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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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

Inositol polyphosphate 1-phosphatase, an enzyme of the phosphatidylinositol signaling pathway, hydrolyzes the 1-phosphate from inositol 1,4-bisphosphate and inositol 1,3,4-trisphosphate. We have used indirect immunofluorescence microscopy, Western blot analysis, and enzyme assays to determine the cellular localization of the enzyme. We find that the enzyme is present, but not exclusively, in the nucleus of Madin-Darby bovine kidney cells, and also in COS-7 and HeLa cells that were transiently transfected with a cDNA encoding bovine inositol polyphosphate 1-phosphatase. DNA synthesis, as measured in COS-7 and HeLa cells transiently over-expressing enzyme, was reduced 50% in cells transfected with wild-type enzyme compared with nontransfected cells or cells transfected with an inactive mutant form of the enzyme. These data demonstrate that this response is mediated by one of the substrates or products of inositol polyphosphate 1-phosphatase. We propose that overexpressed inositol polyphosphate 1-phosphatase degrades a stimulatory inositol phosphate(s) and thereby inhibits DNA synthesis.

Severe mutations within the norpA gene of Drosophila abolish the photoreceptor potential and render the fly blind by deleting phospholipase C, an essential component of the phototransduction pathway. To study the membrane association of phospholipase C, we have utilized biochemical assays of phospholipase C activity, which predominant measurable phospholipase C activity in head homogenates has been shown to be encoded by norpA , as well as antisera generated against the major gene product of norpA to examine its subcellular distribution before and during phototransduction. We find that both phospholipase C activity and the norpA protein are predominantly associated with membrane fractions in heads of both light- and dark-adapted flies. Moreover, phospholipase C activity as well as norpA protein can be easily extracted from membrane preparations of light- or dark-adapted flies using high salt, indicating that the norpA protein is peripherally localized on the membrane. These data suggest that the norpA encoded phospholipase C of Drosophila is a permanent peripheral membrane protein. If this is indeed the case, then it would mean that the reversible redistribution of phospholipase C from the cytosol to the membrane, as observed in epidermal growth factor receptor stimulation of mammalian phospholipase C /gg , is not a universal mechanism utilized by all types of phosphatidylinositol-specific phospholipase C.

The modulation of a brain phosphoinositide-specific phospholipase C-alpha activity was studied using a variety of compounds of different charge. Detergents such as sodium deoxycholate and cetyltrimethylammonium bromide stimulated the phospholipase C activity when used alone but when used together the effects were not additive. Spermine was an effective inhibitor of the enzyme activity while the cationic peptide, Melittin, had no effect. The inositol phosphates produced by hydrolysis with phosphoinositide-specific phospholipase C were inhibitory while diacylglycerol and inositol did not affect the phospholipase activity. Myelin basic protein, which was previously shown to stimulate phospholipase C activity by 2.5-fold, did not interact with the anionic inositol phosphatases to any significant extent. Thus we concluded that the mechanism of stimulation was not due to relief of product inhibition. Crosslinking studies with the photoactivatable reagent, N-hydroxysuccinimidyl-4-azidosalicylic acid, showed that peptide 24-33 of myelin basic protein, which stimulated the activity almost as much as the native protein, interacted specifically with the phospholipase C. Thus the mechanism by which myelin basic protein stimulated the enzyme appeared to be through specific protein-protein interaction.

The expression of phospholipase C isozymes and phosphatidylinositol 4-kinase in the rat facial nucleus was studied using in situ hybridization at various times after unilateral crushing and resectioning the facial nerve. The level of phospholipase C alpha messenger RNA increased from three days to one week after the operation. On the other hand, an apparent reduction in the level of phospholipase C beta 1 occurred from three days to one week after resection. After either crushing or resection, phospholipase C gamma 1 messenger RNA levels were not noticeably changed. As phosphatidylinositol 4-kinase is the rate-limiting enzyme for the production of phosphatidylinositol 4,5-bisphosphate, which is the preferred substrate for phospholipase C, we investigated the expression of phosphatidylinositol 4-kinase messenger RNA. The level of phosphatidylinositol 4-kinase messenger RNA was decreased one day after axonal injury. Among phospholipase C isozymes, phospholipase C alpha is up-regulated. As the structure of phospholipase C alpha is different from other isozymes, phospholipase C alpha is supposed to have a different function. The present unique up-regulation of phospholipase C alpha may suggest a novel function in nerve regeneration. Phospholipase C beta 1 is down-regulated, as is phosphatidylinositol 4-kinase. This suggests that the signal transmission system using a G-linked receptor is broken down after nerve injury. On the other hand, phospholipase C gamma 1, which is related to the receptor tyrosine kinase, does not demonstrate any transcriptional regulation after nerve injury.

Liver homogenates phosphorylated inositol 1,4,5,6-tetrakisphosphate exclusively to inositol 1,3,4,5,6-pentakisphosphate. Approximately 30% of this phosphorylating activity was associated with the particulate fraction of the cell, in contrast to the inositol 3,4,5,6-tetrakisphosphate 1-kinase, which was 90% soluble. This soluble 1-kinase activity was resolved from the soluble activity that phosphorylated inositol 1,4,5,6-tetrakisphosphate by anion-exchange chromatography. The two phosphorylating activities were also found to be differentially inhibited by inositol 1,3,4-trisphosphate (IC50 for 3-kinase100 microM; IC50 for 1-kinase1 microM). Thus, we have demonstrated that inositol 1,4,5,6-tetrakisphosphate is phosphorylated directly by a 3-kinase, and inositol 3,4,5,6-tetrakisphosphate is not an obligatory intermediate, in contrast to one previous model (Oliver, K. G., Putney, J. W., Jr., Obie, J. F., and Shears, S. B. (1992) J. Biol. Chem. 267, 21528-21534). Inositol 1,4,5,6-tetrakisphosphate 3-kinase was inhibited by inositol 1,3,4,6-tetrakisphosphate (IC50, 1 microM). Soluble inositol 1,4,5,6-tetrakisphosphate 3-kinase and inositol 1,4,5-trisphosphate 3-kinase were resolved by anion-exchange chromatography. Furthermore, cDNA clones of two isozymes of inositol 1,4,5-trisphosphate 3-kinase from rat and human brain did not phosphorylate inositol 1,4,5,6-tetrakisphosphate. Thus, these two 3-kinase activities are performed by distinct enzymes.

A method for assessing stimulated phosphoinositide turnover by measurement of the liponucleotide CDP-diacylglycerol is presented. The phosphoinositide signal transduction pathway consists of a sequence of reactions in which the second messengers Inositol 1,4,5-triphosphate and diacylglycerol are recycled back to phosphatidylinositol (PtdIns), which then serves to replenish the initial hydrolyzed substrate, phosphatidylinositol 4,5-bis-phosphate. Receptor-stimulated inositol lipid turnover is most commonly assessed by measurement of the accumulation of [ 3 H]inositol-labeled inositol phosphates in the presence of Li + . The latter blocks Inositol monophosphatase and thus can lead to a depletion of intracellular inositol. Because inositol is required for resynthesis of PtdIns, the immediate precursor of PtdIns, CDP-diacylglycerol, also accumulates in the presence of agonist and Li + . Measurement of radiolabeling of this liponucleotide following Incorporation of [ 3 H]cytidine thus forms the basis for an alternative assay for Inositol lipid turnover. The general applicability of this method may be limited, since, In brain slices, not all receptors exhibit CDP-diacylglycerol responses that are consistent with their inositol phosphate responses. In addition, in cultured neural cells, growth in inositol-free, chemically defined medium is required to maximize the Li + -dependent CDP-diacylglycerol response. A major advantage of this method may be its ability to provide insight Into the regulation of phosphoinositide turnover since this method uniquely reflects slowing of the regenerative cycle. Such in vitro studies may have relevance to the in vivo action of Li + as a psychotherapeutic agent.

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol. NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated. Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold). The increases correlated with the levels of enzyme expression obtained in each cell type. The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection. The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered. Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.

In order to study the regulatory mechanisms of phospholipase C-gamma (PLC-gamma) via the intrinsic SH2/SH3 region (Z region), two recombinant Z proteins, rP45Z and rP38Z, derived from rat PLC-gamma 1 and PLC-gamma 2, respectively, were purified from the inclusion bodies of Escherichia coli. We examined their direct effects on phosphoinositide hydrolysis induced by four different PLC isoforms purified from bovine brain and thymus, and found that both of these Z proteins suppress the enzyme activity of all four PLC isoforms in a dose-dependent manner. This suppressive effect is very potent and stoichiometric. The kinetics studies indicate that the suppression is non-competitive. This suppression is eliminated by treatment with proteases but is not affected by heat treatment at 95 degrees C for 15 min, indicating that the primary structure might be important for the action of Z proteins. Comparative studies suggested that two Z proteins but not Src and phosphatidylinositol 3-kinase possess, adjacent to their SH2 and SH3 motifs, a phospholipase C inhibitor (PCI) region that strongly suppresses their phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity. A series of synthetic peptides identical with the sequence of the proposed PCI region, including an octamer, YRKMRLRY, inhibited PIP2 hydrolysis induced by four different phospholipase C isoforms. These results demonstrate that both types of phospholipase C-gamma contain the PCI sequence which is responsible for the inhibition of PIP2 hydrolysis, indicating that phospholipase C-gamma is a self-regulating enzyme.

The preparation of a phosphatidylinositol analog lacking the axial 2-hydroxyl of the inositol ring is described. The compound is a usefulk mecanistic probe for the phosphatidylinositol specifc phospholipase C.

The preceding paper describes purification and properties of a 150-kDa polyphosphoinositide-specific phospholipase C from a cytosolic fraction of turkey erythrocytes (Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507). Turkey erythrocytes express a P2Y-purinergic receptor that employs an unidentified G-protein to activate phospholipase C (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890; Cooper, C. L., Morris, A. J., and Harden, T. K. (1989) J. Biol. Chem. 264, 6202-6206). This paper describes receptor and G-protein regulation of the purified turkey erythrocyte phospholipase C after reconstitution of the enzyme using [3H]inositol pre-labeled turkey erythrocyte ghosts as acceptor membranes. These membranes contain polyphosphoinositides labeled to high specific radioactivity and display reduced responsiveness of their endogenous phospholipase C to P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of purified enzyme had no effect on basal inositol phosphate production, but markedly increased P2Y-purinergic receptor agonist and guanine nucleotide-dependent accumulation of inositol phosphates. Reconstitution of 5 ng of purified phospholipase C with 10 micrograms of acceptor membrane protein produced half-maximal effects, and maximal activity was observed with reconstitution of 100 ng of purified enzyme. Agonist and guanine nucleotide-regulated phospholipase C activity measured using a reconstitution assay co-purified with phospholipase C activity detected using exogenously provided phosphatidylinositol 4,5-bisphosphate during purification of the 150-kDa protein. Only the maximal rate of inositol phosphate formation attained upon activation was increased in the presence of the purified phospholipase C. K0.5 values for adenosine 5'-O-(2-thiodiphosphate), guanosine 5'-3-O-(thio)triphosphate, and A1F4- activation of the purified enzyme were the same as for the endogenous phospholipase C activity of the acceptor membranes.

Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus. In these molecules, the phosphodiester bond of phosphatidylinositol hydrolyzed by the phospholipase was replaced by a phosphonate linkage and a simpler hydrophobic group replaced the diacylglycerol moiety. One of the phosphonates also contained a carboxylate functional group suitable for matrix attachment. All three synthetic phosphonates inhibited the phospholipase C activity in a concentration-dependent manner, with the analogue most closely resembling the structure of the natural substrate, phosphatidylinositol, being the most potent inhibitor. The data indicate that phosphonate analogues of phosphatidylinositol may be useful for study of phospholipase C and other proteins interacting with myo-inositol phospholipids.

R 59 022 (6-[2-[4-[(4-fluorophenyl) phenylmethylene)-1-piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-alpha] pyrimidin-5-one) was found to inhibit diacylglycerol kinase in human red blood cell membranes at concentrations where polyphosphoinositide phosphodiesterase, phosphatidylinositol kinase, and phosphatidylinositol 4-phosphate kinase activity remained unaffected. The concentration needed for half-maximal inhibition (IC50) was 2.8 +/- 1.5 X 10(-6) M for the kinase acting on endogenous diacylglycerol and 3.3 +/- 0.4 X 10(-6) M when 1-oleoyl-2-acetylglycerol (OAG) was added exogenously as substrate. In intact platelets, R 59 022 inhibits the phosphorylation of OAG to 1-oleoyl-2-acetylglyceryl-3-phosphoric acid (OAPA) (IC50: 3.8 +/- 1.2 X 10(-6) M); concomitantly the stimulation of protein kinase C activity by OAG was amplified. When in platelets inositol lipid turnover is accelerated by thrombin, further addition of R 59 022 results in a marked elevation of diacylglycerol levels, a decreased formation of phosphatidic acid and an increased protein kinase C activity as compared with the controls. It is concluded that in studies on the signal-transducing system coupled to inositol lipid metabolism R 59 022 might occupy a role comparable to cyclic AMP phosphodiesterase inhibitors, since it potentiates the effect of the putative second messenger diacylglycerol by preventing its rapid metabolism.

Treatment of cultured cells with phospholipase C causes increased rates of hydrolysis of cellular phosphatidylcholine and increased rates of incorporation of choline into phosphatidylcholine. The fate of the diacylglycerol produced by the phospholipase C hydrolysis was examined in two cell lines, Chinese hamster ovary and HeLa. In the former cells, turnover of the glycerol moiety of phosphatidylcholine was not enhanced by phospholipase C treatment, indicating that the phospholipase C-generated diacylglycerol was recycled into new phosphatidylcholine. In HeLa cells, turnover of the glycerol backbone of phosphatidylcholine was enhanced by phospholipase C treatment, and the increased rate of turnover of the glycerol moiety was similar to that of the phosphate moiety. Thus, the fate of diacylglycerol generated at the plasma membrane was demonstrated to differ in these two cell lines. Incorporation of precursors of diacylglycerol into phosphatidylcholine was not enhanced by phospholipase C treatment in either cell line.

The subcellular distribution of diacylglycerol- and monoacylglycerol-lipases has been studied in human platelets. Using a fractionation procedure on Percoll gradient (Perret B., Chap H. and Douste-Blazy L. (1979) Biochim. Biophys. Acta 556, 434–446), the enzyme activity displayed the same profile as that of [ 3 H]concanavalin A, a plasma membrane marker. This result was confirmed with highly purified platelet plasma membranes prepared by adsorption onto polyethylenimine-bonded polyacrylamide beads (Kinoshita T., Nachman R.L. and Minick R. (1979) J. Cell Biol. 82, 688–696). Studies with isolated membranes or crude homogenate revealed that the enzyme requires calcium or magnesium and displays an optimal pH of 6.2, showing that it is able to hydrolyse diacylglycerol under conditions where phosphatidylinositol-specific phospholipase C is fully active. Using diacylglycerol labelled in the 1- or 2-position, it was found that the two fatty acids are released at the same rate, which is supported by the lack of monoacylglycerol accumulation and by the observation that monoacylglycerol is hydrolysed at a 20-fold faster rate than diacylglycerol. Increasing concentrations of Mg-ATP promote the conversion of diacylglycerol into phosphatidic acid by diacylglycerol kinase, but only high concentrations become inhibitory for diacylglycerol lipase. These results are discussed in the light of our former hypothesis that arachidonic acid release from platelet phospholipids might occur through the sequential action of a phosphatidylinositol-specific phospholipase C coupled to a diacylglycerol lipase (Mauco G., Chap H.,Simon M.F. and Douste-Blazy L. (1978) Biochimie 60, 553–561). The possible role of this enzyme in the regulation of the activity of protein kinase C is also emphasized.

Phosphoinositides of human, rabbit, rat, and turkey erythrocytes were radiolabeled by incubation of intact cells with [32P]Pi. Guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and NaF, which are known activators of guanine nucleotide regulatory proteins, caused a large increase in [32P]inositol phosphate release from plasma membranes derived from turkey erythrocytes, but had no effect on inositol phosphate formation by plasma membranes prepared from the mammalian erythrocytes. High performance liquid chromatography analysis indicated that inositol bisphosphate, inositol 1,3,4-trisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate all increased by 20-30-fold during a 10-min incubation of turkey erythrocyte membranes with GTP gamma S. The increase in inositol phosphate formation was accompanied by a similar decrease in radioactivity in phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). GTP gamma S increased inositol phosphate formation with a K0.5 of 600 nM; guanosine 5'-(beta, gamma-imido)trisphosphate was 50-75% as efficacious as GTP gamma S and expressed a K0.5 of 36 microM. Although GTP alone had little effect on inositol phosphate formation, it blocked GTP gamma S-stimulated inositol phosphate formation, as did guanosine 5'-O-(2-thiodiphosphate). Turkey erythrocytes were also shown to express phosphatidylinositol synthetase activity in that incubation of cells with [3H] inositol resulted in incorporation of radiolabel into phosphatidylinositol, PIP, and PIP2. Incubation of membranes derived from [3H]inositol-labeled erythrocytes with GTP gamma S resulted in large increases in [3H] inositol phosphate formation and corresponding decreases in radiolabel in PIP and PIP2. The data suggest that, in contrast to mammalian erythrocytes, the turkey erythrocyte expresses a guanine nucleotide-binding protein that regulates phospholipase C, and as such, should provide a useful model system for furthering our understanding of hormonal regulation of this enzyme.

Diacylglycerol kinase is though to play a central role in the metabolism of diacylglycerol second messengers in agonist-stimulated cells. A series of diacylglycerol analogs were tested for their ability to act as substrates or inhibitors of diacylglycerol kinase with the goal of determining the substrate specificity of the enzyme, and of discovering inhibitors. Screening of these compounds was performed using a partially purified diacylglycerol kinase from pig brain. Modified assays for this enzyme using co-sonicated mixtures of diacylglycerol and anionic phospholipids were developed. This enzyme was found to be quite specific for sn-1,2-diacylglycerol (KM 24 microM for dioctanoyl-glycerol). Among the analogs investigated, only 1,2-dioctanoyl-2-amino-1,3-propanediol was utilized at a significant rate. Two analogs, dioctanoylethylene glycol (KI 58 microM) and 1-monooleoylglycerol (KI 91 microM), were potent inhibitors in vitro. These compounds were tested for effects on diacylglycerol formation and metabolism in thrombin-stimulated human platelets. Dioctanoylethylene glycol inhibited diacylglycerol phosphorylation in platelets (70-100% at 100 microM) leading to a longer-lived diacylglycerol signal. This compound may be a useful tool for studies of diacylglycerol kinase in other cell types. 1-Monooleoylglycerol treatment elevated diacylglycerol levels up to 4-fold in unstimulated platelets and up to 10-fold in thrombin-stimulated platelets. The implications with regard to the pathways of diacylglycerol metabolism in human platelets are discussed.

Phosphatidylinositol 4,5-bisphosphate has recently gained prominence as the central component of a receptor transduction process which generates inositol 1,4,5-trisphosphate and diacylglycerol in stimulated cells. Both of these products of phospholipid metabolism have intracellular second messenger functions with diacylglycerol formation leading to activation of protein kinase C and inositol 1,4,5-trisphosphate stimulating Ca(2+) release from intracellular stores in the endoplasmic reticulum. There is mounting evidence that the phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate is coupled to activated receptors by a guanylnucleotide binding protein, analogous to Ns and Ni which couple stimulatory and inhibitory hormone receptors to adenylate cyclase. Most of the key elements of this signalling mechanism have been found in the nervous system and so too has an entirely novel and unexpected inositol phosphate ester, inositol 1,3,4,5-tetrakisphosphate, whose function is not yet known. Phosphatidylinositol 4,5-bisphosphate breakdown, detected as the accumulation of inositol phosphates in agonist-stimulated nervous tissue preparations, is a functional response that has been useful in assessing the relevance of receptors identified by radioligand binding assays, and which provides an essential link between receptor occupation and responses such as neurotransmitter release and modulation of neuronal excitability.

One of the developmentally induced gene products that is essential for chemotaxis of Dictyostelium amoebae is a cyclic nucleotide phosphodiesterase. The enzyme can be secreted or exist in a membrane bound form. This enzyme is missing in the mutant HPX235 which, as a consequence, does not aggregate unless exogenous cAMP phosphodiesterase is supplied. We have introduced multiple copies of the cloned phosphodiesterase gene into mutant amoebae and restored aggregation. The formation of anatomically correct fruiting bodies, which does not occur when exogenous enzyme is added, is also restored by transformation with the gene. The construct that we have used gives rise only to secreted phosphodiesterase and therefore the membrane bound form of the enzyme is not absolutely required for normal aggregation and morphogenesis.

Rat renal cortical and medullary slices incorporate [14C]arachidonate into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerols. The percent distribution of [14C]arachidonate among the various phospholipids is similar in renal cortex and medulla, although the total amount of radioactively labeled phospholipids is higher in the renal medulla. Subsequent incubation of prelabeled slices in the presence of deoxycholate induces a loss of radioactivity from [14C]phosphatidylinositol, with a concomitant increase in 1,2-[14C]diacylglycerol. Neutral lipids are not affected. The degradation of phosphatidylinositol to [14C]diacylglycerol indicates the presence of phospholipase C activity. Renal medulla seems to be more sensitive to deoxycholate than the renal cortex. Deoxycholate also induces slightly the disappearance of some 14C radioactivity from phosphatidylethanolamine and phosphatidylcholine, which might reflect activation of phospholipase A2. The activity of the phospholipase C could constitute the first step in the sequence of reactions that leads to the release of arachidonic acid.

The intracellular concentrations of polyphosphoinositides and inositol phosphates were determined, and their role in growth factor-initiated cell division was investigated in a Chinese hamster ovary cell inositol auxotroph (CHO-K1-Ins). Metabolic labeling experiments during inositol starvation of CHO-K1-Ins cells showed that 1) the lipid-linked inositol component was maintained at the expense of the soluble inositol pool, 2) the decreasing cellular content of phosphatidylinositol was replaced by phosphatidylglycerol, and 3) the concentrations of inositol polyphosphates and polyphosphoinositides were conserved at the expense of inositol and phosphatidylinositol. These data show that homeostatic mechanisms exist for the maintenance of the polyphosphoinositide and inositol phosphate pools at the expense of inositol and phosphatidylinositol. The addition of alpha-thrombin to growth-arrested (serum-starved) CHO-K1-Ins cells stimulated the incorporation of [3H]thymidine into DNA to the same extent as that observed following serum readdition. gamma-Thrombin was also an effective mitogen, but active site-inhibited alpha-thrombin was not. Both alpha- and gamma-thrombin, but not catalytic site-inhibited alpha-thrombin, initiated phosphatidylinositol turnover in vivo and increased phosphatidylinositol 4,5-bisphosphate phospholipase C activity in vitro. Serum and insulin were potent CHO-K1-Ins cell mitogens, but neither triggered phosphatidylinositol turnover in vivo nor activated phospholipase C in vitro. The activation of phospholipase C plays a determinant role in thrombin-initiated cell cycle progression in Chinese hamster ovary cells, although other growth factor-signaling pathways exist that are independent of polyphosphoinositide catabolism.

Cyclic inositol-1,2-phosphate was found to be present in tissue culture cells -SV40 transformed- at an approximate concentration of 10−6 molar. Of the water soluble radioactive split products of phosphatidylinositol metabolism more than 90% represents glycerylphosphorylinositol. In pulse chase experiments with [3H]glycerol and 32P-phosphate changes in the 3 H 32 P ratio of phosphatidylinositol were investigated during the biphasic decomposition of this lipid. On the basis of the results obtained, phosphatidylinositol turnover and a model of information exchange between membranes are discussed.

Thrombin, nucleotides, and chelators elicited a phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity that was associated with human platelet membranes. Both alpha- and gamma-thrombin enhanced phospholipase C activity, whereas active site-inhibited alpha-thrombin did not stimulate PtdIns-P2 hydrolysis. PtdIns-P2 phospholipase C was also activated by nucleoside triphosphates, citrate, EDTA, and NaF. Magnesium was an inhibitor of PtdIns-P2 hydrolysis stimulated by nucleotides and chelators. Only PtdIns-P2 was degraded by the phospholipase C activated by alpha-thrombin, nucleotides, and chelators. The soluble fraction phospholipase C activity was also stimulated at low protein concentrations by nucleotides; however, soluble fraction phospholipase C activity cleaved both PtdIns-P2 and phosphatidylinositol 4-phosphate and was inhibited by chelators, suggesting the presence of a different enzyme in this compartment. The pH optimum for the membrane-associated phospholipase C in the presence of alpha-thrombin or nucleotides was 6.0, and the PtdIns-P2 phospholipase C was inhibited by neomycin and high detergent concentrations. Guanine nucleotides did not synergistically activate phospholipase C in the presence of alpha-thrombin. The characteristics of the membrane-associated PtdIns-P2 phospholipase C suggest that this enzyme is involved in platelet activation by the low-affinity alpha- or gamma-thrombin-dependent pathway.

We studied the influence of platelet-derived growth factor (PDGF) on diacylglycerol phosphorylation in Swiss 3T3 cells. Rates of incorporation of 32P into phosphatidic acid (PA) and phosphatidylinositol (PtdIns) were determined in prelabeled cells into which sn-1,2-didecanoylglycerol (diC10) had been introduced. PDGF stimulated the formation of [32P]PA and -PtdIns from endogenous substrates but decreased the formation of [32P]PA10 and -PtdIns10. Direct measurements of diacylglycerol phosphorylation in lysates of quiescent and stimulated cells showed that PDGF stimulated the phosphorylation of endogenous diacylglycerol 2-fold in parallel with diacylglycerol accumulation but decreased by 50% the phosphorylation of diC10. Total diacylglycerol kinase activity, measured in a mixed micellar assay, was not changed by PDGF treatment. The maximum activity of diacylglycerol kinase exceeded that needed to phosphorylate all of the endogenous diacylglycerol, suggesting that the PDGF-dependent increase in diacylglycerol mass would account for the increase in PA formation. The increased mass of diacylglycerol also could explain the inhibition of diC10 phosphorylation, via substrate competition. The predominant species of endogenous diacylglycerol was 1-stearoyl-2-arachidonoyl-glycerol (18:0/20:4 diacylglycerol). In mixed micelles, the rate of phosphorylation of 18:0/20:4 diacylglycerol was 8-fold higher than that of diC10, and the 18:0/20:4 species competed with diC10 for phosphorylation. Studies showed that a membrane-bound enzyme accounted for the PDGF effect on PA formation; there was no evidence for translocation of cytosolic enzyme to the membrane. The results support these conclusions: 1) PDGF stimulates the phosphorylation of cellular diacylglycerol by promoting a transient accumulation of this lipid. 2) The stimulated phosphorylation is catalyzed by a diacylglycerol kinase that preferentially phosphorylates 18:0/20:4 diacylglycerol over diC10. 3) The diacylglycerol kinase responsible for the PDGF effect is membrane-bound.

We recently described the insulin-dependent release of a carbohydrate substance from plasma membranes which regulated certain intracellular enzymes (Saltiel, A. R., and Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5793-5797). This enzyme-modulating substance appeared to arise from the phosphodiesterase hydrolysis of a novel inositol-containing glycolipid. This is supported by observations that insulin stimulated the rapid generation of [3H]myristate-labeled diacylglycerol in cultured BC3Hl myocytes. Myristoyl diacylglycerol production in these cells was unaffected by epinephrine, although arachidonate-labeled diacylglycerol was rapidly produced in response to stimulation by this alpha-1 adrenergic agent. The production of distinct species of diacylglycerol was apparently due to hormonally specific hydrolysis of different precursors. A novel glycolipid was identified on silica TLC or high pressure liquid chromatography which served as a substrate for the insulin-stimulated phosphodiesterase reaction. This glycolipid was metabolically labeled with radioactive inositol, glucosamine, and myristic acid, suggesting a phosphatidylinositol (PI)-glycan structure. Treatment of this glycolipid with a PI-specific phospholipase C resulted in the generation of two products: an inositol phosphate-glycan which modulated the activity of the low Km cAMP phosphodiesterase and myristoyl diacylglycerol. Insulin caused the rapid hydrolysis of the PI-glycan, which was then apparently resynthesized. These data further suggest that insulin stimulates the activity of a phospholipase C which selectively hydrolyzes a novel PI-glycan, releasing a carbohydrate enzyme modulator as well as a unique species of diacylglycerol.

The subcellular distribution of the enzymes which phosphorylate phosphatidylinositol sequentially to form phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was investigated in rat liver. We demonstrate that whilst phosphatidylinositol kinase is present in Golgi, lysosomes and plasma membranes, the kinase that forms phosphatidylinositol 4,5-bisphosphate is localised predominantly at the plasma membrane. The role of the inositol lipid kinases in cell function is discussed.

The regulatory effect of inositol on inositol-1-phosphate synthase in Neurospora crassa strains was studied. Inositol represses enzyme production in the cultures of the wild type and that of the thermosensitive inositol-requiring mutant grown at 22 degrees C. Enzyme activity as well as the quantity of enzyme protein decreased sharply in both strains by increasing concentrations of inositol in the medium. Inositol-requiring strains used in our experiments can be divided into two groups. The first group produces a protein related immunologically to inositol phosphate synthase, but which is enzymatically inactive. The synthesis of this defective enzyme was also repressed by inositol. In the second group, this protein was found to be completely lacking, in both the thermosensitive mutant grown at 37 degrees C, and in a strain requiring inositol due to a reciprocal translocation. The thermostability and the cross immunoelectrophoresis of the enzyme suggest that in the case of the thermosensitive inositol-requiring mutant, the mutation did not occur in the structural gene of the enzyme, but its regulation was probably affected.

Platelets, and a variety of other cells, rapidly hydrolyze the phosphoinositides in response to stimulation by agonists. One of the products of hydrolysis of phosphatidylinositol 4,5-diphosphate is inositol 1,4,5-trisphosphate, which recently has been suggested to mediate intracellular Ca2+ mobilization. We have found that human platelets contain an enzyme that degrades inositol 1,4,5-trisphosphate. We have isolated this soluble enzyme and find that it hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate (Km = 30 microM, Vmax = 5.3 microM/min/mg of protein). The products of the reaction are inositol 1,4-diphosphate and phosphate. The apparent molecular weight of the enzyme is 38,000 as determined both by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol. This enzyme is specific for inositol 1,4,5-trisphosphate. Other water soluble inositol phosphates as well as phosphorylated sugars are not hydrolyzed, while the only inositol containing phospholipid hydrolyzed is phosphatidylinositol 4,5-diphosphate at a rate less than 1% that for inositol 4,5-trisphosphate. The inositol 1,4,5-trisphosphate 5-phosphomonoesterase requires Mg2+ for activity and is inhibited by Ca2+, Ki = 70 microM. Li+, up to 40 mM, has no effect on enzyme activity. The duration and magnitude of any inositol 1,4,5-trisphosphate response in stimulated platelets may be determined by the activity of this enzyme.