genes because decreased phosphatidylinositol synthesis would affect PA levels.

Gq family members of heterotrimeric G protein activate beta isoforms of phospholipase C that hydrolyzes phosphatidylinositol phosphate to diacylglycerol and inositol trisphosphate, leading to the protein kinase C activation and intracellular Ca(2+) mobilization, respectively. To understand the functions and regulatory mechanisms of Gq-signaling pathways, we first introduce the Galphaq-interacting proteins, which function as the effectors and the modulators of Gq. Next, we describe the Pasteurella multocida toxin and YM-254890, which are useful tools to investigate Gq signaling as activator and inhibitor, respectively. Finally, we discuss the physiological function of Gq in developmental brain, especially in neural progenitor cells.

The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca2+ or Mg2+ ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 °C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent Km = 6.5 mol %) and saturation kinetics with respect to CTP (apparent Km = 0.3 mm). dCTP was both a substrate (apparent Km = 0.4 mm) and competitive inhibitor (apparent Ki = 0.4 mm) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.

We investigated coupling of OX(1) receptors to phospholipase activation and diacylglycerol generation in Chinese hamster ovary (CHO) cells using both biochemical and fluorescence "real-time" methods. The results indicate that at lowest orexin-A concentrations (highest potency), diacylglycerol generated results from phospholipase D activity. At 10-100-fold higher orexin-A concentrations, phospholipase C is activated, likely hydrolyzing phosphatidylinositol (PI) or phosphatidylinositol monophosphate (PIP) but not phosphatidylinositol bisphosphate (PIP(2)). At further 7-fold higher orexin-A concentrations, PIP(2) is hydrolyzed, releasing both diacylglycerol and inositol-1,4,5-trisphosphate. Thus, OX(1) orexin receptors connect to multiple phospholipase activities, apparently composed of at least one phospholipase D and two different phospholipase C activities. At low agonist concentrations, diacylglycerol and phosphatidic acid are the preferred products, and interestingly, it seems that even the primarily activated phospholipase C mainly works to increase diacylglycerol and not inositol-1,4,5-trisphosphate.

The phosphatidylinositol phosphate signaling pathway is involved in many crucial cellular functions. The myo-inositol polyphosphate 5-phosphatases (5PTases) (E.C. 3.1.3.56) comprise a large protein family that hydrolyze 5-phosphates from a variety of phosphatidylinositol phosphate and inositol phosphate substrates. We previously reported that the At5PTase11 enzyme (At1g47510), which is one of the smallest predicted 5PTases found in any organism, encodes an active 5PTase whose activity is restricted to tris- and bis-, but not mono-phosphorylated phosphatidylinositol phosphate substrates containing a 5-phosphate. This is in contrast to other unrestricted Arabidopsis 5PTases, which also hydrolyze tris- and bis inositol phosphate molecules. To further explore the function of At5PTase11, we have characterized two T-DNA mutants in the At5PTase11 gene, and have complemented this mutant. Seed from 5ptase11 mutants germinate slower than wildtype seed and mutant seedlings have decreased hypocotyl growth as compared to wildtype seedlings when grown in the dark. This phenotype is the opposite of the increased hypocotyl growth phenotype previously described for other 5ptase mutants defective in inositol phosphate-specific 5PTase enzymes. By labeling the endogenous myo-inositol pool in 5ptase11 mutants, we correlated these hypocotyl growth changes with a small increase in the 5PTase11 substrate, phosphatidylinositol (4,5) bisphosphate, and decreases in the potential products of 5PTase11, phosphatidylinositol (3) phosphate and phosphatidylinositol (4) phosphate. Surprisingly, we also found that dark-grown 5ptase11 mutants contain increases in inositol (1,4,5) trisphosphate and an inositol bisphosphate that is not a substrate for recombinant 5PTase11. We present a model for regulation of hypocotyl growth by specific molecules found in this pathway.

Rhythmic excitation of a rabbit myelin nerve increased diacylglycerol (DAG) content from 1.53 to 2.17 microg/mg lipids. Inhibition of phosphoinositide-specific phospholipase C decreased DAG content. This suggests involvement of this enzyme in processes accompanying rhythmic excitation. The increase in membrane potential of the nerve fiber (K+-depolarization) was accompanied by increase in DAG and phosphatidylinositol monophosphate and decrease in phosphatidylinositol triphosphate and phosphatidylinositol diphosphate content. Treatment of the nerve with DAG or a protein kinase C activator increased (45)Ca influx by 40%, whereas treatment with an inhibitor of this enzyme, polymyxin, inhibited this parameter by 34%. The role of phosphoinositides and protein kinase C in the regulation of Ca2+ transport during rhythmic excitation of the myelin nerve is discussed.

Some ion channels are regulated by inositol phospholipids and by the products of cleavage by phospholipase C (PLC). KCNQ channels (Kv7) require membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) and are turned off when muscarinic receptors stimulate cleavage of PIP(2) by PLC. We test whether diacylglycerols are also important in the regulation of KCNQ2/KCNQ3 channels using electrophysiology and fluorescent translocation probes as indicators for PIP(2) and diacylglycerol in tsA cells. The cells are transfected with M(1) muscarinic receptors, channel subunits, and translocation probes. Although they cause translocation of a fluorescent probe with a diacylglycerol-binding C1 domain, exogenously applied diacylglycerol (oleoyl-acetyl-glycerol and dioctanoyl glycerol) and phorbol ester do not mimic or occlude the suppression of KCNQ current by muscarinic agonist. Blocking the metabolism of endogenous diacylglycerol by inhibiting diacylglycerol kinase with R59022 or R59949 slows the decay of diacylglycerol twofold but does not mimic or occlude muscarinic regulation and recovery of current. Blocking diacylglycerol lipase with RHC-80267 also does not occlude muscarinic modulation of current. We conclude that the diacylglycerol produced during activation of PLC, any activation of protein kinase C that it may stimulate, and downstream products of its metabolism are not essential players in the acute muscarinic modulation of KCNQ channels.

Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.

Inositol hexakisphosphate and other inositol high polyphosphates have diverse and critical roles in eukaryotic regulatory pathways. Inositol 1,3,4-trisphosphate 5/6-kinase catalyzes the rate-limiting step in inositol high polyphosphate synthesis in animals. This multifunctional enzyme also has inositol 3,4,5,6-tetrakisphosphate 1-kinase and other activities. The structure of an archetypal family member, from Entamoeba histolytica, has been determined to 1.2 A resolution in binary and ternary complexes with nucleotide, substrate, and product. The structure reveals an ATP-grasp fold. The inositol ring faces ATP edge-on such that the 5- and 6-hydroxyl groups are nearly equidistant from the ATP gamma-phosphate in catalytically productive phosphoacceptor positions and explains the unusual dual site specificity of this kinase. Inositol tris- and tetrakisphosphates interact via three phosphate binding subsites and one solvent-exposed site that could in principle be occupied by 18 different substrates, explaining the mechanisms for the multiple specificities and catalytic activities of this enzyme.

Inositol depletion is an acute response to lithium and valproate, and aberrant inositol metabolism has been demonstrated in bipolar patients. While it is unlikely that the therapeutic response to inositol depleting drugs is associated with inositol depletion per se, the role of inositol as a metabolic sensor for the secretory, unfolded protein response and glucose response pathways suggests that treatment with inositol depleting drugs may lead to dramatic changes in complex cellular processes. Surprisingly, almost nothing is known about how inositol biosynthesis is regulated in human cells. In contrast, genetic, molecular, and biochemical studies in yeast for over three decades have elucidated intricate mechanisms underlying regulation of inositol biosynthesis in this model eukaryote. INO1 , the gene encoding 1D-myo-inositol 3-phosphate (MIP) synthase, is the most highly regulated of the genes required for phospholipid biosynthesis. This enzyme, which catalyzes the rate limiting step in inositol biosynthesis, is highly conserved from yeast to humans. A recent study has shown that the human INO1 gene is functional in yeast and complements the inositol deficiency of the yeast ino1 mutant. Similar to the yeast enzyme, human MIP synthase activity is decreased in vivo in the presence of valproate. Functional conservation of MIP synthase from yeast to humans underscores the power of the yeast model in understanding the effects of anti-bipolar drugs on inositol synthesis. Because inositol is a metabolic sensor for a variety of signal transduction pathways, MIP synthase may be an important target for new mood stabilizing drugs.

Diacylglycerol is an essential second messenger in mammalian cells. The most prominent intracellular targets of diacylglycerol and the functionally analogous phorbol esters belong to the protein kinase C family, but at least five alternative types of high affinity diacylglycerol/phorbol ester receptors are known: protein kinase D, diacylglycerol kinases alpha, beta, and gamma, RasGRPs, chimaerins, and Munc13s. These function independently of protein kinase C isozymes, and form a network of signaling pathways in the diacylglycerol second messenger system that regulates processes as diverse as gene transcription, lipid signaling, cytoskeletal dynamics, intracellular membrane trafficking, or neurotransmitter release.

The phosphatidylinositol turnover assay is used widely to measure activation, and inhibition, of G(q)-linked G-protein-coupled receptors. Cells expressing the receptor of interest are labeled by feeding with tritiated myo-inositol. The label is incorporated into cellular phosphatidylinositol 4,5-bisphosphate, which, upon agonist binding to the receptor, is hydrolyzed by phospholipase C to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol. In the presence of Li(+), dephosphorylation of IP(3) to inositol is blocked, and the mass of soluble inositol phosphates is a quantitative readout of receptor activation. Current protocols for this assay all involve an anion-exchange chromatography step to separate radiolabeled inositol phosphates from radiolabeled inositol, making the assay cumbersome and difficult to automate. We now describe a scintillation proximity assay to measure soluble inositol phosphate mass in cell extracts, thus obviating the need for the standard chromatography step. The method uses positively charged yttrium silicate beads that bind inositol phosphates, but not inositol. We have used this assay to measure activation of recombinant and endogenous muscarinic acetylcholine receptors and activation of recombinant neuropeptide FF2 receptor coupled to IP(3) production by coexpression of a chimeric G protein. Further, we demonstrate the use and functional validity of this assay in a semiautomated, 384-well format, by characterizing the muscarinic receptor antagonists pirenzepine and atropine.

The enzyme inositol oxygenase (myo-inositol : oxygen oxidoreductase; E.C. 1.13.99.1) is a monooxygenase that converts inositol into glucuronic acid in the presence of molecular oxygen. This enzyme is integrated into a pathway leading to either degradation and energy production or the biosynthesis of precursors for polysaccharides. The enzyme was purified from the yeast Cryptococcus lactativorus by a five-step chromatography procedure. The purified enzyme shows a molecular mass of 37 kDa on SDS–PAGE, similar to the estimation of the size of the native enzyme determined by size exclusion chromatography. Peptides of the inositol oxygenase protein derived from a tryptic digest were sequenced de novo by nanoelectrospray tandem mass spectrometry. Using degenerate oligonucleotides, the corresponding gene was cloned from first strand cDNA. The open reading frame encodes a 315 amino acid polypeptide with a predicted molecular mass of 36.9 kDa. Inositol oxygenase is a single copy gene in C. lactativorus. It has close homologues in other fungi such as Cryptococcus neoformans and Neurospora crassa. Biochemical characterization of the enzyme showed a pH optimum of 6–6.5 and a temperature optimum of 30 °C. Myo-inositol is the only accepted substrate with a Km of ca. 5 mM. The enzyme contains a Fe-centre but the enzyme activity is resistant to KCN. Copyright © 2003 John Wiley & Sons, Ltd.

Myo-inositol exerts behavioral effects in animal models of psychiatric disorders and is effective in clinical trials in psychiatric patients. Interestingly, epi-inositol exerts behavioral effects similar to myo-inositol, even though epi-inositol is not a substrate for synthesis of phosphatidylinositol. We postulated that the behavioral effects of epi-inositol may be due to its effects on gene expression. Yeast INO1expression was measured in northern blots. INM1 was determined by beta-galactosidase activity in a strain containing the fusion gene INM1-lacZintegrated into the genome. Epi-inositol affects regulation of expression of the INO1 gene (encoding inositol-1-P synthase), even though it cannot support growth of an inositol auxotroph (suggesting that, as in mammalian cells, it is not incorporated into phosphatidylinositol). Like myo-inositol, although to a lesser extent, epi-inositol causes a significant reduction in INO1 expression, and reverses the lithium- or valproate-induced increase in INO1 expression. However, it does not affect regulation of INM1 (encoding inositol monophosphatase), the expression of which is up-regulated by myo-inositol. The observed regulatory effects of epi-inositol on expression of the most highly regulated gene in the inositol biosynthetic pathway may help to explain how this inositol isomer can exert behavioral effects without being incorporated into phosphatidylinositol.

The synthesis of inositol provides precursors of inositol lipids and inositol phosphates that are pivotal for cell signaling. Mood stabilizers lithium and valproic acid, used for treating bipolar disorder, cause cellular inositol depletion, which has been proposed as a therapeutic mechanism of action of both drugs. Despite the importance of inositol, the requirement for inositol synthesis in neuronal cells is not well understood. Here, we examined inositol effects on proliferation of SK-N-SH neuroblastoma cells. The essential role of inositol synthesis in proliferation is underscored by the findings that exogenous inositol was dispensable for proliferation, and inhibition of inositol synthesis decreased proliferation. Interestingly, the inhibition of inositol synthesis by knocking down INO1, which encodes inositol-3-phosphate synthase, the rate-limiting enzyme of inositol synthesis, led to the inactivation of GSK-3α by increasing the inhibitory phosphorylation of this kinase. Similarly, the mood stabilizer valproic acid effected transient decreases in intracellular inositol, leading to inactivation of GSK-3α. As GSK-3 inhibition has been proposed as a likely therapeutic mechanism of action, the finding that inhibition of inositol synthesis results in the inactivation of GSK-3α suggests a unifying hypothesis for mechanism of mood-stabilizing drugs. Inositol is an essential metabolite that serves as a precursor for inositol lipids and inositol phosphates. We report that inhibition of the rate-limiting enzyme of inositol synthesis leads to the inactivation of glycogen synthase kinase (GSK) 3α by increasing inhibitory phosphorylation of this kinase. These findings have implications for the therapeutic mechanisms of mood stabilizers and suggest that inositol synthesis and GSK 3α activity are intrinsically related.

Diacylglycerol kinase plays a central role in the metabolism of diacylglycerol by converting diacylglycerol into phosphatidic acid thus initiating resynthesis of phosphatidylinositols. Diacylglycerol is a known second messenger reversibly activating protein kinase C. In addition, diacylglycerol is a potential precursor for polyunsaturated fatty acids. We describe the identification and molecular analysis of a novel type III Drosophila diacylglycerol kinase isoform, DGKepsilon. Drosophila DGKepsilon is mapped to the cytological position 49C1-3. DGKepsilon mRNA is 1.9 kb in length and is broadly distributed throughout development in different cells, primordia and organs, including testes. In embryogenesis, the transcripts are enriched in the cells, which are in S-phase or undergoing endoreplication. Comparison of the Drosophila DGKepsilon with the human homologue revealed that the first zinc finger-like motif is specific for the type III isoform. Although the testis-specific diacylglycerol kinase activity is dependent upon the dose of DGKepsilon gene, the deletion of DGKepsilon does not modulate the total cellular diacylglycerol level. In spite of a proposed key role of diacylglycerol kinase in termination of the diacylglycerol signal, overexpression of a DGKepsilon transgene in flies under the control of a yeast upstream activating sequence promoter does not disrupt normal development in Drosophila.

Extracellular stimuli elicit cellular responses through generation of intracellular second messengers. The lipid second messenger diacylglycerol is produced following activation of the phosphoinositide signalling system. Diacylglycerol is the physiological activator of protein kinase C but also interacts indirectly with other signalling molecules such as small G proteins. Diacylglycerol kinases convert diacylglycerol to phosphatidic acid so terminating signalling through diacylglycerol. However, phosphatidic acid itself has a lipid second messenger role, with targets distinct from those of its precursor diacylglycerol. Therefore, diacylglycerol kinases occupy a central position in signal transduction and regulation of their activity is crucial to cellular function. A family of nine mammalian diacylglycerol kinases have been identified. Their structural diversity and complex pattern of tissue expression suggests that they function in distinct cellular processes. In addition to the plasma membrane, diacylglycerol kinases are found at the nucleus and cytoskeleton and translocation between subcellular compartments occurs with agonist stimulation. In small arteries diacylglycerol kinase activity is increased by adrenergic stimulation implying a role in vascular smooth muscle responses. Due to their role as key regulators of protein kinase C activity diacylglycerol kinases may play a role in the cardiovascular changes that occur in hypertension and as such could represent novel therapeutic targets.

An Arabidopsis thaliana gene (AtLPP1) was isolated on the basis that it was transiently induced by ionizing radiation. The putative AtLPP1 gene product showed homology to the yeast and mammalian lipid phosphate phosphatase enzymes and possessed a phosphatase signature sequence motif. Heterologous expression and biochemical characterization of the AtLPP1 gene in yeast showed that it encoded an enzyme (AtLpp1p) that exhibited both diacylglycerol pyrophosphate phosphatase and phosphatidate phosphatase activities. Kinetic analysis indicated that diacylglycerol pyrophosphate was the preferred substrate for AtLpp1p in vitro. A second Arabidopsis gene (AtLPP2) was identified based on sequence homology to AtLPP1 that was also heterologously expressed in yeast. The AtLpp2p enzyme also utilized diacylglycerol pyrophosphate and phosphatidate but with no preference for either substrate. The AtLpp1p and AtLpp2p enzymes showed differences in their apparent affinities for diacylglycerol pyrophosphate and phosphatidate as well as other enzymological properties. Northern blot analyses showed that the AtLPP1 gene was preferentially expressed in leaves and roots, whereas the AtLPP2 gene was expressed in all tissues examined. AtLPP1, but not AtLPP2, was regulated in response to various stress conditions. The AtLPP1 gene was transiently induced by genotoxic stress (gamma ray or UV-B) and elicitor treatments with mastoparan and harpin. The regulation of the AtLPP1 gene in response to stress was consistent with the hypothesis that its encoded lipid phosphate phosphatase enzyme may attenuate the signaling functions of phosphatidate and/or diacylglycerol pyrophosphate that form in response to stress in plants.

Phospholipase C and two inositol polyphosphate (IP) kinases constitute a signaling pathway that regulates nuclear messenger RNA export through production of inositol hexakisphosphate (IP6). The inositol 1,4,5-trisphosphate kinase of this pathway inSaccharomyces cerevisiae, designated Ipk2, was found to be identical to Arg82, a regulator of the transcriptional complex ArgR-Mcm1. Synthesis of inositol 1,4,5,6-tetrakisphosphate, but not IP6, was required for gene regulation through ArgR-Mcm1. Thus, the phospholipase C pathway produces multiple IP messengers that modulate distinct nuclear processes. The results reveal a direct mechanism by which activation of IP signaling may control gene expression.

1L-myo-inositol (inositol) is vital for the biogenesis of mycothiol, phosphatidylinositol and glycosylphosphatidylinositol anchors linked to complex carbohydrates in Mycobacterium tuberculosis. All these cellular components are thought to play important roles in host-pathogen interactions and in the survival of the pathogen within the host. However, the inositol biosynthetic pathway in M. tuberculosis is not known. To delineate the pathways for inositol formation, we employed a unique combination of tertiary structure prediction and yeast-based functional assays. Here, we describe the identification of the gene for mycobacterial INO1 that encodes inositol-1-phosphate synthase distinct in many respects from the eukaryotic analogues.

Inositol monophosphatases (IMPases) are lithium-sensitive enzymes that participate in the inositol cycle of calcium signalling and in inositol biosynthesis. Two open reading frames (YHR046c and YDR287w) with homology to animal and plant IMPases are present in the yeast genome. The two recombinant purified proteins were shown to catalyse inositol-1-phosphate hydrolysis sensitive to lithium and sodium. A double gene disruption had no apparent growth defect and was not auxotroph for inositol. Therefore, lithium effects in yeast cannot be explained by inhibition of IMPases and inositol depletion, as suggested for animal systems. Overexpression of yeast IMPases increased lithium and sodium tolerance and reduced the intracellular accumulation of lithium. This phenotype was blocked by a null mutation in the cation-extrusion ATPase encoded by the ENA1/PMR2A gene, but it was not affected by inositol supplementation. As overexpression of IMPases increased intracellular free Ca2+, it is suggested that yeast IMPases are limiting for the optimal operation of the inositol cycle of calcium signalling, which modulates the Ena1 cation-extrusion ATPase.

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.

Phosphatidylinositol synthase (CDP-diacylglycerol:myo-inositol 3-phosphatidyl-transferase, EC 2.7.8.11) is a 24-kDa membrane-bound enzyme. It is present in all mammalian cells and is localized predominantly to the endoplasmic reticulum. The enzyme performs the last step in the de novo biosynthesis of the phospholipid phosphatidylinositol by catalyzing the condensation of CDP-diacylglycerol and myo-inositol to form the products phosphatidylinositol and CMP. Phosphatidylinositol, apart from being an essential membrane phospholipid, is involved in protein membrane anchoring and is the precursor for the second messengers inositol-tri-phosphate and diacylglycerol.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits 'interfacial activation' toward the water-soluble substrate myo-inositol 1,2-(cyclic)phosphate [Zhou et al. (1997) Biochemistry 36, 347-355]. The activation of PI-PLC enzyme is optimal with PC or PE interfaces. NMR experiments (TRNOE and 31P line width analyses) were carried out to investigate the interaction of PI-PLC with activator amphiphiles. These studies showed that the enzyme had high affinity for phosphatidylcholine (or PE) molecules with dissociation constants of 0.5 and 0.3 mM for diC6PC and diC7PC, respectively. TRNOE cross-peaks of bound PC were confirmed to represent intramolecular relaxation pathways using partially perdeuterated PC molecules consistent with a single molecule binding tightly. The large activation by a PC interface can be explained by a single PC molecule binding specifically to PI-PLC and anchoring the enzyme-lipid complex to the interface. Other interfaces, such as micellar diC8PS, can activate PI-PLC about 2-3-fold; however, the monomers of these detergents showed little affinity for the enzyme as measured by TRNOE or 31P NMR line widths. The 3.6-fold activation produced by polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (compared to the 15-fold activation generated by nonpolymerized PC vesicles) was comparable to the nonspecific activation of other detergents. This confirmed that single-PC molecule binding was allosteric and anchored the enzyme in the interface. The conformation of interfacially activated enzyme is discussed in term of the stabilization of a critical surface loop and helix B observed with weak intensity in the X-ray crystal structure.

Large enhancements (maximum of 82-fold in terms of enzyme efficiency, Vmax/Km) of bacterial PI-PLC cyclic phosphodiesterase activity were observed in the presence of organic solvents miscible in water (dimethyl sulfoxide, dimethylformamide, and 2-propanol). In general, organic solvents lowered the Km for myo-inositol 1,2-cyclic phosphate (cIP) and increased Vmax substantially. This kinetic effect was similar to that obtained with phosphatidylcholine micelles and bilayers in an aqueous assay system for cyclic inositol phosphate hydrolysis [Zhou, C., et al. (1997) Biochemistry 36, 347-355]. Solvent properties were examined to determine which ones correlated with the activation of PI-PLC toward cIP in each solvent. Activation correlated best with the solvent polarity as measured by ET(30); no significant correlation was observed with solution surface tension, the bulk dielectric constant (epsilon), 1/epsilon (a measure of the strength of charge interactions), or the Hildebrand solubility parameter. The sigmoidal curve of the enzyme activity versus solvent polarity was consistent with the solvent promoting a transition in the enzyme from a low-activity to a high-activity form. Possible candidates for this change, including enzyme dimerization, helix B/loop stabilization, and dehydration of the active site, are discussed.

Stimulation of cells with certain agonists often activates both phospholipases C and D. These generate diacylglycerol and phosphatidate, respectively, although the two lipids are also apparently interconvertable through the actions of phosphatidate phosphohydrolase and diacylglycerol kinase. Diacylglycerol activates protein kinase C while one role for phosphatidate is the activation of actin stress fiber formation. Therefore, if the two lipids are interconvertable, it is theoretically possible that an uncontrolled signaling loop could arise. To address this issue structural analysis of diacylglycerol, phosphatidate, and phosphatidylbutanol (formed in the presence of butan-1-ol) from both Swiss 3T3 and porcine aortic endothelial cells was performed. This demonstrated that phospholipase C activation generates primarily polyunsaturated species while phospholipase D activation generates saturated/monounsaturated species. In the endothelial cells, where phospholipase D was activated by lysophosphatidic acid independently of phospholipase C, there was no activation of protein kinase C. Thus we propose that only polyunsaturated diacylglycerols and saturated/monounsaturated phosphatidates function as intracellular messengers and that their interconversion products are inactive.

To elucidate the signaling mechanisms associated with keratinocyte differentiation, we studied in vitro phospholipase C-mediated signal transduction, which results in the generation of inositol phosphates, comparing proliferating versus differentiated HaCaT cells, a human keratinocyte line. Bradykinin- or A23187-induced formation of inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol monophosphates, as determined by anion exchange high performance liquid chromatography, were found to be highest in the early logarithmic growth phase of the cells. In more highly differentiated HaCaT cells, which expressed maximal amounts of the differentiation marker involucrin, inositol phosphate formation was reduced to about one third of that in proliferating cells. Thin layer chromatography of membrane phosphatidylinositol phosphates revealed that this reduction was associated with a steady decrease in phospholipase C substrates. Immunoblot analysis of phospholipase C isozymes, however, and of expression of Gq alpha, the G protein subunit that activates phospholipase C beta, revealed no decrease during the differentiation phase. The results suggest that the inositol-phospholipid signal transduction pathway is involved in keratinocyte proliferation and in the induction of differentiation, with attenuated signal transduction activity via phospholipase C-coupled receptors in more differentiated keratinocytes.

Inositol 3,4,5,6-tetrakisphosphate is a novel intracellular signal that regulates calcium-dependent chloride conductance (Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092-14097). The molecular mechanisms that regulate the cellular levels of this signal are not characterized. To pursue this problem we have now studied the 1-kinase that deactivates inositol 3,4,5,6-tetrakisphosphate. The enzyme was purified from rat liver 1600-fold with a 1% yield. The native molecular mass was determined to be 46 kDa by gel filtration. The Km values for inositol 3,4,5,6-tetrakisphosphate and ATP were 0. 3 and 10.6 microM, respectively. The kinase was unaffected by either protein kinase A or protein kinase C. Increases in Ca2+ concentration from 0.1 to 1-2 microM inhibited activity by 10-20%. Most importantly, inositol 1,3,4-trisphosphate was shown to be a potent (Ki = 0.2 microM), specific, and competitive inhibitor of the 1-kinase. Our new kinetic data show that typical receptor-dependent adjustments in cellular levels of inositol 1,3,4-trisphosphate provide a mechanism by which the concentration of inositol 3,4,5,6-tetrakisphosphate is dependent on changes in phospholipase C activity. These conclusions also provide a new perspective to our understanding of the physiological importance of the pathway of inositol phosphate turnover initiated by the inositol 1,4, 5-trisphosphate 3-kinase.

Diacylglycerol is a key regulator of cell physiology, controlling the membrane recruitment and activation of signaling molecules. Accordingly, diacylglycerol generation and metabolism are strictly controlled, allowing for localized regulation of its concentration. While the increased production of diacylglycerol upon receptor triggering is well recognized, the modulation of diacylglycerol metabolism by diacylglycerol kinases (DGKs) is less characterized. Some agonists induce DGK activation and recruitment to the plasma membrane, promoting diacylglycerol metabolism to phosphatidic acid. Conversely, several reports indicate that signaling pathways that selectively inhibits DGK isoforms can enhance cellular diacylglycerol levels and signal transduction. For example, the impairment of DGKθ activity by RhoA binding to the catalytic domain represents a conserved mechanism controlling diacylglycerol signaling from Caenorhabditis elegans motoneurons to mammalian hepatocytes. Similarly, DGKα activity is inhibited in lymphocytes by TCR signaling, thus contributing to a rise in diacylglycerol concentration for downstream signaling. Finally, DGKμ activity is inhibited by ischemia–reperfusion-generated reactive oxygen species in airway endothelial cells, promoting diacylglycerol-mediated ion channel opening and edema. In those systems, DGKs provide a gatekeeper function by blunting diacylglycerol levels or possibly establishing permissive domains for diacylglycerol signaling. In this review, I discuss the possible general relevance of DGK inhibition to enhanced diacylglycerol signaling.

Phospholipases are enzymes that use phospholipids as substrate and are classified in three major classes A, C and D based on the reaction they catalyse. Phosphatidylinositol-specific Phospholipase C enzymes utilize phosphatidylinositol 4,5-bisphosphate as substrate and cleave the bond between the glycerol and the phosphate to produce important second messenger such as inositol trisphosphate and diacylglycerol. The Phospholipase C members are the most well-known phospholipases for their role in lipid signalling and cell proliferation and comprise 13 isoforms classified in 6 distinct sub-families. In particular, signalling activated by Phospholipase C γ, mostly activated by receptor and non-receptor tyrosine kinases, is well characterized in different cell systems. Increasing evidence suggest that Phospholipase C γ plays a key role in cell migration and invasion. Because of its role in cell growth and invasion, aberrant Phospholipase C γ signalling can contribute to carcinogenesis. A major challenge facing investigators who seek to target Phospholipase C γ directly is the fact that it is considered an “undruggable” protein. Indeed, isoform specificity and toxicity represents a big hurdle in the development of Phospholipase C γ small molecule inhibitors. Therefore, a future development in the field could be the identification of interacting partners as therapeutic targets that could be more druggable than Phospholipase C γ.

Some key elements of signal transduction have been identified within the nucleus and demonstrated to be responsive to specific agonists in numerous cell types. In particular, mitogenic stimuli have been reported to induce a transient increase of the nuclear phospholipase C beta 1 activity, causing the release of inositide-derived second messengers, whereas differentiating stimuli induced a decrease of the enzyme activity and an increase of nuclear phosphatidylinositol 4,5-bisphosphate (PIP2). Recently, we reported evidence, in human osteosarcoma Saos-2 cell lines, on the presence of specific nuclear phospholipase C isoforms and on the activation of phospholipase C beta 1 in the nucleus following the exposure to interleukin-1 alpha. In this study we report immunocytochemical ultrastructural evidence on quantitative variations of PIP2 and phospholipase C beta 1 amounts in the nucleus of Saos-2 cells at different times of exposure to interleukin-1 alpha. After short periods of culture in the presence of the agonist, the intranuclear amount of PIP2 is decreased, while a translocation of phospholipase C beta 1 occurs from the cytoplasm to the nucleus, in correspondence with the increased hydrolyzing activity of the enzyme. After longer periods of incubation with interleukin-1 alpha, on the other hand, the intranuclear amount of PIP2 is restored to initial level, while the amount of phospholipase C beta 1 is increased both at the nuclear and cytoplasmic level, when its activation is no longer effective. The results, compared with those obtained in other cell types responsive to given agonists, account for a cell-specific modulation of signal transduction based on polyphosphoinositide breakdown at the nuclear level.

PI-specific PLC enzymes are a key component of phosphatidylinositol-mediated signaling pathways since the hydrophobic product, diacylglycerol, activates protein kinase C and the water-soluble product, inositol trisphosphate, is involved in Ca2+ mobilization. Nonspecific, or PC-PLC, enzymes can generate diacylglycerol without Ca2+ mobilization. A series of inhibitors, both lipophilic and water-soluble, have been synthesized to target each of these two classes of PLC enzymes. Design of the inhibitors was based on proposed enzyme mechanisms and available crystal structures. The solution conformations of the lipophilic phospholipid analogs, (diheptanoylphosphatidyl(2-O-methyl)inositol for PI-PLC and a dihexanoyl-sn-(3-N-benzylaminoglycero)phosphoramidocholine for PC-PLC, have been determined using NMR methodology and the interaction of these compounds with bacterial enzymes has been examined. Water-soluble inhibitors include strained cyclic phosphonates for PI-PLC and vanadate for PC-PLC. An eventual goal of this work is to generate compounds that specifically target each type of intracellular PLC activity.

Previous studies have demonstrated a strict extracellular Ca2+ dependence for the G0 to G1 and G1 to S transition in growth factor-treated T51B rat liver cells that is associated with increased levels of protein kinase C activity. Consequently, we have examined these cells for changes in phospholipid-derived second messengers in response to epidermal growth factor (EGF) and thrombin in order to determine which signals are generated during the initiation of the G0 to G1 transition. Thrombin is coupled to a phosphoinositide hydrolyzing phospholipase C, as we have found a rapid Ca2+-independent increase in the levels of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), inositol 1,4-bisphosphate (Ins[1,4]P2), and inositol 4-monophosphate (Ins[4]P), as well as a concomitant, transient elevation in diacylglycerol. No changes in either intracellular or extracellular choline metabolites, or an increase in DNA synthesis, were found in response to thrombin. By contrast, treatment of T51B cells with EGF results in a slower, more prolonged extracellular Ca2+-dependent increase in both [3H]-glycerol radiolabeled diacylglycerol, and diacylglycerol mass, an increase in choline release into the extracellular medium, and eventually a substantial DNA synthesis. We were, however, unable to detect any changes in phosphatidylinositol (Ptdlns) turnover, either by accumulation of inositol phosphates or by changes in phospholipids in response to EGF. These results indicate that DNA synthesis can readily occur in the absence of stimulated Ptdlns turnover, and that Ptdlns turnover is not sufficient in itself or necessary to induce DNA synthesis and is not necessary for a Ca2+-dependent increase in diacylglycerol. Moreover, we have demonstrated that the extracellular Ca2+-dependent increase in diacylglycerol levels in response to EGF is associated with an increase in extracellular choline release, which is indicative of an activation of a phosphatidylcholine-linked phospholipase D. These results suggest that diacylglycerol sources other than Ptdlns's may be important in the extracellular Ca2+-dependent regulation of EGF-mediated cell replication. © 1995 Wiley-Liss, Inc.

Ganglioside IV 3 (NeuAc) 2 , II 3 (NeuAc) 2 -GgOse 4 Cer (GQ Ib , which induces terminal differentiation in keratinocytes, was previously found to enhance the mass content of inositol 1,4,5-trisphosphate and intracellular calcium concentration ([Ca ++ ] i ), peaking at 30 seconds. In the present study, the biphasic accumulation of 1,2 diacylglycerol, i.e., the first transient and the second sustained phase, was observed in cultured human keratinocytes stimulated by GQ 1b . On the other hand, II 3 NeuAc-LacCer (GM 3 ), which inhibits keratinocyte proliferation without inducing differentiation, did not cause diacylglycerol formation, Phosphatidylethanol, produced by transphosphatidylation and a potential marker for phospholipase D activity, was produced by the exposure to GQ 1b in the presence of ethanol. The second sustained phase of diacylglycerol was repressed by ethanol, indicating that the diacylglycerol-formation pathway via phospholipase D followed by phosphatidic acid phosphohydrolase would in part account for the second diacylglycerol phase. Furthermore, this second phase of GQ 1b -induced diacylglycerol generation was reduced by pretreatment with propranolol, an inhibitor of phosphatidic acid phosphohydrolase. In addition, the levels of[ 3 H] choline, a direct metabolite of the phospholipase D pathway, were elevated within 1 min after GQ 1b addition and then sustained for at least 20 min. Taken together, the results suggest that the phospholipase D pathway may contribute to the second phase of diacylglycerol formation, which might be involved in differentiation.

We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem. 148, 593-602]. PIP synthase in the pathway is a promising target for the development of new anti-mycobacterium drugs. In the present study, we evaluated the characteristics of the PIP synthase of Mycobacterium tuberculosis. Four types of compounds were chemically synthesized based on the assumption that structural homologues of inositol 1-phosphate, a PIP synthase substrate, would act as PIP synthase inhibitors, and the results confirmed that all synthesized compounds inhibited PIP synthase activity. The phosphonate analogue of inositol 1-phosphate (Ino-C-P) had the greatest inhibitory effect among the synthesized compounds examined. Kinetic analysis indicated that Ino-C-P acted as a competitive inhibitor of inositol 1-phosphate. The IC(50) value for Ino-C-P inhibition of the PIP synthase activity was estimated to be 2.0 mM. Interestingly, Ino-C-P was utilized in the same manner as the normal PIP synthase substrate, leading to the synthesis of a phosphonate analogue of PIP (PI-C-P), which had a structure similar to that of the natural product, PIP. In addition, PI-C-P had high inhibitory activity against PIP synthase.