Science.gov

Sample records for phospholipase-c pi-plc family

  1. Biochemical characterization of the tomato phosphatidylinositol-specific phospholipase C (PI-PLC) family and its role in plant immunity.

    PubMed

    Abd-El-Haliem, Ahmed M; Vossen, Jack H; van Zeijl, Arjan; Dezhsetan, Sara; Testerink, Christa; Seidl, Michael F; Beck, Martina; Strutt, James; Robatzek, Silke; Joosten, Matthieu H A J

    2016-09-01

    Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca(2+) ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

  2. Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance.

    PubMed

    Vossen, Jack H; Abd-El-Haliem, Ahmed; Fradin, Emilie F; van den Berg, Grardy C M; Ekengren, Sophia K; Meijer, Harold J G; Seifi, Alireza; Bai, Yuling; ten Have, Arjen; Munnik, Teun; Thomma, Bart P H J; Joosten, Matthieu H A J

    2010-04-01

    The perception of pathogen-derived elicitors by plants has been suggested to involve phosphatidylinositol-specific phospholipase-C (PI-PLC) signalling. Here we show that PLC isoforms are required for the hypersensitive response (HR) and disease resistance. We characterised the tomato [Solanum lycopersicum (Sl)] PLC gene family. Six Sl PLC-encoding cDNAs were isolated and their expression in response to infection with the pathogenic fungus Cladosporium fulvum was studied. We found significant regulation at the transcriptional level of the various SlPLCs, and SlPLC4 and SlPLC6 showed distinct expression patterns in C. fulvum-resistant Cf-4 tomato. We produced the encoded proteins in Escherichia coli and found that both genes encode catalytically active PI-PLCs. To test the requirement of these Sl PLCs for full Cf-4-mediated recognition of the effector Avr4, we knocked down the expression of the encoding genes by virus-induced gene silencing. Silencing of SlPLC4 impaired the Avr4/Cf-4-induced HR and resulted in increased colonisation of Cf-4 plants by C. fulvum expressing Avr4. Furthermore, expression of the gene in Nicotiana benthamiana enhanced the Avr4/Cf-4-induced HR. Silencing of SlPLC6 did not affect HR, whereas it caused increased colonisation of Cf-4 plants by the fungus. Interestingly, Sl PLC6, but not Sl PLC4, was also required for resistance to Verticillium dahliae, mediated by the transmembrane Ve1 resistance protein, and to Pseudomonas syringae, mediated by the intracellular Pto/Prf resistance protein couple. We conclude that there is a differential requirement of PLC isoforms for the plant immune response and that Sl PLC4 is specifically required for Cf-4 function, while Sl PLC6 may be a more general component of resistance protein signalling.

  3. Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction.

    PubMed

    Martelli, A M; Fiume, R; Faenza, I; Tabellini, G; Evangelista, C; Bortul, R; Follo, M Y; Falà, F; Cocco, L

    2005-10-01

    Several studies have demonstrated the existence of an autonomous intranuclear phospho-inositide cycle that involves the activation of nuclear PI-PLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PI-PLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-beta1. Nuclear PI-PLC-beta1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-beta1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-beta1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.

  4. Heterotrimeric Gα subunit from wheat (Triticum aestivum), GA3, interacts with the calcium-binding protein, Clo3, and the phosphoinositide-specific phospholipase C, PI-PLC1.

    PubMed

    Khalil, Hala Badr; Wang, Zhejun; Wright, Justin A; Ralevski, Alexandra; Donayo, Ariel O; Gulick, Patrick J

    2011-09-01

    The canonical Gα subunit of the heterotrimeric G protein complex from wheat (Triticum aestivum), GA3, and the calcium-binding protein, Clo3, were revealed to interact both in vivo and in vitro and Clo3 was shown to enhance the GTPase activity of GA3. Clo3 is a member of the caleosin gene family in wheat with a single EF-hand domain and is induced during cold acclimation. Bimolecular Fluorescent Complementation (BiFC) was used to localize the interaction between Clo3 and GA3 to the plasma membrane (PM). Even though heterotrimeric G-protein signaling and Ca²⁺ signaling have both been shown to play a role in the response to environmental stresses in plants, little is known about the interaction between calcium-binding proteins and Gα. The GAP activity of Clo3 towards GA3 suggests it may play a role in the inactivation of GA3 as part of the stress response in plants. GA3 was also shown to interact with the phosphoinositide-specific phospholipase C, PI-PLC1, not only in the PM but also in the endoplasmic reticulum (ER). Surprisingly, Clo3 was also shown to interact with PI-PLC1 in the PM and ER. In vitro analysis of the protein-protein interaction showed that the interaction of Clo3 with GA3 and PI-PLC1 is enhanced by high Ca²⁺ levels. Three-way affinity characterizations with GA3, Clo3 and PI-PLC1 showed the interaction with Clo3 to be competitive, which suggests that Clo3 may play a role in the Ca²⁺-triggered feedback regulation of both GA3 and PI-PLC1. This hypothesis was further supported by the demonstration that Clo3 has GAP activity with GA3.

  5. 3Beta-hydroxy-6-aza-cholestane and related analogues as phosphatidylinositol specific phospholipase C (PI-PLC) inhibitors with antitumor activity.

    PubMed

    Xie, W; Peng, H; Zalkow, L H; Li, Y H; Zhu, C; Powis, G; Kunkel, M

    2000-04-01

    6-Aza steroid analogues were synthesized as PI-PLC inhibitors. The most active compound, 3beta-hydroxy-6-aza-cholestane (1) showed potent PI-PLC inhibition (IC50 = 1.8 microM), similar to that of the commercially available steroid analogue U73122 (IC50 = 1-2.1 microM). Compound 1 exhibited significant growth inhibition effects (IC50 = 1.3 microM in each case) against MCF-7 and HT-29 cancer cells in in vitro cell culture. Compound 1 also inhibited the in vitro adhesion and transmigration of HT-1080 fibrosarcoma cells at 2.5 and 5.0 microM, respectively. In vivo, compound 1, at 1 mg/kg/day, reduced the volume of MCF-7 tumors in xenograft models, without weight loss in mice. Structure activity relationships of this series of compounds revealed that a hydrophobic cholesteryl side chain, 3beta-hydroxy group and a C-6 nitrogen containing a hydrogen atom at position-6 are crucial for activity. N-Maleic amidoacid derivative 11 also exhibited weak inhibition (IC50 = 16.2 microM).

  6. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    PubMed

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  7. Plant phospholipase C family: Regulation and functional role in lipid signaling.

    PubMed

    Singh, Amarjeet; Bhatnagar, Nikita; Pandey, Amita; Pandey, Girdhar K

    2015-08-01

    Phospholipase C (PLC), a major membrane phospholipid hydrolyzing enzyme generates signaling messengers such as diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) in animals, and their phosphorylated forms such as phosphatidic acid (PA) and inositol hexakisphosphate (IP6) are thought to regulate various cellular processes in plants. Based on substrate specificity, plant PLC family is sub-divided into phosphatidylinositol-PLC (PI-PLC) and phosphatidylcholine-PLC (PC-PLC) groups. The activity of plant PLCs is regulated by various factors and the major ones include, Ca(2+) concentration, phospholipid substrate, post-translational modifications and interacting proteins. Most of the PLC members have been localized at the plasma membrane, suited for their function of membrane lipid hydrolysis. Several PLC members have been implicated in various cellular processes and signaling networks, triggered in response to a number of environmental cues and developmental events in different plant species, which makes them potential candidates for genetically engineering the crop plants for stress tolerance and enhancing the crop productivity. In this review article, we are focusing mainly on the plant PLC signaling and regulation, potential cellular and physiological role in different abiotic and biotic stresses, nutrient deficiency, growth and development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Genome-Wide Analysis and Expression Profiling of the Phospholipase C Gene Family in Soybean (Glycine max)

    PubMed Central

    Zhou, Yonggang; Dong, Jinye; Chen, Huan; Dong, Yuanyuan; Wang, Nan; Li, Xiaowei; Li, Haiyan

    2015-01-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate to produce diacylglycerol and inositol 1,4,5-trisphosphate. It plays an important role in plant development and abiotic stress responses. However, systematic analysis and expression profiling of the phospholipase C (PLC) gene family in soybean have not been reported. In this study, 12 putative PLC genes were identified in the soybean genome. Soybean PLCs were found on chromosomes 2, 11, 14 and 18 and encoded 58.8–70.06 kD proteins. Expression pattern analysis by RT-PCR demonstrated that expression of the GmPLCs was induced by PEG, NaCl and saline-alkali treatments in roots and leaves. GmPLC transcripts accumulated specifically in roots after ABA treatment. Furthermore, GmPLC transcripts were analyzed in various tissues. The results showed that GmPLC7 was highly expressed in most tissues, whereas GmPLC12 was expressed in early pods specifically. In addition, subcellular localization analysis was carried out and confirmed that GmPLC10 was localized in the plasma membrane in Nicotiana benthamiana. Our genomic analysis of the soybean PLC family provides an insight into the regulation of abiotic stress responses and development. It also provides a solid foundation for the functional characterization of the soybean PLC gene family. PMID:26421918

  9. 1p36.32 rearrangements and the role of PI-PLC η2 in nervous tumours.

    PubMed

    Lo Vasco, Vincenza Rita

    2011-07-01

    Deletions in the distal region of the short arm of chromosome 1 (1p36) are widely diffuse, both in congenital 1p36 Deletion Syndrome and as somatic abnormalities in tumours. Rearrangements in 1p36 have been described in a broad spectrum of human neoplasias in addition to other chromosomal abnormalities. In neuroblastomas, wide hemizygous deletions in 1p36.23-1p36.32 have been described suggesting that the 1p36 region contains a tumour-suppressor gene involved in malignancy. A role for phosphoinositide (PI)-specific phospholipase C (PLC) η2, whose gene maps on 1p36.32, was suggested. PI-PLC η2 belongs to a family of enzymes related to the phosphoinositide signalling pathway, which provide an important intracellular signalling system involved in a variety of cell functions such as hormone secretion, neurotransmitter signal transduction, cell growth, membrane trafficking, ion channel activity, regulation of the cytoskeleton, cell cycle control and apoptosis. Expression of PI-PLC η2 occurs after birth and continues throughout the life. Synapse formation occurs during a short period of postnatal development. Thus, it is likely that PI-PLC η2 acts in formation and maintenance of the neuronal network in the brain. The fact that PI-PLC η2, a highly neuron-specific isozyme, is abundantly expressed in the postnatal brain suggests the importance of PI-PLC η2 in formation and maintenance of the neuronal network in the postnatal brain. Further studies are required to verify the possible involvement of PI-PLC η2 mutation/deletion in central nervous tumour tissues presenting abnormalities of the 1p36 chromosomal band.

  10. Inhibition of phosphatidylinositol-specific phospholipase C: studies on synthetic substrates, inhibitors and a synthetic enzyme.

    PubMed

    Vizitiu, D; Kriste, A G; Campbell, A S; Thatcher, G R

    1996-01-01

    Enzyme inhibition studies on phosphatidylinositol-specific phospholipase C (PI-PLC) from B. Cereus were performed in order to gain an understanding of the mechanism of the PI-PLC family of enzymes and to aid inhibitor design. Inhibition studies on two synthetic cyclic phosphonate analogues (1,2) of inositol cyclic-1:2-monophosphate (cIP), glycerol-2-phosphate and vanadate were performed using natural phosphatidylinositol (PI) substrate in Triton X100 co-micelles and an NMR assay. Further inhibition studies on PI-PLC from B. Cereus were performed using a chromogenic, synthetic PI analogue (DPG-PI), an HPLC assay and Aerosol-OT (AOT), phytic acid and vanadate as inhibitors. For purposes of comparison, a model PI-PLC enzyme system was developed employing a synthetic Cu(II)-metallomicelle and a further synthetic PI analogue (IPP-PI). The studies employing natural PI substrate in Triton X100 co-micelles and synthetic DPG-PI in the absence of surfactant indicate three classes of PI-PLC inhibitors: (1) active-site directed inhibitors (e.g. 1,2); (2) water-soluble polyanions (e.g. tetravanadate, phytic acid); (3) surfactant anions (e.g. AOT). Three modes of molecular recognition are indicated to be important: (1) active site molecular recognition; (2) recognition at an anion-recognition site which may be the active site, and; (3) interfacial (or hydrophobic) recognition which may be exploited to increase affinity for the anion-recognition site in anionic surfactants such as AOT. The most potent inhibition of PI-PLC was observed by tetravanadate and AOT. The metallomicelle model system was observed to mimic PI-PLC in reproducing transesterification of the PI analogue substrate to yield cIP as product and in showing inhibition by phytic acid and AOT.

  11. Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with lipopolysaccharide.

    PubMed

    Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Fumagalli, Lorenzo; Cocco, L

    2010-04-01

    Signal transduction pathways, involved in cell cycle and activities, depend on various components including lipid signalling molecules, such as phosphoinositides and related enzymes. Many evidences support the hypothesis that inositol lipid cycle is involved in astrocytes activation during neurodegeneration. Previous studies investigated the pattern of expression of phosphoinositide-specific phospholipase C (PI-PLC) family isoforms in astrocytes, individuating in cultured neonatal rat astrocytes, supposed to be quiescent cells, the absence of some isoforms, accordingly to their well known tissue specificity. The same study was conducted in cultured rat astrocytoma C6 cells and designed a different pattern of expression of PI-PLCs in the neoplastic counterpart, accordingly to literature suggesting a PI signalling involvement in tumour progression. It is not clear the role of PI-PLC isoforms in inflammation; recent data demonstrate they are involved in cytokines production, with special regard to IL-6. PI-PLCs expression in LPS treated neonatal rat astrocytes performed by using RT-PCR, observed at 3, 6, 18 and 24 h intervals, expressed: PI-PLC beta1, beta4 and gamma1 in all intervals analysed; PI-PLC delta1 at 6, 18 and 24 h; PI-PLC delta3 at 6 h after treatment. PI-PLC beta3, delta4 and epsilon, present in untreated astrocytes, were not detected after LPS treatment. Immunocytochemical analysis, performed to visualize the sub-cellular distribution of the expressed isoforms, demonstrated different patterns of localisation at different times of exposure. These observations suggest that PI-PLCs expression and distribution may play a role in ongoing inflammation process of CNS.

  12. The plant non-specific phospholipase C gene family. Novel competitors in lipid signalling.

    PubMed

    Pokotylo, Igor; Pejchar, Přemysl; Potocký, Martin; Kocourková, Daniela; Krčková, Zuzana; Ruelland, Eric; Kravets, Volodymyr; Martinec, Jan

    2013-01-01

    Non-specific phospholipases C (NPCs) were discovered as a novel type of plant phospholipid-cleaving enzyme homologous to bacterial phosphatidylcholine-specific phospholipases C and responsible for lipid conversion during phosphate-limiting conditions. The six-gene family was established in Arabidopsis, and growing evidence suggests the involvement of two articles NPCs in biotic and abiotic stress responses as well as phytohormone actions. In addition, the diacylglycerol produced via NPCs is postulated to participate in membrane remodelling, general lipid metabolism and cross-talk with other phospholipid signalling systems in plants. This review summarises information concerning this new plant protein family and focusses on its sequence analysis, biochemical properties, cellular and tissue distribution and physiological functions. Possible modes of action are also discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Expression pattern and sub-cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U-73122 in rat astrocytoma cells.

    PubMed

    Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Panetta, Barbara; Fumagalli, Lorenzo; Cocco, Lucio

    2010-07-01

    Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellular distribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells.

  14. Phosphoinositide-specific phospholipase C in oat roots: association with the actin cytoskeleton.

    PubMed

    Huang, Chiung-Hua; Crain, Richard C

    2009-10-01

    Phosphoinositide-specific phospholipase C (PI-PLC) activities are involved in mediating plant cell responses to environmental stimuli. Two variants of PI-PLC have been partially purified from the roots of oat seedlings; one cytosolic and one particulate. Although the cytosolic enzyme was significantly purified, the activity still co-migrated with a number of other proteins on heparin HPLC and also on size-exclusion chromatography. The partially purified PI-PLC was tested by Western blotting, and we found that actin and actin-binding proteins, profilin and tropomyosin, co-purified with cytosolic phospholipase C. After a non-ionic detergent (Triton X-100) treatment, PI-PLC activities still remained with the actin cytoskeleton. The effects of phalloidin and F-buffer confirmed this association; these conditions, which favor actin polymerization, decreased the release of PI-PLC from the cytoskeleton. The treatments of latrunculin and G-buffer, the conditions that favor actin depolymerization, increased the release of PI-PLC from the cytoskeleton. These results suggest that oat PI-PLC associates with the actin cytoskeleton.

  15. Ca2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C.

    PubMed

    King-Keller, Sharon; Moore, Christina A; Docampo, Roberto; Moreno, Silvia N J

    2015-05-01

    We characterized a phosphoinositide phospholipase C (PI-PLC) from the procyclic form (PCF) of Trypanosoma brucei. The protein contains a domain organization characteristic of typical PI-PLCs, such as X and Y catalytic domains, an EF-hand calcium-binding motif, and a C2 domain, but it lacks a pleckstrin homology (PH) domain. In addition, the T. brucei PI-PLC (TbPI-PLC) contains an N-terminal myristoylation consensus sequence found only in trypanosomatid PI-PLCs. A peptide containing this N-terminal domain fused to green fluorescent protein (GFP) was targeted to the plasma membrane. TbPI-PLC enzymatic activity was stimulated by Ca(2+) concentrations below the cytosolic levels in the parasite, suggesting that the enzyme is constitutively active. TbPI-PLC hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2), with a higher affinity for PIP2. We found that modification of a single amino acid in the EF-hand motif greatly affected the protein's Ca(2+) sensitivity and substrate preference, demonstrating the role of this motif in Ca(2+) regulation of TbPI-PLC. Endogenous TbPI-PLC localizes to intracellular vesicles and might be using an intracellular source of PIP2. Knockdown of TbPI-PLC expression by RNA interference (RNAi) did not result in growth inhibition, although enzymatic activity was still present in parasites, resulting in hydrolysis of PIP2 and a contribution to the inositol 1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) pathway. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. The ability of Listeria monocytogenes PI-PLC to facilitate escape from the macrophage phagosome is dependent on host PKCbeta.

    PubMed

    Poussin, Mathilde A; Leitges, Michael; Goldfine, Howard

    2009-01-01

    Listeria monocytogenes are facultative intracellular pathogenic bacteria that can infect macrophages as well as non-professional phagocytes. After entry in the host cell, the bacteria escape from the phagosome into the cytoplasm. In murine macrophages and in cell lines derived from these cells, escape of L. monocytogenes from the phagosome is absolutely dependent on listeriolysin O (LLO) and facilitated by a secreted phosphatidylinositol-specific phospholipase C (PI-PLC). Work in this laboratory has previously demonstrated a LLO and PI-PLC-dependent translocation of host PKCbeta isoforms. Pharmacological inhibition of PKCbeta resulted in a significant reduction in permeabilization of the phagosome, and in the number of bacteria reaching the cytosol. These findings led to the prediction that the bacterial PI-PLC promotes escape through the production of diacylglycerol leading to the activation of host PKCbeta. To test this hypothesis, bone marrow-derived macrophages (BMMf) obtained from PKCbeta knockout (PKCbetaKO) or C57Bl/6 mice were infected with L. monocytogenes. We observed that wild-type L. monocytogenes escapes from the phagosome of PKCbetaKO BMMf as well as from C57Bl/6 BMMf. However, in PKCbetaKO BMMf, L. monocytogenes uses a PI-PLC-independent, but phosphatidylcholine-preferring PLC (PC-PLC)-dependent pathway to facilitate escape. These findings strongly support the hypothesis that PI-PLC promotes escape through mobilization of host PKCbeta.

  17. Revealing Transient Interactions between Phosphatidylinositol-specific Phospholipase C and Phosphatidylcholine--Rich Lipid Vesicles

    NASA Astrophysics Data System (ADS)

    Yang, Boqian; He, Tao; Grauffel, Cédric; Reuter, Nathalie; Roberts, Mary; Gershenson, Anne

    2013-03-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes transiently interact with target membranes. Previous fluorescence correlation spectroscopy (FCS) experiments showed that Bacillus thuringiensis PI-PLC specifically binds to phosphatidylcholine (PC)-rich membranes and preferentially interacts with unilamellar vesicles that show larger curvature. Mutagenesis studies combined with FCS measurements of binding affinity highlighted the importance of interfacial PI-PLC tyrosines in the PC specificity. All-atom molecular dynamics simulations of PI-PLC performed in the presence of a PC membrane indicate these tyrosines are involved in specific cation-pi interactions with choline headgroups. To further understand those transient interactions between PI-PLC and PC-rich vesicles, we monitor single fluorescently labeled PI-PLC proteins as they cycle on and off surface-tethered small unilamellar vesicles using total internal reflection fluorescent microscopy. The residence times on vesicles along with vesicle size information, based on vesicle fluorescence intensity, reveal the time scales of PI-PLC membrane interactions as well as the curvature dependence. The PC specificity and the vesicle curvature dependence of this PI-PLC/membrane interaction provide insight into how the interface modulates protein-membrane interactions. This work was supported by the National Institute of General Medical Science of the National Institutes of Health (R01GM060418).

  18. Two families of extracellular phospholipase C genes are present in aspergilli.

    PubMed

    Tuckwell, Danny; Lavens, Sandra E; Birch, Mike

    2006-10-01

    Fungi secrete extracellular enzymes to enable them to harvest nutrients from the environment. In the case of pathogenic fungi these enzymes can also be pathogenesis factors. Here we report the identification in fungi of a complex family of extracellular phospholipase C (PLC) enzymes, homologous to the Pseudomonas aeruginosa PLCH_PSEAE. Database searches and phylogenetic analysis showed that the PLCs clustered into two groups with different evolutionary histories. One group, subdivided into PLC-A, -B, -C and -D, was found only in aspergilli and Neosartorya fischeri. Each species only ever showed three of the four PLCs except N. fischeri which had all four PLCs plus duplicate PLC-A, -B and -C genes. Modelling studies indicated that these PLCs had mechanistic similarities to phosphoesterases and aryl sulphatases, but that they probably did not differ in substrate specificity. The second group, PLC-E, was seen in a wider range of fungi including some species of aspergilli and was always found in a head-to-head arrangement with a copper oxidase, similar to the laccases. The PLC genes appear to have arisen from separate gene transfer events from bacteria or lower eukaryotes. Thus, aspergilli have acquired PLCs twice in the course of evolution.

  19. Phosphatidylinositol-specific phospholipase C of Bacillus cereus: cloning, sequencing, and relationship to other phospholipases.

    PubMed Central

    Kuppe, A; Evans, L M; McMillen, D A; Griffith, O H

    1989-01-01

    The phosphatidylinositol (PI)-specific phospholipase C (PLC) of Bacillus cereus was cloned into Escherichia coli by using monoclonal antibody probes raised against the purified protein. The enzyme is specific for hydrolysis of the membrane lipid PI and PI-glycan-containing membrane anchors, which are important structural components of one class of membrane proteins. The protein expressed in E. coli comigrated with B. cereus PI-PLC in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as detected by immunoblotting, and conferred PI-PLC activity on the host. This enzyme activity was inhibited by PI-PLC-specific monoclonal antibodies. The nucleotide sequence of the PI-PLC gene suggests that this secreted bacterial protein is synthesized as a larger precursor with a 31-amino-acid N-terminal extension to the mature enzyme of 298 amino acids. From analysis of coding and flanking sequences of the gene, we conclude that the PI-PLC gene does not reside next to the gene cluster of the other two secreted phospholipases C on the bacterial chromosome. The deduced amino acid sequence of the B. cereus PI-PLC contains a stretch of significant similarity to the glycosylphosphatidylinositol-specific PLC of Trypanosoma brucei. The conserved peptide is proposed to play a role in the function of these enzymes. Images PMID:2509427

  20. Structure-activity relationship of aza-steroids as PI-PLC inhibitors.

    PubMed

    Xie, W; Peng, H; Kim, D I; Kunkel, M; Powis, G; Zalkow, L H

    2001-05-01

    A number of aza-steroids were synthesized as potent phosphatidylinositol phospholipase C (PI-PLC) inhibitors. The epimeric mixtures 22,25-diazacholesterol (8a) and 3beta-hydroxy-22,25-diazacholestane (8b) were among the most active of these inhibitors, with IC(50) values of 7.4 and 7.5 microM, respectively. The 20alpha epimer, 8a2 (IC(50)=0.64 microM), whose stereochemistry at C-20 coincides with that of cholesterol, was found 50 times more potent than the 20beta epimer, 8a1 (IC(50)=32.2 microM). In diaza-estrone derivatives, the 3-methoxy group on the aromatic A-ring of 23 exhibited moderate PI-PLC inhibitory activity (IC(50)=19.7 microM), while compound with a free hydroxyl group (21) was inactive. However, in diaza-pregnane derivatives, epimers with a 3-hydroxyl group (8a, IC(50)=7.4 microM) exhibited more potent PI-PLC inhibitory activity than their counterparts with 3-methoxyl group on the non-aromatic A-ring (26, IC(50)=17.4 microM). We have illustrated in our previous publication that 3-hydroxyl-6-aza steroids are potent PI-PLC inhibitors.(3) However, simultaneous presence of the 6-aza and 22,25-diaza moieties in one molecule as in 13, led to loss of activity. Epimeric mixture 8a showed selective growth inhibition effects in the NCI in vitro tumor cell screen with a mean GI(50) value (MG-MID) of 5.75 microM for 54 tumors.

  1. Salicylic acid modulates levels of phosphoinositide dependent-phospholipase C substrates and products to remodel the Arabidopsis suspension cell transcriptome

    PubMed Central

    Ruelland, Eric; Pokotylo, Igor; Djafi, Nabila; Cantrel, Catherine; Repellin, Anne; Zachowski, Alain

    2014-01-01

    Basal phosphoinositide-dependent phospholipase C (PI-PLC) activity controls gene expression in Arabidopsis suspension cells and seedlings. PI-PLC catalyzes the production of phosphorylated inositol and diacylglycerol (DAG) from phosphoinositides. It is not known how PI-PLC regulates the transcriptome although the action of DAG-kinase (DGK) on DAG immediately downstream from PI-PLC is responsible for some of the regulation. We previously established a list of genes whose expression is affected in the presence of PI-PLC inhibitors. Here this list of genes was used as a signature in similarity searches of curated plant hormone response transcriptome data. The strongest correlations obtained with the inhibited PI-PLC signature were with salicylic acid (SA) treatments. We confirm here that in Arabidopsis suspension cells SA treatment leads to an increase in phosphoinositides, then demonstrate that SA leads to a significant 20% decrease in phosphatidic acid, indicative of a decrease in PI-PLC products. Previous sets of microarray data were re-assessed. The SA response of one set of genes was dependent on phosphoinositides. Alterations in the levels of a second set of genes, mostly SA-repressed genes, could be related to decreases in PI-PLC products that occur in response to SA action. Together, the two groups of genes comprise at least 40% of all SA-responsive genes. Overall these two groups of genes are distinct in the functional categories of the proteins they encode, their promoter cis-elements and their regulation by DGK or phospholipase D. SA-regulated genes dependent on phosphoinositides are typical SA response genes while those with an SA response that is possibly dependent on PI-PLC products are less SA-specific. We propose a model in which SA inhibits PI-PLC activity and alters levels of PI-PLC products and substrates, thereby regulating gene expression divergently. PMID:25426125

  2. Differences in the release of 5'-nucleotidase and alkaline phosphatase from plasma membrane of several cell types by PI-PLC.

    PubMed

    Zekri, M; Harb, J; Bernard, S; Poirier, G; Devaux, C; Meflah, K

    1989-01-01

    1. We have compared the effect of phosphatidyl inositol specific phospholipase C (PI-PLC) on the attachment of both 5'-nucleotidase and alkaline phosphatase to the liver plasma membrane from different species. 2. Our results demonstrate differences in the susceptibilities of both enzymes to PI-PLC treatment in relation to their origin. 3. These results were confirmed by immunoblotting using polyclonal anti-5'-nucleotidase antibodies. 4. In addition, in a single animal, susceptibility of both enzymes to PI-PLC treatment is different from one tissue to another. 5. The different percentages of released enzymes could be explained either by a polymorphism in the anchoring of these proteins at the cell surface membrane, or by a different steric hindrance or environment at the cleavage site itself.

  3. The structure of a calcium-dependent phosphoinositide-specific phospholipase C from Pseudomonas sp. 62186, the first from a Gram-negative bacterium.

    PubMed

    Moroz, Olga V; Blagova, Elena; Lebedev, Andrey A; Nørgaard, Allan; Segura, Dorotea R; Blicher, Thomas H; Brask, Jesper; Wilson, Keith S

    2017-01-01

    Bacterial phosphoinositide-specific phospholipases C (PI-PLCs) are the smallest members of the PI-PLC family, which includes much larger mammalian enzymes responsible for signal transduction as well as enzymes from protozoan parasites, yeast and plants. Eukaryotic PI-PLCs have calcium in the active site, but this is absent in the known structures of Gram-positive bacteria, where its role is instead played by arginine. In addition to their use in a number of industrial applications, the bacterial enzymes attract special interest because they can serve as convenient models of the catalytic domains of eukaryotic enzymes for in vitro activity studies. Here, the structure of a PI-PLC from Pseudomonas sp. 62186 is reported, the first from a Gram-negative bacterium and the first of a native bacterial PI-PLC with calcium present in the active site. Solution of the structure posed particular problems owing to the low sequence identity of available homologous structures. Its dependence on calcium for catalysis makes this enzyme a better model for studies of the mammalian PI-PLCs than the previously used calcium-independent bacterial PI-PLCs.

  4. Phosphoinositide-specific phospholipase C in health and disease.

    PubMed

    Cocco, Lucio; Follo, Matilde Y; Manzoli, Lucia; Suh, Pann-Ghill

    2015-10-01

    Phospholipases are widely occurring and can be found in several different organisms, including bacteria, yeast, plants, animals, and viruses. Phospholipase C (PLC) is a class of phospholipases that cleaves phospholipids on the diacylglycerol (DAG) side of the phosphodiester bond producing DAGs and phosphomonoesters. Among PLCs, phosphoinositide-specific PLC (PI-PLC) constitutes an important step in the inositide signaling pathways. The structures of PI-PLC isozymes show conserved domains as well as regulatory specific domains. This is important, as most PI-PLCs share a common mechanism, but each of them has a peculiar role and can have a specific cell distribution that is linked to a specific function. More importantly, the regulation of PLC isozymes is fundamental in health and disease, as there are several PLC-dependent molecular mechanisms that are associated with the activation or inhibition of important physiopathological processes. Moreover, PI-PLC alternative splicing variants can play important roles in complex signaling networks, not only in cancer but also in other diseases. That is why PI-PLC isozymes are now considered as important molecules that are essential for better understanding the molecular mechanisms underlying both physiology and pathogenesis, and are also potential molecular targets useful for the development of innovative therapeutic strategies.

  5. Comparative diagnostic efficacy of recombinant LLO and PI-PLC-based ELISAs for detection of listeriosis in animals.

    PubMed

    Suryawanshi, Rahul D; Malik, Satya Veer Singh; Jayarao, Bhushan; Chaudhari, Sandeep P; Savage, Emily; Vergis, Jess; Kurkure, Nitin V; Barbuddhe, Sukhadeo B; Rawool, Deepak B

    2017-06-01

    The present study for the first time evaluates the serodiagnostic efficacy of two recombinant antigens namely, listeriolysin O (rLLO) and phosphatidyl-inositol phospholipase C (rPI-PLC). Indirect ELISA with the above recombinant antigens was used on samples collected from bovines (n=106), goats (n=138) and pigs (n=92) having either a history of abortion, emaciation and/or apparently healthy animals. Isolation of Listeria was attempted from the blood samples using USDA-FSIS method. On screening of test sera by rLLO-based ELISA, antibodies against anti-listeriolysin O (ALLO) were observed in goats (22.46%), bovines (15.10%) and pigs (16.31%). As advocated, after adsorption of positive serum samples with streptolysin O (SLO), the seropositivity for ALLO was marginally reduced (p>0.05) in goats (21.73%) and bovines (10.38%), whereas, in pigs the reduction (5.43%) was significant (p<0.05). On the contrary, rPI-PLC-based ELISA revealed higher non-specific seropositivity for antilisterial antibodies in goats (45.65%), bovines (31.13%) and pigs (8.69%). Further, on comparing the seropositivity with isolation rate, of the 16 animals that were culturally-positive for L. monocytogenes, 15 showed ALLO positivity in unadsorbed as well as SLO-adsorbed sera by rLLO-based ELISA, however, rPI-PLC-based ELISA could detect seropositivity in only 5 animals. Moreover, rPI-PLC-based ELISA also showed seropositivity in those animals (7/30) that were culturally positive for other Listeria spp. In conclusion, rLLO can serve as a better antigen than rPI-PLC in ELISA for the serodiagnosis of listeriosis in animals; however, prior adsorption of test sera with SLO is required to avoid false positive results. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Emerging roles of phosphoinositide-specific phospholipases C in the ciliates Tetrahymena and Paramecium.

    PubMed

    Leondaritis, George; Galanopoulou, Dia

    2011-09-01

    Phospholipases C (PLCs) that hydrolyze inositol phospholipids regulate vital cellular functions in both eukaryotic and prokaryotic organisms. The PLC superfamily consists of eukaryotic phosphoinositide-specific PLCs (PI-PLCs), bacterial PLCs and trypanosomal PLCs.1 PI-PLCs hydrolyze phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)) to produce inositol-1,4,5-trisphosphate (Ins1,4,5P(3)) and constitute a hallmark feature of eukaryotic cells. In metazoa, this reaction is coupled to receptor signaling via specific PI-PLC isoforms and results in acute increase of cytosolic Ca(2+) levels by Ins1,4,5P(3)-sensitive Ca(2+) channels (IP(3)-receptors, IP3Rs).2 A striking result of many studies so far has been the presence of a single PI-PLC gene in all unicellular eukaryotes investigated, as opposed to expansion of PI-PLC isoforms in metazoa;3 this has suggested that a single housekeeping PI-PLC represents an archetypal and simplified form of PI-PLC signaling.3 Several studies however have noted a unique expansion of PI-PLC/IP3R pathway components in ciliates.4,5 In a recent paper we showed the presence of multiple functional PI-PLC genes in Tetrahymena thermophila and biochemical characterization, pharmacological studies and study of their expression patterns suggested that they are likely to serve distinct non-redundant roles.4 In this report we discuss these studies and how they advance our understanding of PI-PLC functions in ciliates.

  7. Emerging roles of phosphoinositide-specific phospholipases C in the ciliates Tetrahymena and Paramecium

    PubMed Central

    Leondaritis, George

    2011-01-01

    Phospholipases C (PLCs) that hydrolyze inositol phospholipids regulate vital cellular functions in both eukaryotic and prokaryotic organisms. The PLC superfamily consists of eukaryotic phosphoinositide-specific PLCs (PI-PLCs), bacterial PLCs and trypanosomal PLCs.1 PI-PLCs hydrolyze phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2) to produce inositol-1,4,5-trisphosphate (Ins1,4,5P3) and constitute a hallmark feature of eukaryotic cells. In metazoa, this reaction is coupled to receptor signaling via specific PI-PLC isoforms and results in acute increase of cytosolic Ca2+ levels by Ins1,4,5P3-sensitive Ca2+ channels (IP3-receptors, IP3Rs).2 A striking result of many studies so far has been the presence of a single PI-PLC gene in all unicellular eukaryotes investigated, as opposed to expansion of PI-PLC isoforms in metazoa;3 this has suggested that a single housekeeping PI-PLC represents an archetypal and simplified form of PI-PLC signaling.3 Several studies however have noted a unique expansion of PI-PLC/IP3R pathway components in ciliates.4,5 In a recent paper we showed the presence of multiple functional PI-PLC genes in Tetrahymena thermophila and biochemical characterization, pharmacological studies and study of their expression patterns suggested that they are likely to serve distinct non-redundant roles.4 In this report we discuss these studies and how they advance our understanding of PI-PLC functions in ciliates. PMID:22046467

  8. Bacterial phospholipases C.

    PubMed Central

    Titball, R W

    1993-01-01

    A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C. PMID:8336671

  9. End-product diacylglycerol enhances the activity of PI-PLC through changes in membrane domain structure.

    PubMed

    Ahyayauch, Hasna; Sot, Jesús; Collado, M Isabel; Huarte, Nerea; Requejo-Isidro, José; Alonso, Alicia; Goñi, Félix M

    2015-04-07

    Diacylglycerol (DAG)-induced activation of phosphatidylinositol-phospholipase C (PI-PLC) was studied with vesicles containing PI, either pure or in mixtures with dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, sphingomyelin, or galactosylceramide, used as substrates. At 22°C, DAG at 33 mol % increased PI-PLC activity in all of the mixtures, but not in pure PI bilayers. DAG also caused an overall decrease in diphenylhexatriene fluorescence polarization (decreased molecular order) in all samples, and increased overall enzyme binding. Confocal fluorescence microscopy of giant unilamellar vesicles of all of the compositions under study, with or without DAG, and quantitative evaluation of the phase behavior using Laurdan generalized polarization, and of enzyme binding to the various domains, indicated that DAG activates PI-PLC whenever it can generate fluid domains to which the enzyme can bind with high affinity. In the specific case of PI/dimyristoyl phosphatidylcholine bilayers at 22°C, DAG induced/increased enzyme binding and activation, but no microscopic domain separation was observed. The presence of DAG-generated nanodomains, or of DAG-induced lipid packing defects, is proposed instead for this system. In PI/galactosylceramide mixtures, DAG may exert its activation role through the generation of small vesicles, which PI-PLC is known to degrade at higher rates. In general, our results indicate that global measurements obtained using fluorescent probes in vesicle suspensions in a cuvette are not sufficient to elucidate DAG effects that take place at the domain level. The above data reinforce the idea that DAG functions as an important physical agent in regulating membrane and cell properties.

  10. Mammalian phospholipase C.

    PubMed

    Kadamur, Ganesh; Ross, Elliott M

    2013-01-01

    Phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate (PIP(2)) to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG). DAG and IP(3) each control diverse cellular processes and are also substrates for synthesis of other important signaling molecules. PLC is thus central to many important interlocking regulatory networks. Mammals express six families of PLCs, each with both unique and overlapping controls over expression and subcellular distribution. Each PLC also responds acutely to its own spectrum of activators that includes heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, Ca(2+), and phospholipids. Mammalian PLCs are autoinhibited by a region in the catalytic TIM barrel domain that is the target of much of their acute regulation. In combination, the PLCs act as a signaling nexus that integrates numerous signaling inputs, critically governs PIP(2) levels, and regulates production of important second messengers to determine cell behavior over the millisecond to hour timescale.

  11. Acylation-dependent Export of Trypanosoma cruzi Phosphoinositide-specific Phospholipase C to the Outer Surface of Amastigotes*

    PubMed Central

    de Paulo Martins, Vicente; Okura, Michael; Maric, Danijela; Engman, David M.; Vieira, Mauricio; Docampo, Roberto; Moreno, Silvia N. J.

    2010-01-01

    Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid-modified in its N terminus and localizes to the plasma membrane of amastigotes. Here, we show that TcPI-PLC is located onto the extracellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino acids are necessary and sufficient to target the fused GFP to the outer surface of the parasite. Mutagenesis of the predicted acylated residues confirmed that myristoylation of a glycine residue in the 2nd position and acyl modification of a cysteine in the 4th but not in the 8th or 15th position of the coding sequence are required for correct plasma membrane localization in T. cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the cysteine at the 8th position increased its flagellar localization. When expressed as fusion constructs with GFP, the N-terminal 6 and 10 amino acids fused to GFP are predominantly located in the cytosol and concentrated in a compartment that co-localizes with a Golgi complex marker. The N-terminal 20 amino acids of TcPI-PLC associate with lipid rafts when dually acylated. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell. PMID:20647312

  12. Arabidopsis phosphatidylinositol-phospholipase C2 (PLC2) is required for female gametogenesis and embryo development.

    PubMed

    Di Fino, Luciano M; D'Ambrosio, Juan Martín; Tejos, Ricardo; van Wijk, Ringo; Lamattina, Lorenzo; Munnik, Teun; Pagnussat, Gabriela C; Laxalt, Ana M

    2017-04-01

    AtPLC2 is an essential gene in Arabidopsis, since it is required for female gametogenesis and embryo development. AtPLC2 might play a role in cell division during embryo-sac development and early embryogenesis. Phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in signal transduction during plant development and in the response to various biotic- and abiotic stresses. The Arabidopsis PI-PLC gene family is composed of nine members, named PLC1 to PLC9. Here, we report that PLC2 is involved in female gametophyte development and early embryogenesis. Using two Arabidopsis allelic T-DNA insertion lines with different phenotypic penetrations, we observed both female gametophytic defects and aberrant embryos. For the plc2-1 mutant (Ws background), no homozygous plants could be recovered in the offspring from self-pollinated plants. Nonetheless, plc2-1 hemizygous mutants are affected in female gametogenesis, showing embryo sacs arrested at early developmental stages. Allelic hemizygous plc2-2 mutant plants (Col-0 background) present reduced seed set and embryos arrested at the pre-globular stage with abnormal patterns of cell division. A low proportion (0.8%) of plc2-2 homozygous mutants was found to escape lethality and showed morphological defects and disrupted megagametogenesis. PLC2-promoter activity was observed during early megagametogenesis, and after fertilization in the embryo proper. Immunolocalization studies in early stage embryos revealed that PLC2 is restricted to the plasma membrane. Altogether, these results establish a role for PLC2 in both reproductive- and embryo development, presumably by controlling mitosis and/or the formation of cell-division planes.

  13. Comprehensive genomic analysis and expression profiling of phospholipase C gene family during abiotic stresses and development in rice.

    PubMed

    Singh, Amarjeet; Kanwar, Poonam; Pandey, Amita; Tyagi, Akhilesh K; Sopory, Sudhir K; Kapoor, Sanjay; Pandey, Girdhar K

    2013-01-01

    Phospholipase C (PLC) is one of the major lipid hydrolysing enzymes, implicated in lipid mediated signaling. PLCs have been found to play a significant role in abiotic stress triggered signaling and developmental processes in various plant species. Genome wide identification and expression analysis have been carried out for this gene family in Arabidopsis, yet not much has been accomplished in crop plant rice. An exhaustive in-silico exploration of rice genome using various online databases and tools resulted in the identification of nine PLC encoding genes. Based on sequence, motif and phylogenetic analysis rice PLC gene family could be divided into phosphatidylinositol-specific PLCs (PI-PLCs) and phosphatidylcholine- PLCs (PC-PLC or NPC) classes with four and five members, respectively. A comparative analysis revealed that PLCs are conserved in Arabidopsis (dicots) and rice (monocot) at gene structure and protein level but they might have evolved through a separate evolutionary path. Transcript profiling using gene chip microarray and quantitative RT-PCR showed that most of the PLC members expressed significantly and differentially under abiotic stresses (salt, cold and drought) and during various developmental stages with condition/stage specific and overlapping expression. This finding suggested an important role of different rice PLC members in abiotic stress triggered signaling and plant development, which was also supported by the presence of relevant cis-regulatory elements in their promoters. Sub-cellular localization of few selected PLC members in Nicotiana benthamiana and onion epidermal cells has provided a clue about their site of action and functional behaviour. The genome wide identification, structural and expression analysis and knowledge of sub-cellular localization of PLC gene family envisage the functional characterization of these genes in crop plants in near future.

  14. Comprehensive Genomic Analysis and Expression Profiling of Phospholipase C Gene Family during Abiotic Stresses and Development in Rice

    PubMed Central

    Singh, Amarjeet; Kanwar, Poonam; Pandey, Amita; Tyagi, Akhilesh K.; Sopory, Sudhir K.; Kapoor, Sanjay; Pandey, Girdhar K.

    2013-01-01

    Background Phospholipase C (PLC) is one of the major lipid hydrolysing enzymes, implicated in lipid mediated signaling. PLCs have been found to play a significant role in abiotic stress triggered signaling and developmental processes in various plant species. Genome wide identification and expression analysis have been carried out for this gene family in Arabidopsis, yet not much has been accomplished in crop plant rice. Methodology/Principal Findings An exhaustive in-silico exploration of rice genome using various online databases and tools resulted in the identification of nine PLC encoding genes. Based on sequence, motif and phylogenetic analysis rice PLC gene family could be divided into phosphatidylinositol-specific PLCs (PI-PLCs) and phosphatidylcholine- PLCs (PC-PLC or NPC) classes with four and five members, respectively. A comparative analysis revealed that PLCs are conserved in Arabidopsis (dicots) and rice (monocot) at gene structure and protein level but they might have evolved through a separate evolutionary path. Transcript profiling using gene chip microarray and quantitative RT-PCR showed that most of the PLC members expressed significantly and differentially under abiotic stresses (salt, cold and drought) and during various developmental stages with condition/stage specific and overlapping expression. This finding suggested an important role of different rice PLC members in abiotic stress triggered signaling and plant development, which was also supported by the presence of relevant cis-regulatory elements in their promoters. Sub-cellular localization of few selected PLC members in Nicotiana benthamiana and onion epidermal cells has provided a clue about their site of action and functional behaviour. Conclusion/Significance The genome wide identification, structural and expression analysis and knowledge of sub-cellular localization of PLC gene family envisage the functional characterization of these genes in crop plants in near future. PMID

  15. In vitro distribution and characterization of membrane-associated PLD and PI-PLC in Brassica napus.

    PubMed

    Novotná, Zuzana; Martinec, Jan; Profotová, Bronislava; Zdárová, Stĕpánka; Kader, Jean-Claude; Valentová, Olga

    2003-02-01

    Two types of phospholipid degrading enzyme, phospholipase D (PLD; EC 3.1.4.4) and phosphatidyl- inositol-specific phospholipase C (PIP(2)-PLC; PI-PLC 3.1.4.11) were studied during the development of seeds and plants of Brassica napus. PLD exhibits two types of activity; polyphosphoinositide-requiring (PIP(2)-dependent PLD) and polyphosphoinositide-independent requiring millimolar concentrations of calcium (PLDalpha). Significantly different patterns of activity profiles were found for soluble and membrane-associated forms of all three enzymes within both processes. Membrane-associated PIP(2)-dependent PLD activity shows the opposite trend when compared to PLDalpha, while the highest PI-PLC activity appears in the same stages of development of seeds and plants as for PLDalpha. In subcellular fractions of hypocotyls of young plants, phospholipases were localized predominantly on plasma membranes. The biochemical characteristics (Ca(2+), pH) of all three enzymes associated with plasma membrane vesicles, isolated by partitioning in an aqueous dextran- polyethylene glycol two-phase system, are also described. Direct interaction of PLDalpha with G-proteins under in vitro conditions was not confirmed.

  16. Crystallization, optimization and preliminary X-ray characterization of a metal-dependent PI-PLC from Streptomyces antibioticus

    PubMed Central

    Jackson, Michael R.; Selby, Thomas L.

    2012-01-01

    A recombinant metal-dependent phosphatidylinositol-specific phospholipase C (PI-PLC) from Streptomyces antibioticus has been crystallized by the hanging-drop method with and without heavy metals. The native crystals belonged to the orthorhombic space group P222, with unit-cell parameters a = 41.26, b = 51.86, c = 154.78 Å. The X-ray diffraction results showed significant differences in the crystal quality of samples soaked with heavy atoms. Additionally, drop pinning, which increases the surface area of the drops, was also used to improve crystal growth and quality. The combination of heavy-metal soaks and drop pinning was found to be critical for producing high-quality crystals that diffracted to 1.23 Å resolution. PMID:23143254

  17. Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato1

    PubMed Central

    Kopka, Joachim; Pical, Christophe; Gray, Julie E.; Müller-Röber, Bernd

    1998-01-01

    Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 μm Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner. PMID:9449844

  18. Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion.

    PubMed

    Coonrod, S A; Naaby-Hansen, S; Shetty, J; Shibahara, H; Chen, M; White, J M; Herr, J C

    1999-03-15

    The effect of phosphatidyinositol-specific phospholipase C (PI-PLC) on mouse sperm-egg interaction was investigated in this study to determine if glycosyl-phosphatidylinositol (GPI)-anchored proteins are involved in mammalian fertilization. When both sperm and zona-intact oocytes were pretreated with a highly purified preparation of PI-PLC and coincubated, there was no significant effect on sperm-zona pellucida binding; however, fertilization was reduced from 59.6% (control group) to 2.8% (treatment group). A similar reduction in fertilization rates was found when zona-intact oocytes were treated with PI-PLC and washed prior to incubation with untreated sperm. The effect of PI-PLC on sperm binding and fusion with zona-free oocytes was then investigated. Treatment of sperm with PI-PLC had no significant effect on sperm-egg binding or fusion. However, treatment of eggs with PI-PLC significantly reduced sperm-egg binding and fusion from 6.2 bound and 2.1 fused sperm per egg in the control group to 2.1 bound and 0.02 fused sperm per egg in the treatment group. This decrease in sperm-egg binding and fusion depended on the dose of PI-PLC employed, with a maximal inhibitory effect on binding and fusion at 5 and 1 U/ml, respectively. PI-PLC-treated oocytes could be artificially activated by calcium ionophore, demonstrating that the oocytes were functionally viable following treatment. Furthermore, treatment of oocytes with PI-PLC did not reduce the immunoreactivity of the non-GPI-anchored egg surface integrin, alpha6beta1. Taken together, these observations support the hypothesis that PI-PLC affects fertilization by specifically releasing GPI-anchored proteins from the oolemma. In order to identify the oolemmal GPI-anchored proteins involved in fertilization, egg surface proteins were labeled with sulfo-NHS biotin, treated with PI-PLC, and analyzed by two-dimensional gel electrophoresis followed by avidin blotting. A prominent high-molecular-weight protein cluster

  19. Phosphoinositide-specific Phospholipase C β1 gene deletion in bipolar disorder affected patient.

    PubMed

    Lo Vasco, Vincenza Rita; Longo, Lucia; Polonia, Patrizia

    2013-03-01

    The involvement of phosphoinositides (PI) signal transduction pathway and related molecules, such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, in the pathophysiology of mood disorders is corroborated by a number of recent evidences. Our previous works identified the deletion of PLCB1 gene, which codifies for the PI-PLC β1 enzyme, in 4 out 15 patients affected with schizophrenia, and no deletion both in major depression affected patients and in normal controls. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with bipolar disorder. Deletion of PLCB1 was identified in one female patient.

  20. A plasma-membrane linker for the phosphoinositide-specific phospholipase C in tobacco plants.

    PubMed

    Nakamura, Kimiyo; Sano, Hiroshi

    2009-01-01

    We previously screened genes that were transcriptionally activated during the early stage of wound response in tobacco plants (Nicotiana tabacum), and isolated a particular clone, which encoded a membrane-located protein, designated as NtC7. Upon overexpression in tobacco plants, NtC7 conferred a marked tolerance to osmotic stress, suggesting it to be involved in maintenance of osmotic adjustments. In this study, we searched for proteins which interact with NtC7 by the yeast two-hybrid screening, and isolated a clone encoding phosphoinositide-specific phospholipase C, designated as NtPI-PLC. Physical interaction between NtC7 and C2 domain of NtPI-PLC was confirmed by the pull-down assay. Expression of fused protein to green-fluorescence protein in onion epidermal cell layers indicated both proteins to predominantly localize to the plasma membrane. Their interaction in planta was shown by the bimolecular fluorescence complementation, which exhibited a clear fluorescence of reconstituted yellow fluorescence protein. Transcripts of NtC7 and NtPI-PLC were markedly increased 30 to 60 min after wounding. PI-PLC is one of key enzymes in metabolism of inositol phospholipids, which function in signal transduction and also in response to stresses including osmotic changes. It was shown to localize to plasma-membrane and, to a lesser extent, to cytosol. However, molecular mechanism of membrane localization has remained to be determined, because of the apparent lack of domains for membrane association. The present results suggest that one of such mechanisms is tethering NtPI-PLC to the plasma membrane through interaction with NtC7, which possesses a transmembrane domain at the C-terminus.

  1. A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation.

    PubMed Central

    Payne, W E; Fitzgerald-Hayes, M

    1993-01-01

    We identified a putative Saccharomyces cerevisiae homolog of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the delta class of PI-PLC enzymes. The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segregation. plc1-1 cells showed a 10-fold increase in aberrant chromosome segregation compared with the wild type. Molecular analysis revealed that PLC1 encodes a predicted protein of 101 kDa with approximately 50 and 26% identity to the highly conserved X and Y domains of PI-PLC isozymes from humans, bovines, rats, and Drosophila melanogaster. The putative yeast protein also contains a consensus EF-hand domain that is predicted to bind calcium. Interestingly, the temperature-sensitive and chromosome missegregation phenotypes exhibited by plc1-1 cells were partially suppressed by exogenous calcium. Images PMID:8391635

  2. Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils

    PubMed Central

    White, Mark J.; Boyd, Jeffrey M.

    2014-01-01

    Staphylococcus aureus is an important human pathogen that employs a large repertoire of secreted virulence factors to promote disease pathogenesis. Many strains of S. aureus possess a plc gene that encodes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) capable of hydrolyzing PI and cleaving glycosyl-PI (GPI)-linked proteins from cell surfaces. Despite being secreted by virulent staphylococci, the contribution of PI-PLC to the capacity of S. aureus to cause disease remains undefined. Our goal in these studies was to understand PI-PLC in the context of S. aureus biology. Among a collection of genetically diverse clinical isolates of S. aureus, community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 secreted the most PI-PLC. Screening a collection of two-component system (TCS) mutants of S. aureus, we identified both the agr quorum-sensing system and the SrrAB TCS to be positive regulators of plc gene expression. Real-time PCR and PI-PLC enzyme assays of the TCS mutants, coupled with SrrA promoter binding studies, demonstrated that SrrAB was the predominant transcriptional activator of plc. Furthermore, plc regulation was linked to oxidative stress both in vitro and in vivo in a SrrAB-dependent manner. A Δplc mutant in a CA-MRSA USA300 background exhibited a survival defect in human whole blood and in isolated neutrophils. However, the same mutant strain displayed no survival defect in murine models of infection or murine whole blood. Overall, these data identify potential links between bacterial responses to the host innate immune system and to oxidative stress and suggest how PI-PLC could contribute to the pathogenesis of S. aureus infections. PMID:24452683

  3. Nuclear Phosphoinositide-Specific Phospholipase C β1 Controls Cytoplasmic CCL2 mRNA Levels in HIV-1 gp120-Stimulated Primary Human Macrophages

    PubMed Central

    Purificato, Cristina; Sabbatucci, Michela; Podo, Franca; Ramoni, Carlo; Gessani, Sandra; Fantuzzi, Laura

    2013-01-01

    HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of CCL2 from macrophages. In turn, this chemokine acts as an autocrine factor enhancing viral replication. In this study, we show for the first time that phosphoinositide-specific phospholipase C (PI-PLC) is required for the production of CCL2 triggered by gp120 in macrophages. Using a combination of confocal laser-scanner microscopy, pharmacologic inhibition, western blotting and fluorescence-activated cell sorter analysis, we demonstrate that gp120 interaction with CCR5 leads to nuclear localization of the PI-PLC β1 isozyme mediated by mitogen-activated protein kinase ERK-1/2. Notably, phosphatidylcholine-specific phospholipase C (PC-PLC), previously reported to be required for NF-kB-mediated CCL2 production induced by gp120 in macrophages, drives both ERK1/2 activation and PI-PLC β1 nuclear localization induced by gp120. PI-PLC β1 activation through CCR5 is also triggered by the natural chemokine ligand CCL4, but independently of ERK1/2. Finally, PI-PLC inhibition neither blocks gp120-mediated NF-kB activation nor overall accumulation of CCL2 mRNA, whereas it decreases CCL2 transcript level in the cytoplasm. These results identify nuclear PI-PLC β1 as a new intermediate in the gp120-triggered PC-PLC-driven signal transduction pathway leading to CCL2 secretion in macrophages. The finding that a concerted gp120-mediated signaling involving both PC- and PI-specific PLCs is required for the expression of CCL2 in macrophages suggests that this signal transduction pathway may also be relevant for the modulation of viral replication in these cells. Thus, this study may contribute to identify novel targets for therapeutic intervention in HIV-1 infection. PMID:23555755

  4. Nuclear phosphoinositide-specific phospholipase C β1 controls cytoplasmic CCL2 mRNA levels in HIV-1 gp120-stimulated primary human macrophages.

    PubMed

    Spadaro, Francesca; Cecchetti, Serena; Purificato, Cristina; Sabbatucci, Michela; Podo, Franca; Ramoni, Carlo; Gessani, Sandra; Fantuzzi, Laura

    2013-01-01

    HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of CCL2 from macrophages. In turn, this chemokine acts as an autocrine factor enhancing viral replication. In this study, we show for the first time that phosphoinositide-specific phospholipase C (PI-PLC) is required for the production of CCL2 triggered by gp120 in macrophages. Using a combination of confocal laser-scanner microscopy, pharmacologic inhibition, western blotting and fluorescence-activated cell sorter analysis, we demonstrate that gp120 interaction with CCR5 leads to nuclear localization of the PI-PLC β1 isozyme mediated by mitogen-activated protein kinase ERK-1/2. Notably, phosphatidylcholine-specific phospholipase C (PC-PLC), previously reported to be required for NF-kB-mediated CCL2 production induced by gp120 in macrophages, drives both ERK1/2 activation and PI-PLC β1 nuclear localization induced by gp120. PI-PLC β1 activation through CCR5 is also triggered by the natural chemokine ligand CCL4, but independently of ERK1/2. Finally, PI-PLC inhibition neither blocks gp120-mediated NF-kB activation nor overall accumulation of CCL2 mRNA, whereas it decreases CCL2 transcript level in the cytoplasm. These results identify nuclear PI-PLC β1 as a new intermediate in the gp120-triggered PC-PLC-driven signal transduction pathway leading to CCL2 secretion in macrophages. The finding that a concerted gp120-mediated signaling involving both PC- and PI-specific PLCs is required for the expression of CCL2 in macrophages suggests that this signal transduction pathway may also be relevant for the modulation of viral replication in these cells. Thus, this study may contribute to identify novel targets for therapeutic intervention in HIV-1 infection.

  5. Inhibition of the phosphatidylinositol-specific phospholipase C from Bacillus cereus by a monoclonal antibody binding to a region with sequence similarity to eukaryotic phospholipases.

    PubMed

    Kuppe, A; Hedberg, K K; Volwerk, J J; Griffith, O H

    1990-10-22

    Bacterial phosphatidylinositol-specific phospholipases C (PI-PLC) display similar substrate specificity as their eukaryotic counterparts involved in signal transduction of insulin and Ca2(+)-mobilizing hormones, and are used in the study of the novel glycosylphosphatidylinositol-protein anchors (GPI-anchors). For the investigation of structure-function aspects of the PI-PLC secreted from Bacillus cereus cells, a panel of murine monoclonal antibodies was generated and shown to be specific for the PI-PLC polypeptide in enzyme-linked immunosorbent assays and Western blots. Two of the monoclonals inhibited reactions catalyzed by the bacterial enzyme in vitro: hydrolysis of phosphatidylinositol and the release of bovine erythrocyte acetylcholinesterase from its GPI-anchor. At saturating concentrations of inhibitory antibody only a few percent of the enzyme activity remained. The epitope recognized by one of the inhibitory antibodies, A72-24, was mapped by proteolytic digestion, protein sequencing, and Western blotting of the generated fragments. The data indicate that at least part of the epitope resides within an 8 kDa-stretch of the bacterial PI-PLC (Gln-45 - Lys-122). Essentially the same segment of the bacterial polypeptide has previously been shown to display limited amino acid sequence similarity with several eukaryotic PI-specific phospholipases C (Kuppe, A., Evans, L.M., McMillen, D.A. and Griffith, O.H. (1989) J. Bacteriol. 171, 6077-6083). The results reported here suggest that the conserved peptide of these enzymes may contain functionally important residues.

  6. N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana.

    PubMed

    Otterhag, L; Sommarin, M; Pical, C

    2001-05-25

    Phosphoinositide-specific phospholipase C's (PI-PLCs) are ubiquitous in eukaryotes, from plants to animals, and catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In animals, four distinct subfamilies of PI-PLCs have been identified, and the three-dimensional structure of one rat isozyme, PLC-delta1, determined. Plants appear to contain only one gene family encoding PI-PLCs. The catalytic properties of plant PI-PLCs are very similar to those of animal enzymes. However, very little is known about the regulation of plant PI-PLCs. All plant PI-PLCs comprise three domains, X, Y and C2, which are also conserved in isoforms from animals and yeast. We here show that one PI-PLC isozyme from Arabidopsis thaliana, AtPLC2, is predominantly localized in the plasma membrane, and that the conserved N-terminal domain may represent an EF-hand domain that is required for catalytic activity but not for lipid binding.

  7. Crystallization, optimization and preliminary X-ray characterization of a metal-dependent PI-PLC from Streptomyces antibioticus

    SciTech Connect

    Jackson, Michael R.; Selby, Thomas L.

    2012-10-30

    A recombinant metal-dependent phosphatidylinositol-specific phospholipase C (PI-PLC) fromStreptomyces antibioticushas been crystallized by the hanging-drop method with and without heavy metals. The native crystals belonged to the orthorhombic space groupP222, with unit-cell parametersa= 41.26,b= 51.86,c = 154.78 Å. The X-ray diffraction results showed significant differences in the crystal quality of samples soaked with heavy atoms. Additionally, drop pinning, which increases the surface area of the drops, was also used to improve crystal growth and quality. The combination of heavy-metal soaks and drop pinning was found to be critical for producing high-quality crystals that diffracted to 1.23 Å resolution.

  8. Fibroblast growth factor acts upon the transcription of phospholipase C genes in human umbilical vein endothelial cells.

    PubMed

    Lo Vasco, Vincenza Rita; Leopizzi, Martina; Puggioni, Chiara; Della Rocca, Carlo; Businaro, Rita

    2014-03-01

    Besides the control of calcium levels, the phosphoinositide-specific phospholipases C (PI-PLCs), the main players in the phosphoinositide signalling pathway, contribute to a number of cell activities. The expression of PI-PLCs is strictly tissue specific and evidence suggests that it varies under different conditions, such as tumour progression or cell activation. In previous studies, we obtained a complete panel of expression of PI-PLC isoforms in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for endothelial cells (EC), and demonstrated that the expression of the PLC genes varies under inflammatory stimulation. The fibroblast growth factor (FGF) activates the PI-PLC γ1 isoform. In the present study, PI-PLC expression in FGF-treated HUVEC was performed using RT-PCR, observed 24 h after stimulation. The expression of selected genes after stimulation was perturbed, suggesting that FGF affects gene transcription in PI signalling as a possible mechanism of regulation of its activity upon the AkT-PLC pathway. The most efficient effects of FGF were recorded in the 3-6-h interval. To understand the complex events progressing in EC might provide useful insights for potential therapeutic strategies. The opportunity to manipulate the EC might offer a powerful tool of considerable practical and clinical importance.

  9. Biochemical and genetic evidence for the presence of multiple phosphatidylinositol- and phosphatidylinositol 4,5-bisphosphate-specific phospholipases C in Tetrahymena.

    PubMed

    Leondaritis, George; Sarri, Theoni; Dafnis, Ioannis; Efstathiou, Antonia; Galanopoulou, Dia

    2011-03-01

    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+) regulatory axis in ciliates.

  10. Biochemical and Genetic Evidence for the Presence of Multiple Phosphatidylinositol- and Phosphatidylinositol 4,5-Bisphosphate-Specific Phospholipases C in Tetrahymena▿‡

    PubMed Central

    Leondaritis, George; Sarri, Theoni; Dafnis, Ioannis; Efstathiou, Antonia; Galanopoulou, Dia

    2011-01-01

    Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], produce the Ca2+-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 Ca2+. One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca2+, 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)P2 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)P3-Ca2+ regulatory axis in ciliates. PMID:21169416

  11. Plant phosphoinositide-dependent phospholipases C: variations around a canonical theme.

    PubMed

    Pokotylo, Igor; Kolesnikov, Yaroslav; Kravets, Volodymyr; Zachowski, Alain; Ruelland, Eric

    2014-01-01

    Phosphoinositide-specific phospholipase C (PI-PLC) cleaves, in a Ca(2+)-dependent manner, phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) into diacylglycerol (DAG) and inositol triphosphate (IP3). PI-PLCs are multidomain proteins that are structurally related to the PI-PLCζs, the simplest animal PI-PLCs. Like these animal counterparts, they are only composed of EF-hand, X/Y and C2 domains. However, plant PI-PLCs do not have a conventional EF-hand domain since they are often truncated, while some PI-PLCs have no EF-hand domain at all. Despite this simple structure, plant PI-PLCs are involved in many essential plant processes, either associated with development or in response to environmental stresses. The action of PI-PLCs relies on the mediators they produce. In plants, IP3 does not seem to be the sole active soluble molecule. Inositol pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) also transmit signals, thus highlighting the importance of coupling PI-PLC action with inositol-phosphate kinases and phosphatases. PI-PLCs also produce a lipid molecule, but plant PI-PLC pathways show a peculiarity in that the active lipid does not appear to be DAG but its phosphorylated form, phosphatidic acid (PA). Besides, PI-PLCs can also act by altering their substrate levels. Taken together, plant PI-PLCs show functional differences when compared to their animal counterparts. However, they act on similar general signalling pathways including calcium homeostasis and cell phosphoproteome. Several important questions remain unanswered. The cross-talk between the soluble and lipid mediators generated by plant PI-PLCs is not understood and how the coupling between PI-PLCs and inositol-kinases or DAG-kinases is carried out remains to be established. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  12. Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.

    PubMed Central

    Flick, J S; Thorner, J

    1993-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses. Images PMID:8395015

  13. Nuclear envelope assembly is promoted by phosphoinositide-specific phospholipase C with selective recruitment of phosphatidylinositol-enriched membranes.

    PubMed

    Byrne, Richard D; Barona, Teresa M; Garnier, Marie; Koster, Grielof; Katan, Matilda; Poccia, Dominic L; Larijani, Banafshé

    2005-04-15

    Nuclear envelope (NE) formation in a cell-free egg extract proceeds by precursor membrane vesicle binding to chromatin in an ATP-dependent manner, followed by a GTP-induced NE assembly step. The requirement for GTP in the latter step of this process can be mimicked by addition of bacterial PI-PLC [phosphoinositide (PtdIns)-specific phospholipase C]. The NE assembly process is here dissected in relation to the requirement for endogenous phosphoinositide metabolism, employing recombinant eukaryotic PI-PLC, inhibitors and direct phospholipid analysis using ESI-MS (electrospray ionization mass spectrometry). PtdIns (phosphatidylinositol) species analysis by ESI-MS indicates that the chromatin-bound NE precursor vesicles are enriched for specific PtdIns species. Moreover, during GTP-induced precursor vesicle fusion, the membrane vesicles become partially depleted of the PtdIns 18:0/20:4 species. These data indicate that eukaryotic PI-PLC can support NE formation, and the sensitivity to exogenous recombinant PtdIns-5-phosphatases shows that the endogenous PLC hydrolyses a 5-phosphorylated species. It is shown further that the downstream target of this DAG (diacylglycerol) pathway does not involve PKC (protein kinase C) catalytic function, but is mimicked by phorbol esters, indicating a possible engagement of one of the non-PKC phorbol ester receptors. The results show that ESI-MS can be used as a sensitive means to measure the lipid composition of biological membranes and their changes during, for example, membrane fusogenic events. We have exploited this and the intervention studies to illustrate a pivotal role for PI-PLC and its product DAG in the formation of NEs.

  14. Nuclear envelope assembly is promoted by phosphoinositide-specific phospholipase C with selective recruitment of phosphatidylinositol-enriched membranes

    PubMed Central

    2004-01-01

    Nuclear envelope (NE) formation in a cell-free egg extract proceeds by precursor membrane vesicle binding to chromatin in an ATP-dependent manner, followed by a GTP-induced NE assembly step. The requirement for GTP in the latter step of this process can be mimicked by addition of bacterial PI-PLC [phosphoinositide (PtdIns)-specific phospholipase C]. The NE assembly process is here dissected in relation to the requirement for endogenous phosphoinositide metabolism, employing recombinant eukaryotic PI-PLC, inhibitors and direct phospholipid analysis using ESI-MS (electrospray ionization mass spectrometry). PtdIns (phosphatidylinositol) species analysis by ESI-MS indicates that the chromatin-bound NE precursor vesicles are enriched for specific PtdIns species. Moreover, during GTP-induced precursor vesicle fusion, the membrane vesicles become partially depleted of the PtdIns 18:0/20:4 species. These data indicate that eukaryotic PI-PLC can support NE formation, and the sensitivity to exogenous recombinant PtdIns-5-phosphatases shows that the endogenous PLC hydrolyses a 5-phosphorylated species. It is shown further that the downstream target of this DAG (diacylglycerol) pathway does not involve PKC (protein kinase C) catalytic function, but is mimicked by phorbol esters, indicating a possible engagement of one of the non-PKC phorbol ester receptors. The results show that ESI-MS can be used as a sensitive means to measure the lipid composition of biological membranes and their changes during, for example, membrane fusogenic events. We have exploited this and the intervention studies to illustrate a pivotal role for PI-PLC and its product DAG in the formation of NEs. PMID:15554872

  15. The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins

    PubMed Central

    Dutta, Mouparna; Ghosh, Anindya S.; Oda, Masataka; Venkatramani, Ravindra; Rao, Basuthkar J.; Dandekar, Abhaya M.; Goñi, Félix M.

    2015-01-01

    The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that

  16. The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins.

    PubMed

    Chakraborty, Sandeep; Rendón-Ramírez, Adela; Ásgeirsson, Bjarni; Dutta, Mouparna; Ghosh, Anindya S; Oda, Masataka; Venkatramani, Ravindra; Rao, Basuthkar J; Dandekar, Abhaya M; Goñi, Félix M

    2013-01-01

    The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that

  17. The Arabidopsis DREB2 genetic pathway is constitutively repressed by basal phosphoinositide-dependent phospholipase C coupled to diacylglycerol kinase.

    PubMed

    Djafi, Nabila; Vergnolle, Chantal; Cantrel, Catherine; Wietrzyñski, Wojciech; Delage, Elise; Cochet, Françoise; Puyaubert, Juliette; Soubigou-Taconnat, Ludivine; Gey, Delphine; Collin, Sylvie; Balzergue, Sandrine; Zachowski, Alain; Ruelland, Eric

    2013-01-01

    Phosphoinositide-dependent phospholipases C (PI-PLCs) are activated in response to various stimuli. They utilize substrates provided by type III-Phosphatidylinositol-4 kinases (PI4KIII) to produce inositol triphosphate and diacylglycerol (DAG) that is phosphorylated into phosphatidic acid (PA) by DAG-kinases (DGKs). The roles of PI4KIIIs, PI-PLCs, and DGKs in basal signaling are poorly understood. We investigated the control of gene expression by basal PI-PLC pathway in Arabidopsis thaliana suspension cells. A transcriptome-wide analysis allowed the identification of genes whose expression was altered by edelfosine, 30 μM wortmannin, or R59022, inhibitors of PI-PLCs, PI4KIIIs, and DGKs, respectively. We found that a gene responsive to one of these molecules is more likely to be similarly regulated by the other two inhibitors. The common action of these agents is to inhibit PA formation, showing that basal PI-PLCs act, in part, on gene expression through their coupling to DGKs. Amongst the genes up-regulated in presence of the inhibitors, were some DREB2 genes, in suspension cells and in seedlings. The DREB2 genes encode transcription factors with major roles in responses to environmental stresses, including dehydration. They bind to C-repeat motifs, known as Drought-Responsive Elements that are indeed enriched in the promoters of genes up-regulated by PI-PLC pathway inhibitors. PA can also be produced by phospholipases D (PLDs). We show that the DREB2 genes that are up-regulated by PI-PLC inhibitors are positively or negatively regulated, or indifferent, to PLD basal activity. Our data show that the DREB2 genetic pathway is constitutively repressed in resting conditions and that DGK coupled to PI-PLC is active in this process, in suspension cells and seedlings. We discuss how this basal negative regulation of DREB2 genes is compatible with their stress-triggered positive regulation.

  18. The Arabidopsis DREB2 genetic pathway is constitutively repressed by basal phosphoinositide-dependent phospholipase C coupled to diacylglycerol kinase

    PubMed Central

    Djafi, Nabila; Vergnolle, Chantal; Cantrel, Catherine; Wietrzyñski, Wojciech; Delage, Elise; Cochet, Françoise; Puyaubert, Juliette; Soubigou-Taconnat, Ludivine; Gey, Delphine; Collin, Sylvie; Balzergue, Sandrine; Zachowski, Alain; Ruelland, Eric

    2013-01-01

    Phosphoinositide-dependent phospholipases C (PI-PLCs) are activated in response to various stimuli. They utilize substrates provided by type III-Phosphatidylinositol-4 kinases (PI4KIII) to produce inositol triphosphate and diacylglycerol (DAG) that is phosphorylated into phosphatidic acid (PA) by DAG-kinases (DGKs). The roles of PI4KIIIs, PI-PLCs, and DGKs in basal signaling are poorly understood. We investigated the control of gene expression by basal PI-PLC pathway in Arabidopsis thaliana suspension cells. A transcriptome-wide analysis allowed the identification of genes whose expression was altered by edelfosine, 30 μM wortmannin, or R59022, inhibitors of PI-PLCs, PI4KIIIs, and DGKs, respectively. We found that a gene responsive to one of these molecules is more likely to be similarly regulated by the other two inhibitors. The common action of these agents is to inhibit PA formation, showing that basal PI-PLCs act, in part, on gene expression through their coupling to DGKs. Amongst the genes up-regulated in presence of the inhibitors, were some DREB2 genes, in suspension cells and in seedlings. The DREB2 genes encode transcription factors with major roles in responses to environmental stresses, including dehydration. They bind to C-repeat motifs, known as Drought-Responsive Elements that are indeed enriched in the promoters of genes up-regulated by PI-PLC pathway inhibitors. PA can also be produced by phospholipases D (PLDs). We show that the DREB2 genes that are up-regulated by PI-PLC inhibitors are positively or negatively regulated, or indifferent, to PLD basal activity. Our data show that the DREB2 genetic pathway is constitutively repressed in resting conditions and that DGK coupled to PI-PLC is active in this process, in suspension cells and seedlings. We discuss how this basal negative regulation of DREB2 genes is compatible with their stress-triggered positive regulation. PMID:23964284

  19. Molecular cytogenetic interphase analysis of Phosphoinositide-specific Phospholipase C β1 gene in paraffin-embedded brain samples of major depression patients.

    PubMed

    Lo Vasco, Vincenza Rita; Polonia, Patrizia

    2012-01-01

    Mood disorders represent a major medical need, as their chronic treatments are not effective in all patients. Literature data suggested that phosphoinositides (PI) signal transduction pathway and related molecules such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, might be involved in the pathophysiology of mood disorders, including major depression. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 gene, which codifies for the PI-PLC β1 enzyme, in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with major depression and in 15 normal controls. No deletions of PLCB1 were identified with the methodology used, which allows to exclude wide gene deletions. The results, the technical aspects of the FISH methodology, and its limitations are discussed.

  20. Plant phosphatidylinositol-specific phospholipase C at the center of plant innate immunity.

    PubMed

    Abd-El-Haliem, Ahmed M; Joosten, Matthieu H A J

    2017-03-01

    Understanding plant resistance to pathogenic microbes requires detailed information on the molecular mechanisms controlling the execution of plant innate immune responses. A growing body of evidence places phosphoinositide-specific phospholipase C (PI-PLC) enzymes immediately downstream of activated immune receptors, well upstream of the initiation of early defense responses. An increase of the cytoplasmic levels of free Ca(2+) , lowering of the intercellular pH and the oxidative burst are a few examples of such responses and these are regulated by PI-PLCs. Consequently, PI-PLC activation represents an early primary signaling switch between elicitation and response involving the controlled hydrolysis of essential signaling phospholipids, thereby simultaneously generating lipid and non-lipid second messenger molecules required for a swift cellular defense response. Here, we elaborate on the signals generated by PI-PLCs and their respective downstream effects, while providing an inventory of different types of evidence describing the involvement of PI-PLCs in various aspects of plant immunity. We project the discussed information into a model describing the cellular events occurring after the activation of plant immune receptors. With this review we aim to provide new insights supporting future research on plant PI-PLCs and the development of plants with improved resistance. © 2017 Institute of Botany, Chinese Academy of Sciences.

  1. Lypopolysaccharide downregulates the expression of selected phospholipase C genes in cultured endothelial cells.

    PubMed

    Lo Vasco, V R; Leopizzi, M; Chiappetta, C; Puggioni, C; Della Rocca, C; Polonia, P; Businaro, R

    2013-08-01

    The signaling system of phosphoinositides (PI) is involved in a variety of cell and tissue functions, including membrane trafficking, ion channel activity, cell cycle, apoptosis, differentiation, and cell and tissue polarity. Recently, PI and related molecules, such as the phosphoinositide-specific phospholipases C (PI-PLCs), main players in PI signaling were supposed to be involved in inflammation. Besides the control of calcium levels, PI-PLCs contribute to the regulation of phosphatydil-inositol bisphosphate metabolism, crucial in cytoskeletal organization. The expression of PI-PLCs is strictly tissue specific and evidences suggest that it varies under different conditions, such as tumor progression or cell activation. In a previous study, we obtained a complete panel of expression of PI-PLC isoforms in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for endothelial cells. In the present study, we analyzed the mRNA concentration of PI-PLCs in lipopolysaccharide (LPS)-treated HUVEC by using the multiliquid bioanalyzer methodology after 3, 6, 24, 48, and 72 h from LPS administration. Marked differences in the expression of most PI-PLC codifying genes were evident.

  2. Silencing of the tomato phosphatidylinositol-phospholipase C2 (SlPLC2) reduces plant susceptibility to Botrytis cinerea.

    PubMed

    Gonorazky, Gabriela; Guzzo, María Carla; Abd-El-Haliem, Ahmed M; Joosten, Matthieu H A J; Laxalt, Ana María

    2016-12-01

    The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated on pathogen attack. We have previously shown that the fungal elicitor xylanase induces a raise of SlPLC2 and SlPLC5 transcripts and that SlPLC2, but not SlPLC5, is required for xylanase-induced expression of defense-related genes. In this work we studied the role of SlPLC2 in the interaction between tomato and the necrotrophic fungus Botrytis cinerea. Inoculation of tomato leaves with B. cinerea increases SlPLC2 transcript levels. We knocked-down the expression of SlPLC2 by virus-induced gene silencing and plant defense responses were analyzed upon B. cinerea inoculation. SlPLC2 silenced plants developed smaller necrotic lesions concomitantly with less proliferation of the fungus. Silencing of SlPLC2 resulted as well in a reduced production of reactive oxygen species. Upon B. cinerea inoculation, transcript levels of the salicylic acid (SA)-defense pathway marker gene SlPR1a were diminished in SlPLC2 silenced plants compared to non-silenced infected plants, while transcripts of the jasmonic acid (JA)-defense gene markers Proteinase Inhibitor I and II (SlPI-I and SlPI-II) were increased. This implies that SlPLC2 participates in plant susceptibility to B. cinerea.

  3. Phospholipase C beta3 is a key component in the Gbetagamma/PKCeta/PKD-mediated regulation of trans-Golgi network to plasma membrane transport.

    PubMed

    Díaz Añel, Alberto M

    2007-08-15

    The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gbetagamma (GTP-binding protein betagamma subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCeta (protein kinase Ceta) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCeta, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gbetagamma, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that betagamma-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCbeta3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCbeta3, which is necessary to activate PKCeta and PKD in that Golgi compartment, via DAG production.

  4. Phospholipase C β3 is a key component in the Gβγ/PKCη/PKD-mediated regulation of trans-Golgi network to plasma membrane transport

    PubMed Central

    Díaz Añel, Alberto M.

    2007-01-01

    The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gβγ (GTP-binding protein βγ subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCη (protein kinase Cη) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCη, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gβγ, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that βγ-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCβ3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCβ3, which is necessary to activate PKCη and PKD in that Golgi compartment, via DAG production. PMID:17492941

  5. Activation of phospholipase C by the alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells.

    PubMed

    Wu, D Q; Lee, C H; Rhee, S G; Simon, M I

    1992-01-25

    High efficiency transient transfection was used to introduce cDNA corresponding to various G protein alpha subunits into Cos-7 cells. The proteins that were subsequently synthesized were detected with specific G protein alpha subunit antipeptide antiserum and were localized in the membrane fraction of the cell. Cells that were prelabeled with the [3H]inositol and transfected with G alpha q and G alpha 11 cDNA showed marked increases in formation of [3H]inositol phosphates after stimulation with aluminum fluoride. Co-transfection with cDNAs corresponding to phosphoinositide specific phospholipase C beta 1 (PI-PLC beta 1) and to G alpha q or G alpha 11 resulted in even higher levels of inositol phosphate formation. The introduction of mutations that convert residue glutamine 209 to leucine in G alpha q and G alpha 11 resulted in persistent activation of PI-PLC and high steady state levels of inositol phosphates. On the other hand, transfection with a variety of other G alpha subunit cDNAs, i.e. G alpha Z, G alpha OA, G alpha OB, transducin, and the glutamine 205 to leucine mutants of G alpha Z and of G alpha OA did not increase inositol phosphate formation. To further test the specificity of G protein activation of PI-PLC, a cell-free system was prepared by using washed membranes of transiently transfected cells and purified PI-PLC beta 1. Membranes derived from G alpha q and G alpha 11, but not G alpha OA transfected cells, showed guanosine 5-O-thiotriphosphate (GTP gamma S)-stimulated PIP2 hydrolysis. The activity seen in the system reconstituted with membranes derived from G alpha 11-transfected cells was blocked by preincubation with specific G alpha 11 antipeptide antibodies. All of these results are consistent with the conclusion that G alpha q and G alpha 11 cDNA encode proteins that in the presence of GTP gamma S specifically activate PI-PLC.

  6. 1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation.

    PubMed Central

    Khare, S; Bolt, M J; Wali, R K; Skarosi, S F; Roy, H K; Niedziela, S; Scaglione-Sewell, B; Aquino, B; Abraham, C; Sitrin, M D; Brasitus, T A; Bissonnette, M

    1997-01-01

    Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-alpha and -betaII in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-gamma (PI-PLC-gamma), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-gamma, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-gamma in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-gamma. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-gamma in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PP1, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-gamma in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time

  7. Listeriolysin O suppresses Phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection

    PubMed Central

    Lam, Grace Y.; Fattouh, Ramzi; Muise, Aleixo M.; Grinstein, Sergio; Higgins, Darren E.; Brumell, John H.

    2012-01-01

    Summary The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L. monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allows L. monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst. PMID:22177565

  8. The activity of phosphoinositide-specific phospholipase C is required for vegetative growth and cell wall regeneration in Coprinopsis cinerea.

    PubMed

    Oh, Young Taek; Ahn, Chun-Seob; Lee, Kyung-Jin; Kim, Jeong-Geun; Ro, Hyeon-Su; Kim, Jae Won; Lee, Chang-Won

    2012-08-01

    Three isotypes of phosphoinositide-specific phospholipase C designated CcPLC1, CcPLC2, and CcPLC3 were identified in Coprinopsis cinerea, through a search of the genome sequence database. The functional role of the PI-PLCs were studied by using U73122, which specifically inhibits the activity of PI-PLC. The specificity of the inhibitor effect was confirmed by using an inactive structural analog U73433. The inhibition of PI-PLCs activity resulted in severely retarded germination of basidiospores and oidia, reduced hyphal growth, knobbly hyphal tips with many irregular side branches, and aberrant (branch-like structure) clamp cells. Furthermore, U73122 definitely inhibited cell wall formation. Here we report that PI-PLCs play important roles in various aspects of C. cinerea biology.

  9. Phosphatidylinositol-specific phospholipase C from Listeria monocytogenes contributes to intracellular survival and growth of Listeria innocua.

    PubMed Central

    Schwan, W R; Demuth, A; Kuhn, M; Goebel, W

    1994-01-01

    Listeria monocytogenes is a facultative intracellular organism that is capable of replicating within macrophage and macrophage-like cells. The species secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) encoded by the plcA gene. A plcA gene from L. monocytogenes was cloned downstream of a gram-positive promoter in the plasmid pWS2-2. To determine what effect plcA would have on intracellular survival when introduced into Listeria innocua, a species that does not growth intracellularly or contain plcA, transformation with the recombinant pWS2-2 plasmid was performed. Phospholipase C activity in Listeria innocua/pWS2-2 was confirmed on a brain heart infusion-phosphatidylinositol agar plate, whereas wild-type L. innocua did not produce PI-PLC activity. Intracellular growth of L. innocua/pWS2-2 was subsequently measured in the macrophage-like cell line J774 by Giemsa staining and viable count determinations at specific time points following infection. The J774 cells infected with wild-type L. innocua showed a falling viable count through 8 h postinfection. Although J774 cells infected with L. innocua/pWS2-2 also initially displayed reduced viable counts, the viable count rose after 6 h postinfection and increased further at 8 h postinfection before a subsequent decline again at 16 h postinfection. Giemsa staining revealed fewer than 6 bacteria in individual macrophage cells at 2 h postinfection, and yet approximately 15% of the J774 cells had 6 to 12 bacteria localized to one area of the macrophage cell after 6 h; moreover, electron micrographs showed that the L. innocua/pWS2-2 cells were replicating inside the phagosome of the host cell. Furthermore, Thoria Sol labeling demonstrated that lysosomes had fused with these phagosomes, and acridine orange staining revealed that the compartments were acidified. These results demonstrate that L. innocua cells transformed with the plasmid-borne plcA gene, and expressing functional PI-PLC, are able to grow

  10. Alopecia in a Viable Phospholipase C Delta 1 and Phospholipase C Delta 3 Double Mutant

    PubMed Central

    Runkel, Fabian; Hintze, Maik; Griesing, Sebastian; Michels, Marion; Blanck, Birgit; Fukami, Kiyoko; Guénet, Jean-Louis; Franz, Thomas

    2012-01-01

    Background Inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG) are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD) forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia), whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. Methodology/Principal Findings We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3mNab) that resulted from the insertion of an intracisternal A particle (IAP) into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3mNab alleles are phenotypically normal. However, the presence of one Plcd3mNab allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9)olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9)olt1Pas and the Plcd3mNab mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. Conclusions/Significance The Plcd3mNab mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface. PMID:22723964

  11. Phospholipase C-dependent phosphoinositide breakdown induced by ELF-EMF in Peganum harmala calli.

    PubMed

    Piacentini, Maria Piera; Piatti, Elena; Fraternale, Daniele; Ricci, Donata; Albertini, Maria Cristina; Accorsi, Augusto

    2004-01-01

    With the aim of examining the response of plant cells to extremely low frequency (ELF) electromagnetic fields (EMF), we investigated the behaviour of the phosphatidylinositol 4,5 bisphosphate (PtdIns 4,5-P(2)) molecule (the precursor of the phosphoinositide signal transduction cascade) by exposing callus cells from Peganum harmala to 50 Hz, 1 gauss EMF for 10 min and by examining the level and the fatty acid composition of PtdIns 4,5-P(2) after the exposure. Our results evidenced a statistically significant decrease in PtdIns 4,5-P(2) concentrations and a different involvement of the constituting fatty acids in the induced breakdown. The manipulation of the lipid-based signalling pathway by phosphoinositide-phospholipase C (PI-PLC) inhibitors (i.e., neomycin, U-73122 and ET-18-OCH(3)) seems to support the hypothesis that, as in animals, also in plants, the cell membrane is the primary impact site of ELF electromagnetic stimulus and that this interaction could probably involve the activation of PI signal transduction pathway including a heterotrimeric G protein.

  12. Secretion of phospholipase C by Pseudomonas aeruginosa.

    PubMed Central

    Stinson, M W; Hayden, C

    1979-01-01

    The conditions necessary for the secretion of phospholipase C (phosphatidylcholine cholinephosphohydrolase) by Pseudomonas aeruginosa were studied. Enzyme secretion by washed cell suspensions required a carbon source and ammonium, potassium, and calcium ions. The calcium requirement could be substituted by magnesium and strontium but not by copper, manganese, cobalt, or zinc. During growth in liquid medium, cells secreted phospholipase C during late logarithmic and early stationary phases. Secretion was repressed by the addition of inorganic phosphate but not by organic phosphates, glucose, or sodium succinate. Studies with tetracycline indicated that de novo protein synthesis was necessary for the secretion of phospholipase C and that the exoenzyme was not released from a preformed periplasmic pool. Similarly, extraction of actively secreting cells with 0.2 M MgCl2 at pH 8.4 solubilized large quantities of the periplasmic enzyme alkaline phosphatase but insignificant amounts of phospholipase C. Bacteria continued to secrete enzyme for nearly 45 min after the addition of inorganic phosphate or rifampin. Images PMID:114487

  13. Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms.

    PubMed

    Estacion, M; Sinkins, W G; Schilling, W P

    2001-01-01

    Patch clamp and fura-2 fluorescence were employed to characterize receptor-mediated activation of recombinant Drosophila TrpL channels expressed in Sf9 insect cells. TrpL was activated by receptor stimulation and by exogenous application of diacylglycerol (DAG) or poly-unsaturated fatty acids (PUFAs). Activation of TrpL was blocked more than 70% by U73122, suggesting that the effect of these agents was dependent upon phospholipase C (PLC). In fura-2 assays, extracellular application of bacterial phosphatidylinositol (PI)-PLC or phosphatidylcholine (PC)-PLC caused a transient increase in TrpL channel activity, the magnitude of which was significantly less than that observed following receptor stimulation. TrpL channels were also activated in excised inside-out patches by cytoplasmic application of mammalian PLC-b2, bacterial PI-PLC and PC-PLC, but not by phospholipase D (PLD). The phospholipases had little or no effect when examined in either whole-cell or cell-attached configurations.TrpL activity was inhibited by addition of phosphatidylinositol-4,5-bisphosphate (PIP2) to excised inside-out membrane patches exhibiting spontaneous channel activity or to patches pre-activated by treatment with PLC. The effect was reversible, specific for PIP2, and was not observed with phosphatidylethanolamine (PE), PI, PC or phosphatidylserine (PS). However, antibodies against PIP2 consistently failed to activate TrpL in inside-out patches. It is concluded that both the hydrolysis of PIP2 and the generation of DAG are required to rapidly activate TrpL following receptor stimulation, or that some other PLC-dependent mechanism plays a crucial role in the activation process.

  14. [Phospholipase C of fungi and staphylococci].

    PubMed

    Zaikina, N A; Robakidze, T N

    1976-01-01

    The activity of phospholipase was studied in the cultural broth and cell extract of 112 strains of fungi and yeasts. The endoenzyme was detected in 19 strains of mycelial fungi, the exoenzyme was found in Mucor hiemalis 50 and Aspergillus niger 117. Phospholipase C of M. hiemalis was purified and compared to phospholipase of staphylococci. The values of Km are 8.9 and 1.07 mM, respectively, for the fungal and staphylococcal enzymes.

  15. Regulation of CD3-induced phospholipase C-gamma 1 (PLC gamma 1) tyrosine phosphorylation by CD4 and CD45 receptors.

    PubMed Central

    Kanner, S B; Deans, J P; Ledbetter, J A

    1992-01-01

    Stimulation of the signal transduction cascade in T cells through the T-cell receptor (CD3) coincides with activation of the phosphatidylinositol-phospholipase C (PI-PLC) pathway. activation of phospholipase C-gamma 1 (PLC gamma 1) occurs through tyrosine phosphorylation in T cells following surface ligation of CD3 receptors with CD3-specific monoclonal antibodies (mAb). Here we show that cross-linking of CD4 molecules with CD3 augments the tyrosine phosphorylation of PLC gamma 1, while co-ligation of CD3 with CD45 (a receptor tyrosine phosphatase) results in reduced PLC gamma 1 tyrosine phosphorylation. Mobilization of intracellular calcium correlated with the extent of PLC gamma 1 tyrosine phosphorylation, indicating that PLC gamma 1 enzymatic activity in T cells may be regulated by its phosphorylation state. The time-course of PLC gamma 1 tyrosine phosphorylation in cells stimulated by soluble anti-CD3 was transient and closely paralleled that of calcium mobilization, while the kinetics in cells stimulated by immobilized anti-CD3 were prolonged. The PI-PLC pathway in T cells was not stimulated by tyrosine phosphorylation of PLC gamma 2, a homologue of PLC gamma 1, demonstrating the strict regulation of PLC gamma isoform usage in CD3-stimulated T cells. A 35,000/36,000 MW tyrosine phosphorylated protein in T cells formed stable complexes with PLC gamma 1, and its tyrosine phosphorylation was co-regulated with that of PLC gamma 1 by CD4 and CD45 receptors. Enzymatic activation and tyrosine phosphorylation of PLC gamma 1 occurs during growth factor stimulation of fibroblasts, where PLC gamma 1 exists in multi-component complexes. The observation that PLC gamma 1 exists in complexes with unique tyrosine phosphorylated proteins in T cells suggests that haematopoietic lineage-specific proteins associated with PLC gamma 1 may play roles in cellular signalling. Images Figure 1 Figure 4 PMID:1533389

  16. The physiological roles of primary phospholipase C.

    PubMed

    Yang, Yong Ryoul; Follo, Matilde Y; Cocco, Lucio; Suh, Pann-Ghill

    2013-09-01

    The roles of phosphoinositide-specific phospholipase C (PLC) have been extensively investigated in diverse cell lines and pathological conditions. Among the PLC isozmes, primary PLCs, PLC-β and PLC-γ, are directly activated by receptor activation, unlike other secondary PLCs (PLC-ɛ, PLC-δ1, and PLC-η1). PLC-β isozymes are activated by G protein couple receptor and PLC-γ isozymes are activated by receptor tyrosine kinase (RTK). Primary PLCs are differentially expressed in different tissues, suggesting their specific roles in diverse tissues and regulate a variety of physiological and pathophysiological functions. Thus, dysregulation of phospholipases contributes to a number of human diseases and primary PLCs have been identified as therapeutic targets for prevention and treatment of diseases. Here we review the roles of primary PLCs in physiology and their impact in pathology.

  17. Primary phospholipase C and brain disorders.

    PubMed

    Yang, Yong Ryoul; Kang, Du-Seock; Lee, Cheol; Seok, Heon; Follo, Matilde Y; Cocco, Lucio; Suh, Pann-Ghill

    2016-05-01

    In the brain, the primary phospholipase C (PLC) proteins, PLCβ, and PLCγ, are activated primarily by neurotransmitters, neurotrophic factors, and hormones through G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). Among the primary PLC isozymes, PLCβ1, PLCβ4, and PLCγ1 are highly expressed and differentially distributed, suggesting a specific role for each PLC subtype in different regions of the brain. Primary PLCs control neuronal activity, which is important for synapse function and development. In addition, dysregulation of primary PLC signaling is linked to several brain disorders including epilepsy, schizophrenia, bipolar disorder, Huntington's disease, depression and Alzheimer's disease. In this review, we included current knowledge regarding the roles of primary PLC isozymes in brain disorders.

  18. Phospholipase C-β in immune cells.

    PubMed

    Kawakami, Toshiaki; Xiao, Wenbin

    2013-09-01

    Great progress has recently been made in structural and functional research of phospholipase C (PLC)-β. We now understand how PLC-β isoforms (β1-β4) are activated by GTP-bound Gαq downstream of G protein-coupled receptors. Numerous studies indicate that PLC-βs participate in the differentiation and activation of immune cells that control both the innate and adaptive immune systems. The PLC-β3 isoform also interplays with tyrosine kinase-based signaling pathways, to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1, with which PLC-β3 and Stat5 form a multi-molecular signaling platform, named SPS complex. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation.

  19. Phospholipase C-β in Immune Cells

    PubMed Central

    Kawakami, Toshiaki; Xiao, Wenbin

    2013-01-01

    Great progress has recently been made in structural and functional research of phospholipase C (PLC)-β. We now understand how PLC-β isoforms (β1-β4) are activated by GTP-bound Gαq downstream of G protein-coupled receptors. Numerous studies indicate that PLC-βs participate in the differentiation and activation of immune cells that control both the innate and adaptive immune systems. The PLC-β3 isoform also interplays with tyrosine kinase-based signaling pathways, to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1, with which PLC-β3 and Stat5 form a multi-molecular signaling platform, named SPS complex. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation. PMID:23981313

  20. Listeriolysin O suppresses phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection.

    PubMed

    Lam, Grace Y; Fattouh, Ramzi; Muise, Aleixo M; Grinstein, Sergio; Higgins, Darren E; Brumell, John H

    2011-12-15

    The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L. monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allow L. monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Involvement of phospholipases C and D in early response to SAR and ISR inducers in Brassica napus plants.

    PubMed

    Profotová, B; Burketová, L; Novotná, Z; Martinec, J; Valentová, O

    2006-01-01

    Phospholipid signaling is an important component in eukaryotic signal transduction pathways. In plants, it plays a key role in growth and development as well as in responses to environmental stresses, including pathogen attack. We investigated the involvement of both phospholipase C (PLC, EC 3.1.4.11) and D (PLD, EC 3.1.4.4) in early responses to the treatment of Brassica napus plants with the chemical inducers of systemic acquired resistance (SAR): salicylic acid (SA), benzothiadiazole (BTH), and with the inducer mediating the induced systemic resistance (ISR) pathway, methyl jasmonate (MeJA). Rapid activation (within 0.5-6 h treatment) of the in vitro activity level was found for phosphatidyl inositol 4,5 bisphosphate (PIP2)-specific PLC (PI-PLC) and three enzymatically different forms of PLD: conventional PLDalpha, PIP2-dependent PLD beta/gamma, and oleate-stimulated PLDdelta. The strongest response was found in case of cytosolic PIP2-dependent PLD beta/gamma after BTH treatment. PLDdelta was identified in B. napus leaves and was very rapidly activated after MeJA treatment with the highest degree of activation compared to the other PLD isoforms. Interestingly, an increase in the amount of protein was observed only for PLDgamma and/or delta after ISR induction, but later than the activation occurred. These results show that phospholipases are involved in very early processes leading to systemic responses in plants and that they are most probably initially first activated on post translational level.

  2. The Phospholipase C Isozymes and Their Regulation

    PubMed Central

    Gresset, Aurelie; Sondek, John

    2013-01-01

    The physiological effects of many extracellular neurotransmitters, hormones, growth factors, and other stimuli are mediated by receptor-promoted activation of phospholipase C (PLC) and consequential activation of inositol lipid signaling pathways. These signaling responses include the classically described conversion of phosphatidylinositol(4,5)P2 to the Ca2+-mobilizing second messenger inositol(1,4,5)P3 and the protein kinase C-activating second messenger diacylglycerol as well as alterations in membrane association or activity of many proteins that harbor phosphoinositide binding domains. The 13 mammalian PLCs elaborate a minimal catalytic core typified by PLC-δ to confer multiple modes of regulation of lipase activity. PLC-β isozymes are activated by Gαq- and Gβγ-subunits of heterotrimeric G proteins, and activation of PLC-γ isozymes occurs through phosphorylation promoted by receptor and non-receptor tyrosine kinases. PLC-ε and certain members of the PLC-β and PLC-γ subclasses of isozymes are activated by direct binding of small G proteins of the Ras, Rho, and Rac subfamilies of GTPases. Recent high resolution three dimensional structures together with biochemical studies have illustrated that the X/Y linker region of the catalytic core mediates autoinhibition of most if not all PLC isozymes. Activation occurs as a consequence of removal of this autoinhibition. PMID:22403074

  3. Differential inhibitory effects of 2-azafluorenones on PI-PLC activation but not on PC-PLC- or PC-PLD-activation induced by histamine, PAF, PMA or A23187 in C6 glioma cells.

    PubMed

    Wang, Hai-Long; Wang, Li-Chuan; Wei, Jiann-Wu

    2013-02-28

    In this study, C6 glioma cells were used to test the effects of 2-azafluorenone and its related compounds on membrane phosphatidylinositol (PI) and phosphatidylcholine (PC) turnover. An increase of [³H]-labeled inositol phosphate (IP1) formation by histamine (100 μM) or A23187 (100 nM) via the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) to breakdown labeled substrate was observed, and this effect could be partially blocked by about half at 100 μM of 2-azafluorenones. Histamine induced the increase of IP1 formation, but failed to cause an increase in extracellularly releasing of [3H]choline metabolites, or intracellular accumulation of [³H]phosphscholine. However, platelet activation factor (PAF) from 0.2 to 1 μM, and phorbol 12-myristate-13-acetate (PMA) at 1 μM caused an increase in extracellularly releasing of [³H]choline metabolites, and intracellular accumulation of [³H]phosphocholine via the activation on phosphatidylcholine (PC)-PLC. These responses of PAF and PMA were not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at high concentration (10⁻⁴ M). A23187 induced an increase of intracellular [³H]choline release via the activation of PCphospholipase D (PLD). This increasing effect of 100 nM A23187 was not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at a high concentration of 10⁻⁴ M. In summary, the inhibitory effect of 2-azafluorenone and its related compound 4-methyl-2-azafluorenone was observed selectively on PIPLC, but not on PC-PLC or PC-PLD based on changes of products after the activation of these enzymes.

  4. Structural and mechanistic comparison of prokaryotic and eukaryotic phosphoinositide-specific phospholipases C.

    PubMed

    Heinz, D W; Essen, L O; Williams, R L

    1998-01-30

    Phosphoinositide-specific phospholipases C (PI-PLCs) are ubiquitous enzymes that catalyse the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol (DAG). Whereas the eukaryotic PI-PLCs play a central role in most signal transduction cascades by producing two second messengers, inositol-1,4,5-trisphosphate and DAG, prokaryotic PI-PLCs are of interest because they act as virulence factors in some pathogenic bacteria. Bacterial PI-PLCs consist of a single domain of 30 to 35 kDa, while the much larger eukaryotic enzymes (85 to 150 kDa) are organized in several distinct domains. The catalytic domain of eukaryotic PI-PLCs is assembled from two highly conserved polypeptide stretches, called regions X and Y, that are separated by a divergent linker sequence. There is only marginal sequence similarity between the catalytic domain of eukaryotic and prokaryotic PI-PLCs. Recently the crystal structures of a bacterial and a eukaryotic PI-PLC have been determined, both in complexes with substrate analogues thus enabling a comparison of these enzymes in structural and mechanistic terms. Eukaryotic and prokaryotic PI-PLCs contain a distorted (beta alpha)8-barrel as a structural motif with a surprisingly large structural similarity for the first half of the (beta alpha)8-barrel and a much weaker similarity for the second half. The higher degree of structure conservation in the first half of the barrel correlates with the presence of all catalytic residues, in particular two catalytic histidine residues, in this portion of the enzyme. The second half contributes mainly to the features of the substrate binding pocket that result in the distinct substrate preferences exhibited by the prokaryotic and eukaryotic enzymes. A striking difference between the enzymes is the utilization of a catalytic calcium ion that electrostatically stabilizes the transition state in eukaryotic enzymes, whereas this role is filled by an analogously positioned arginine in bacterial PI

  5. A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes.

    PubMed

    Huang, Weigang; Hicks, Stephanie N; Sondek, John; Zhang, Qisheng

    2011-03-18

    Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This family of enzymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. Aberrant regulation of PLCs has been implicated in various diseases including cancer and Alzheimer's disease. How, when, and where PLCs are activated under different cellular contexts are still largely unknown. We have developed a fluorogenic PLC reporter, WH-15, that can be cleaved in a cascade reaction to generate fluorescent 6-aminoquinoline. When applied in enzymatic assays with either pure PLCs or cell lysates, this reporter displays more than a 20-fold fluorescence enhancement in response to PLC activity. Under assay conditions, WH-15 has comparable K(m) and V(max) with the endogenous PIP(2). This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high-throughput screening of PLC inhibitors.

  6. A Fluorogenic, Small Molecule Reporter for Mammalian Phospholipase C Isozymes

    PubMed Central

    Huang, Weigang; Hicks, Stephanie N.; Sondek, John; Zhang, Qisheng

    2012-01-01

    Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This family of enzymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. Aberrant regulation of PLCs has been implicated in various diseases including cancer and Alzheimer’s disease. How, when, and where PLCs are activated under different cellular contexts are still largely unknown. We have developed a fluorogenic PLC reporter, WH-15, that can be cleaved in a cascade reaction to generate fluorescent 6-aminoquinoline. When applied in enzymatic assays with either pure PLCs or cell lysates, this reporter displays more than a 20-fold fluorescence enhancement in response to PLC activity. Under assay conditions, WH-15 has comparable Km and Vmax with the endogenous PIP2. This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high throughput screening of PLC inhibitors. PMID:21158426

  7. G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C.

    PubMed

    Offermanns, S; Simon, M I

    1995-06-23

    The murine G-protein alpha-subunit G alpha 15 and its human counterpart G alpha 16 are expressed in a subset of hematopoietic cells, and they have been shown to regulate beta-isoforms of inositide-specific phospholipase C. We studied the ability of a variety of receptors to interact with G alpha 15 and G alpha 16 by cotransfecting receptors and G-protein alpha-subunits in COS-7 cells. Activation of beta 2 adrenergic and muscarinic M2 receptors in cells expressing the receptors alone or together with G alpha q, G alpha 11, or G alpha 14 led to a very small stimulation of endogenous phospholipase C. However, when the receptors were coexpressed with G alpha 15 and G alpha 16, addition of appropriate ligands caused a severalfold increase in inositol phosphate production which was time- and dose-dependent. A similar activation of phospholipase C was observed when several other receptors which were previously shown to couple to members of the Gi and Gs family were coexpressed with G alpha 15/16. In addition, stimulation of inositol phosphate formation via receptors naturally coupled to phospholipase C was enhanced by cotransfection of G alpha 15 and G alpha 16. These data demonstrate that G alpha 15 and G alpha 16 are unique in that they can be activated by a wide variety of G-protein-coupled receptors. The ability of G alpha 15 and G alpha 16 to bypass the selectivity of receptor G-protein interaction can be a useful tool to understand the mechanism of receptor-induced G-protein activation. In addition, the promiscuous behavior of G alpha 15 and G alpha 16 toward receptors may be helpful in finding ligands corresponding to orphan receptors whose signaling properties are unknown.

  8. Plant phosphatidylcholine-hydrolyzing phospholipases C NPC3 and NPC4 with roles in root development and brassinolide signaling in Arabidopsis thaliana.

    PubMed

    Wimalasekera, Rinukshi; Pejchar, Premysl; Holk, André; Martinec, Jan; Scherer, Günther F E

    2010-05-01

    Phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphocholine and diacylglycerol (DAG). PC-PLC has a long tradition in animal signal transduction to generate DAG as a second messenger besides the classical phosphatidylinositol splitting phospholipase C (PI-PLC). Based on amino acid sequence similarity to bacterial PC-PLC, six putative PC-PLC genes (NPC1 to NPC6) were identified in the Arabidopsis genome. RT-PCR analysis revealed overlapping expression pattern of NPC genes in root, stem, leaf, flower, and silique. In auxin-treated P(NPC3):GUS and P(NPC4):GUS seedlings, strong increase of GUS activity was visible in roots, leaves, and shoots and, to a weaker extent, in brassinolide-treated (BL) seedlings. P(NPC4):GUS seedlings also responded to cytokinin with increased GUS activity in young leaves. Compared to wild-type, T-DNA insertional knockouts npc3 and npc4 showed shorter primary roots and lower lateral root density at low BL concentrations but increased lateral root densities in response to exogenous 0.05-1.0 μM BL. BL-induced expression of TCH4 and LRX2, which are involved in cell expansion, was impaired but not impaired in repression of CPD, a BL biosynthesis gene, in BL-treated npc3 and npc4. These observations suggest NPC3 and NPC4 are important in BL-mediated signaling in root growth. When treated with 0.1 μM BL, DAG accumulation was observed in tobacco BY-2 cell cultures labeled with fluorescent PC as early as 15 min after application. We hypothesize that at least one PC-PLC is a plant signaling enzyme in BL signal transduction and, as shown earlier, in elicitor signal transduction.

  9. A novel class of microbial phosphocholine-specific phospholipases C.

    PubMed

    Stonehouse, Martin J; Cota-Gomez, Adela; Parker, Sarah K; Martin, Wesley E; Hankin, Joseph A; Murphy, Robert C; Chen, Weibin; Lim, Kheng B; Hackett, Murray; Vasil, Adriana I; Vasil, Michael L

    2002-11-01

    In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.

  10. Phospholipase C isozymes in the human brain and their changes in Alzheimer's disease.

    PubMed

    Shimohama, S; Sasaki, Y; Fujimoto, S; Kamiya, S; Taniguchi, T; Takenawa, T; Kimura, J

    1998-02-01

    Phosphoinositide-specific phospholipase C is a key enzyme in signal transduction. We have previously demonstrated that an isozyme of phospholipase C, phospholipase C-delta1, accumulates aberrantly in the brains of patients with Alzheimer's disease. In the present study, we examined the property of phospholipase C isozymes in human brains using the methods of chromatofocusing and gel filtration chromatography, and investigated their changes in Alzheimer's disease brains. The chromatofocusing profile of human brain phospholipase C activity on a Mono P HR column demonstrated that phospholipase C-gamma1, exhibiting an isoelectric point value of 5.2, and phospholipase C-delta1, exhibiting isoelectric point values of 5.2 and 4.6, are partly overlapped in their elution. In contrast, the elution profiles of control and Alzheimer's disease brain phospholipase C on Superdex 200 pg column gel filtration chromatography indicated that phospholipase C-gamma1 and phospholipase C-delta1 can be separated with the elution position having a molecular weight of about 240,000 and 140,000, respectively, in the human brain. Using this gel filtration chromatography it was revealed that the phospholipase C-gamma1 activity was significantly decreased and the phospholipase C-delta1 activity was significantly increased in Alzheimer's disease brains compared with controls. These results suggest that the phospholipase C isozymes are differentially involved in Alzheimer's disease.

  11. The PI-PLC inhibitor U-73122 is a potent inhibitor of the SERCA pump in smooth muscle.

    PubMed

    Hollywood, M A; Sergeant, G P; Thornbury, K D; McHale, N G

    2010-07-01

    In this issue MacMillan and McCarron in 2010 demonstrated that the phospholipase C (PLC) inhibitor U-73122 can potently inhibit Ca(2+) release from isolated smooth muscle cells independent of its effect on PLC. Their data suggest that the PLC inhibitor can block the sarcoplasmic/endoplasmic reticulum calcium ATPase pump in smooth muscle and cast doubt on the reliability of U-73122 as the main pharmacological tool to assess the role of the phosphotidyl inositol-PLC pathway in cellular signalling.

  12. Signalling through phospholipase C interferes with clathrin-mediated endocytosis.

    PubMed

    Carvou, Nicolas; Norden, Anthony G W; Unwin, Robert J; Cockcroft, Shamshad

    2007-01-01

    We investigated if phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) hydrolysis by phospholipase C activation through cell surface receptors would interfere with clathrin-mediated endocytosis as recruitment of clathrin assembly proteins is PtdIns(4,5)P2-dependent. In the WKPT renal epithelial cell line, endocytosed insulin and beta2-glycoprotein I (beta2gpI) were observed in separate compartments, although endocytosis of both ligands was clathrin-dependent as demonstrated by expression of the clathrin-binding C-terminal domain of AP180 (AP180-C). The two uptake mechanisms were different as only insulin uptake was reduced when the mu2-subunit of the adaptor complex AP-2 was silenced by RNA interference. ATP receptors are expressed at the apical surface of renal cells and, thus, we examined the effect of extracellular ATP on insulin and beta2gpI uptake. ATP stimulated phospholipase C activity, and also suppressed uptake of insulin, but not beta2gpI. This effect was reversed by the PLC inhibitor U-73122. In polarized cell cultures, insulin uptake was apical, whereas beta2gpI uptake was through the basolateral membrane, thus providing an explanation for selective inhibition of insulin endocytosis by ATP. Taken together, these results demonstrate that stimulation of apical G-protein-coupled P2Y receptors, which are coupled to phospholipase C activation diminishes clathrin-mediated endocytosis without interfering with basolateral endocytic mechanisms.

  13. Phosphatidylinositol Specific Phospholipase C of Plant Stems 1

    PubMed Central

    Pfaffmann, Helmut; Hartmann, Elmar; Brightman, Andrew O.; Morré, D. James

    1987-01-01

    A phosphatidylinositol-specific phospholipase C of plant stems (EC 3.1.4.10) assayed at pH 6.6 and at 30°C cleaved phosphatidylinositol such that more than 85% of the product was inositol-1-phosphate. Other phospholipids were cleaved 5 to 10% or less under these conditions. The phospholipase had both a soluble and a membrane-associated form. The soluble activity accounted for approximately 85 to 90% of the activity and 15% was associated with membranes. The membrane-associated activity was most concentrated in the plasma membranes of hypocotyl segments of both soybean (Glycine max) and bushbean (Phaseolus vulgaris). The plasma membrane location was verified by analysis of highly purified plasma membranes prepared both by aqueous two-phase partitioning and by preparative free-flow electrophoresis and from the quantitation of the activity in all major cell fractions. Internal membranes also contained phospholipase C activity but at specific activity levels of about 0.1 those present in plasma membranes. Golgi apparatus-enriched fractions from which plasma membrane contaminants were removed by two-phase partition contained the activity at specific activity levels 0.2 those of plasma membrane. Both the soluble and the membrane-associated activity was stimulated by calcium but not by calmodulin, either alone or in the presence of calcium. PMID:16665820

  14. Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits.

    PubMed

    Camps, M; Hou, C; Sidiropoulos, D; Stock, J B; Jakobs, K H; Gierschik, P

    1992-06-15

    We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743-748]. To investigate whether this stimulation was due to a soluble alpha subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein beta gamma dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that beta gamma subunits, purified from bovine retinal transducin (beta gamma t), markedly stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by beta gamma t was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin alpha subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. Beta gamma t also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to beta gamma t, but was also observed with highly purified beta gamma subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL-60 granulocytes. Only one of these forms was sensitive to stimulation by beta gamma t, demonstrating that stimulation of phospholipase C by beta gamma subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein beta gamma subunits may play an important and active role in mediating the stimulation of phospholipase C by

  15. The histidine residues of phospholipase C from Bacillus cereus.

    PubMed Central

    Little, C

    1977-01-01

    The inactivation of phospholipase C from Bacillus cereus at pH6 by diethyl pyrocarbonate parallelled the N-ethoxyformylation of a single histidine residue in the enzyme. The inactivation arose from a decrease in the maximum velocity of the enzymic reaction with no effect on the Km value. The inactivation did not apparently alter the ability of the enzyme to bind to a substrate-based affinity gel. The native enzyme contained only one reactive histidine residue. Removal of the two zinc atoms from the enzyme increased the number of reactive histidine residues to five, whereas in the totally denatured enzyme nearly eight such residues were available for reaction with diethyl pyrocarbonate. The enzyme thus appears to contain one histidine residue that is essential for catalytic activity and four that may be involved in co-ordinating the zinc atoms in the structure. PMID:413541

  16. Recent research progress with phospholipase C from Bacillus cereus.

    PubMed

    Lyu, Yan; Ye, Lidan; Xu, Jun; Yang, Xiaohong; Chen, Weiwei; Yu, Hongwei

    2016-01-01

    Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce phosphate monoesters and diacylglycerol. It has many applications in the enzymatic degumming of plant oils. PLC Bc , a bacterial PLC from Bacillus cereus, is an optimal choice for this activity in terms of its wide substrate spectrum, high activity, and approved safety. Unfortunately, its large-scale production and reliable high-throughput screening of PLC Bc remain challenging. Herein, we summarize the research progress regarding PLC Bc with emphasis on the screening methods, expression systems, catalytic mechanisms and inhibitor of PLC Bc . This review hopefully will inspire new achievements in related areas, to promote the sustainable development of PLC Bc and its application.

  17. Phospholipase C-β1 and schizophrenia-related behaviors.

    PubMed

    Koh, Hae-Young

    2013-09-01

    Abnormal expression patterns of phospholipase C-β1(PLC-β1) in specific brain areas of patients with schizophrenia, and its high genetic linkage to the disorder implicated a pathogenetical involvement of PLC-β1 signaling system. The schizophrenia-related behavioral phenotypes displayed in the mutant mice lacking PLC-β1 (PLC-β1 KO) suggested that PLCβ1-linked signaling pathways may be involved in the neural system whose function is disrupted in the pathogenesis of schizophrenia. In the brain, PLC-β1 is known to be linked to muscarinic acetylcholine receptors, metabotropic glutamatergic, serotonergic, and oxytocinergic systems. The objective of this review is to provide an overview of the current knowledge regarding these schizophrenia-related behaviors and discuss the probable ways in which PLC-β1signalling can be involved in the neural mechanisms for each behavior, which may help suggest future directions for research in this area.

  18. Phospholipase C-γ1 involved in brain disorders.

    PubMed

    Jang, Hyun-Jun; Yang, Yong Ryoul; Kim, Jung Kuk; Choi, Jang Hyun; Seo, Young-Kyo; Lee, Yong Hwa; Lee, Jeung Eun; Ryu, Sung Ho; Suh, Pann-Ghill

    2013-01-01

    Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) is an important signaling regulator involved in various cellular processes. In brain, PLC-γ1 is highly expressed and participates in neuronal cell functions mediated by neurotrophins. Consistent with essential roles of PLC-γ1, it is involved in development of brain and synaptic transmission. Significantly, abnormal expression and activation of PLC-γ1 appears in various brain disorders such as epilepsy, depression, Huntington's disease and Alzheimer's disease. Thus, PLC-γ1 has been implicated in brain functions as well as related brain disorders. In this review, we discuss the roles of PLC-γ1 in neuronal functions and its pathological relevance to diverse brain diseases.

  19. Defective phosphatidic acid-phospholipase C signaling in diabetic cardiomyopathy.

    PubMed

    Tappia, Paramjit S; Maddaford, Thane G; Hurtado, Cecilia; Dibrov, Elena; Austria, J Alejandro; Sahi, Nidhi; Panagia, Vincenzo; Pierce, Grant N

    2004-03-26

    The effects of exogenous phosphatidic acid (PA) on Ca2+ transients and contractile activity were studied in cardiomyocytes isolated from chronic streptozotocin-induced diabetic rats. In control cells, 25 microM PA induced a significant increase in active cell shortening and Ca2+ transients. PA increased IP3 generation in the control cardiomyocytes and its inotropic effects were blocked by a phospholipase C inhibitor. In cardiomyocytes from diabetic rats, PA induced a 25% decrease in active cell shortening and no significant effect on Ca2+ transients. Basal and PA-induced IP3 generation in diabetic rat cardiomyocytes was 3-fold lower as compared to control cells. Sarcolemmal membrane PLC activity was impaired. Insulin treatment of the diabetic animals resulted in a partial recovery of PA responses. Our results, therefore, identify an important defect in the PA-PLC signaling pathway in diabetic rat cardiomyocytes, which may have significant implications for heart dysfunction during diabetes.

  20. Multiple roles of phosphoinositide-specific phospholipase C isozymes.

    PubMed

    Suh, Pann-Ghill; Park, Jae-Il; Manzoli, Lucia; Cocco, Lucio; Peak, Joanna C; Katan, Matilda; Fukami, Kiyoko; Kataoka, Tohru; Yun, Sanguk; Ryu, Sung Ho

    2008-06-30

    Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.

  1. Phospholipase C activation is required for cardioprotection by ethanol consumption

    PubMed Central

    Miyamae, Masami; Domae, Naochika; Zhou, Hui-Zhong; Sugioka, Shingo; Diamond, Ivan; Figueredo, Vincent M

    2003-01-01

    Regular alcohol consumption decreases the incidence of myocardial infarction (MI) and improves post-MI survival. It has previously been reported that chronic ethanol exposure induces long-term protection against cardiac ischemia/reperfusion injury, which improves myocardial recovery after MI. Chronic cardioprotection by ethanol requires the activation of myocyte adenosine A1 receptors and sustained intramyocyte translocation of epsilon protein kinase C. A1 receptors activate phospholipase C (PLC). In the present paper, the role of PLC in mediating ethanol’s protective effect against ischemia/reperfusion injury is investigated. Isolated hearts from guinea pigs fed 2.5% ethanol in their water for four months were subjected to ischemia/reperfusion. Hearts from ethanol-treated animals showed improved recovery of left ventricular developed pressure compared with controls (61% versus 38% of baseline, respectively; P<0.05) and decreased necrosis, assessed by the release of creatine kinase (263±18 U/mL × g dry weight versus 360±24 U/mL × g dry weight, respectively; P<0.05). Ethanol protection was abolished by the PLC antagonist, U-73122 (50 nM). These findings suggest that PLC activation is required for ethanol cardioprotection against ischemia/reperfusion injury. PMID:19649218

  2. Stimulation and binding of myocardial phospholipase C by phosphatidic acid.

    PubMed

    Henry, R A; Boyce, S Y; Kurz, T; Wolf, R A

    1995-08-01

    Exposure of adult ventricular myocytes to exogenous natural phosphatidic acid results in the production of inositol phosphates by unknown mechanism(s). We characterized stimulation of myocytic phosphoinositide-specific phospholipase C (PLC) by synthetic dioleoyl phosphatidic acid (PA) as a potential mechanism for modulation of inositol phosphate production. Our data demonstrate that exogenous PA, at 10(-8)-10(-5) M, caused a concentration-dependent increase in inositol 1,4,5-trisphosphate in adult rabbit ventricular myocytes. PA also caused a concentration-dependent increase in in vitro activity of myocytic PLC in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLC-delta 1, the predominant isozyme of PLC expressed in adult rabbit ventricular myocytes, bound to liposomes of PA with high affinity in the presence of EGTA. The phosphomonoester group of PA was critical to in vitro stimulation of myocytic PLC activity and high-affinity binding of PLC-delta 1. We propose that binding of PLC-delta 1 to phosphatidic acid may be a novel mechanism for dynamic membrane association and modulation of PLC in adult ventricular myocytes.

  3. Characterization of two distinct phospholipase C enzymes from Burkholderia pseudomallei.

    PubMed

    Korbsrisate, Sunee; Tomaras, Andrew P; Damnin, Suwat; Ckumdee, Jutturong; Srinon, Varintip; Lengwehasatit, Idsada; Vasil, Michael L; Suparak, Supaporn

    2007-06-01

    Burkholderia pseudomallei is a serious bacterial pathogen that can cause a lethal infection in humans known as melioidosis. In this study two of its phospholipase C (PLC) enzymes (Plc-1 and Plc-2) were characterized. Starting with a virulent strain, two single mutants were constructed, each with one plc gene inactivated, and one double mutant with both plc genes inactivated. The single plc mutants exhibited decreased extracellular PLC activity in comparison to the wild-type strain, thereby demonstrating that the two genes encoded functional extracellular PLCs. Growth comparisons between the wild-type and PLC mutants in egg-yolk-supplemented medium indicated that both PLCs contributed to egg-yolk phospholipid utilization. Both PLCs hydrolysed phosphatidylcholine and sphingomyelin but neither was haemolytic for human erythrocytes. Experimental infections of eukaryotic cells demonstrated that Plc-1 itself had no effect on plaque-forming efficiency but it had an additive effect on increasing the efficiency of Plc-2 to form plaques. Only Plc-2 had a significant role in host cell cytotoxicity. In contrast, neither Plc-1 nor Plc-2 appeared to play any role in multinucleated giant cell (MNGC) formation or induction of apoptotic death in the cells studied. These data suggested that PLCs contribute, at least in part, to B. pseudomallei virulence and support the view that Plc-1 and Plc-2 are not redundant virulence factors.

  4. Effects of Phospholipase C on Fusarium graminearum Growth and Development.

    PubMed

    Zhu, Qili; Zhou, Benguo; Gao, Zhengliang; Liang, Yuancun

    2015-12-01

    Phospholipase C (PLC) plays important roles in regulating various biological processes in eukaryotes. Currently, little is known about the function of PLC in filamentous fungi, especially the plant pathogenic fungi. Fusarium graminearum is the causal agent of Fusarium head blight in many cereal crops. BLAST search revealed that Fusarium genome contains six FgPLC genes. Using quantitative RT-PCR, different FgPLC gene expressions in mycelia were analyzed. To investigate the role of FgPLC in F. graminearum biology, a pharmacological study using a known inhibitor of PLC (U73122) was conducted. Results showed that inhibition of FgPLC resulted in significant alterations of mycelial growth, conidiation, conidial germination, perithecium formation, and expressions of Tri5 and Tri6 genes. As expected, the treatment of F. graminearum with U73343, an inactive analog of U73122, showed no effect on F. graminearum biology. Our results suggested strongly that FgPLC plays important roles in F. graminearum growth and development.

  5. Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C.

    PubMed

    Preuss, I; Kaiser, I; Gehring, U

    2001-10-01

    While searching for a phospholipase C (PLC) specific for phosphatidylcholine in mammalian tissues, we came across such an activity originating from a contamination of Pseudomonas fluorescens. This psychrophilic bacterium was found to contaminate placental extracts upon processing in the cold. The secreted phosphatidylcholine-hydrolyzing PLC was purified by a combination of chromatographic procedures. As substrates, the enzyme preferred dipalmitoyl-phosphatidylcholine and 1-palmitoyl-2-arachidonoyl-phosphatidylcholine over phosphatidylinositol. The active enzyme is a monomer of approximately 40 kDa. As for other bacterial PLCs, the enzyme requires Ca2+ and Zn2+ for activity; dithiothreitol affected the activity due to its chelation of Zn2+, but this inhibition could be compensated for by addition of ZnCl2. The compound D609, described to selectively inhibit phosphatidylcholine-specific PLCs, caused half-inhibition of the P. fluorescens enzyme at approximately 420 microM, while 50-fold lower concentrations similarly affected PLCs from Bacillus cereus and Clostridium perfringens. Partial peptide sequences obtained from the pure P. fluorescens enzyme after tryptic cleavage were used to clone a DNA fragment of 3.5 kb from a P. fluorescens gene library prepared from our laboratory isolate. It contains an ORF of 1155 nucleotides encoding the PLC. There is no significant sequence homology to other PLCs, suggesting that the P. fluorescens enzyme represents a distinct subclass of bacterial PLCs. The protein lacks cysteine residues and consequently contains no disulfide bonds. Interestingly, P. fluorescens reference strain DSMZ 50090 is devoid of the PLC activity described here as well as of the relevant coding sequence.

  6. [Overexpression, purification and characterization of phospholipase C from Acinetobacter calcoaceticus].

    PubMed

    Wang, Yanhong; Zhang, Liang; Gu, Zhenghua; Ding, Zhongyang; Shi, Guiyang

    2014-10-04

    In this study, we constructed two recombinant Escherichia coli strains to produce phospholipase C (PLC) from Acinetobacter calcoaceticus. The recombinant enzymes were purified to homogeneity and characterized. [Methods] We cloned the PLC encoding gene plc1, plc2 from genome DNA of A. calcoaceticus ATCC17902. The amplified fragments were inserted into pET28a(+ to obtain expression plasmids. E. coli BL21 (DE3) harboring the above plasmids were cultivated and induced with isopropyl-beta-D-thiogalactopyranoside to express PLCs. The recombinant PLCs were purified by affinity chromatography and their catalytic properties were characterized. Two PLCs from A. calcoaceticus were cloned and functional expressed in E. coli. The recombinant enzymes have activities of 31,160 +/- 418 U/mg for PLC1 and 13640 +/- 354 U/mg for PLC2, when using p-nitrophenyl phosphorycholine as substrate. The purified PLC1 and PLC2 exhibited optimum temperature at 65 degrees C and 50 degrees C, respectively. Their optimal pH were 8 and 7.5, respectively. PLC2 was stable under 40 degrees C and pH at 8, whereas the residual activity of PLC1 was less than 25% in the same condition. Mg2+ and Ca2+ stimulated two enzymes activity, whereas Zn2. stimulated PLC1 and inhibited PLC2. PLC1 and PLC2 hydrolyzed phosphatidylinositol. It is the first time to express and characterize the PLC gene from A. calcoaceticus ATCC17902. These research results provide reference for the study of food-safety microbiological PLC.

  7. Similar effects of phospholipase C and phorbol ester tumor promoters on primary mouse epidermal cells

    SciTech Connect

    Jeng, A.Y.; Lichti, U.; Strickland, J.E.; Blumberg, P.M.

    1985-11-01

    Interaction of tumor promoting phorbol esters with specific high affinity receptors is probably essential for many of the biological responses elicited by these agents. Since diacylglycerols which can be produced enzymatically from phospholipids by phospholipase C are postulated to be the physiological ligands for the phorbol ester receptor, the authors have examined primary cultures of mouse epidermal basal cells exposed to phospholipase C (Clostridium perfringens) for several biological and biochemical responses characteristic of treatment with 12-O-tetradecanoyl-phorbol-13-acetate, the most potent phorbol ester tumor promoter. Formation of diacylglycerols by treatment with phospholipase C was demonstrated by the dose-dependent release of radioactive diacylglycerols in cells prelabeled with (TH)arachidonic acid. Treatment with phospholipase C led to the morphological changes and to the reduction in epidermal growth factor binding (90%) associated with 12-O-tetradecanoylphorbol-13-acetate treatment. Continuous treatment at the same dose led to the induction of the enzymes ornithine decarboxylase and transglutaminase with a time course and extent similar to the inductions by 12-O-tetradecanoylphorbol-13-acetate. Treatment with phospholipase C yielded substantial suppression of the binding affinity of phorbol-12,13-dibutyrate for its receptors without reduction in total number of binding sites, consistent with the production by phospholipase C of a competitive inhibitor of phorbol ester binding.

  8. Expression of phospholipase C isozymes by murine B lymphocytes.

    PubMed

    Hempel, W M; DeFranco, A L

    1991-06-01

    Cross-linking of membrane (m) Ig, the B cell receptor for Ag, activates protein tyrosine phosphorylation and hydrolysis of phosphotidylinositol 4,5-bisphosphate. The latter signal transduction pathway is an important mediator of antigen receptor engagement. The initial event in this pathway is the activation of phospholipase C (PLC). The identity of the isozyme of PLC used in B cells and the mechanism by which it becomes activated are currently unknown. The cDNA encoding five different isozymes have been cloned. As a first step in identifying the isozyme of PLC that is coupled to mIgM, murine cDNA fragments for the five cloned PLC isozymes were generated by the polymerase chain reaction (PCR), cloned, and used to screen a panel of B cell lines representing different stages of development for PLC mRNA expression. All the B cell lines tested expressed high levels of PLC alpha and PLC gamma 2 mRNA, whereas PLC beta and PLC delta mRNA expression were undetectable by both Northern blot and PCR analysis. PLC gamma 1 had a more complicated pattern of mRNA expression. PLC gamma 1 mRNA expression was lower than that observed for PLC alpha or PLC gamma 2 mRNA and varied widely among different cell lines. The pattern of PLC gamma 1 mRNA expression did not correlate with the developmental stage of the cell lines. The pattern of PLC gamma 1 protein expression in the panel of B cell lines correlated with the pattern of PLC gamma 1 mRNA expression. PLC gamma 1 expression was very low in several B cell lines, despite the fact that these cell lines show mIgM-stimulatable PLC activity. The variable and in some cases very low expression of PLC gamma 1 suggests that it may not be the form of PLC that is activated by mIgM. In contrast, PLC alpha and PLC gamma 2 were abundantly expressed in all B cell lines tested. This observation is consistent with the possibility that PLC alpha or PLC gamma 2 is activated by mIgM.

  9. Activation of human phospholipase C-eta2 by Gbetagamma.

    PubMed

    Zhou, Yixing; Sondek, John; Harden, T Kendall

    2008-04-15

    Phospholipase C-eta2 (PLC-eta2) was recently identified as a novel broadly expressed phosphoinositide-hydrolyzing isozyme [Zhou, Y., et al. (2005) Biochem. J. 391, 667-676; Nakahara, M., et al. (2005) J. Biol. Chem. 280, 29128-29134]. In this study, we investigated the direct regulation of PLC-eta2 by Gbetagamma subunits of heterotrimeric G proteins. Coexpression of PLC-eta2 with Gbeta 1gamma 2, as well as with certain other Gbetagamma dimers, in COS-7 cells resulted in increases in inositol phosphate accumulation. Gbeta 1gamma 2-dependent increases in phosphoinositide hydrolysis also were observed with a truncation mutant of PLC-eta2 that lacks the long alternatively spliced carboxy-terminal domain of the isozyme. To begin to define the enzymatic properties of PLC-eta2 and its potential direct activation by Gbetagamma, a construct of PLC-eta2 encompassing the canonical domains conserved in all PLCs (PH domain through C2 domain) was purified to homogeneity after expression from a baculovirus in insect cells. Enzyme activity of purified PLC-eta2 was quantified after reconstitution with PtdIns(4,5)P 2-containing phospholipid vesicles, and values for K m (14.4 microM) and V max [12.6 micromol min (-1) (mg of protein) (-1)] were similar to activities previously observed with purified PLC-beta or PLC-epsilon isozymes. Moreover, purified Gbeta 1gamma 2 stimulated the activity of purified PLC-eta2 in a concentration-dependent manner similar to that observed with purified PLC-beta2. Activation was dependent on the presence of free Gbeta 1gamma 2 since its sequestration in the presence of Galpha i1 or GRK2-ct reversed Gbeta 1gamma 2-promoted activation. The PH domain of PLC-eta2 is not required for Gbeta 1gamma 2-mediated regulation since a purified fragment encompassing the EF-hand through C2 domains but lacking the PH domain nonetheless was activated by Gbeta 1gamma 2. Taken together, these studies illustrate that PLC-eta2 is a direct downstream effector of Gbetagamma and

  10. The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress.

    PubMed

    Kocourková, Daniela; Krcková, Zuzana; Pejchar, Premysl; Veselková, Stepánka; Valentová, Olga; Wimalasekera, Rinukshi; Scherer, Günther F E; Martinec, Jan

    2011-07-01

    Phosphatidylcholine-hydrolysing phospholipase C, also known as non-specific phospholipase C (NPC), is a new member of the plant phospholipase family that reacts to environmental stresses such as phosphate deficiency and aluminium toxicity, and has a role in root development and brassinolide signalling. Expression of NPC4, one of the six NPC genes in Arabidopsis, was highly induced by NaCl. Maximum expression was observed from 3 h to 6 h after the salt treatment and was dependent on salt concentration. Results of histochemical analysis of P(NPC4):GUS plants showed the localization of salt-induced expression in root tips. On the biochemical level, increased NPC enzyme activity, indicated by accumulation of diacylglycerol, was observed as early as after 30 min of salt treatment of Arabidopsis seedlings. Phenotype analysis of NPC4 knockout plants showed increased sensitivity to salinity as compared with wild-type plants. Under salt stress npc4 plants had shorter roots, lower fresh weight, and reduced seed germination. Expression levels of abscisic acid-related genes ABI1, ABI2, RAB18, PP2CA, and SOT12 were substantially reduced in salt-treated npc4 plants. These observations demonstrate a role for NPC4 in the response of Arabidopsis to salt stress.

  11. The controversial role of phospholipase C epsilon (PLCε) in cancer development and progression

    PubMed Central

    Tyutyunnykova, Anna; Telegeev, Gennady; Dubrovska, Anna

    2017-01-01

    The phospholipase C (PLC) enzymes are important regulators of membrane phospholipid metabolism. PLC proteins can be activated by the receptor tyrosine kinases (RTK) or G-protein coupled receptors (GPCR) in response to the different extracellular stimuli including hormones and growth factors. Activated PLC enzymes hydrolyze phosphoinositides to increase the intracellular level of Ca2+ and produce diacylglycerol, which are important mediators of the intracellular signaling transduction. PLC family includes 13 isozymes belonging to 6 subfamilies according to their domain structures and functions. Although importance of PLC enzymes for key cellular functions is well established, the PLC proteins belonging to the ε, ζ and η subfamilies were identified and characterized only during the last decade. As a largest known PLC protein, PLCε is involved in a variety of signaling pathways and controls different cellular properties. Nevertheless, its role in carcinogenesis remains elusive. The aim of this review is to provide a comprehensive and up-to-date overview of the experimental and clinical data about the role of PLCε in the development and progression of the different types of human and experimental tumors. PMID:28382133

  12. PLC-δ1-Lf, a novel N-terminal extended phospholipase C-δ1.

    PubMed

    Kim, Na Young; Ahn, Sang Jung; Kim, Moo-Sang; Seo, Jung Soo; Kim, Bo Seong; Bak, Hye Jin; Lee, Jin Young; Park, Myoung-Ae; Park, Ju Hyeon; Lee, Hyung Ho; Chung, Joon Ki

    2013-10-10

    Phospholipase C-δ (PLC-δ), a key enzyme in phosphoinositide turnover, is involved in a variety of physiological functions. The widely expressed PLC-δ1 isoform is the best characterized and the most well understood phospholipase family member. However, the functional and molecular mechanisms of PLC-δ1 remain obscure. Here, we identified that the N-terminal region of mouse PLC-δ1 gene has two variants, a novel alternative splicing form, named as long form (mPLC-δ1-Lf) and the previously reported short form (mPLC-δ1-Sf), having exon 2 and exon 1, respectively, while both the gene variants share exons 3-16 for RNA transcription. Furthermore, the expression, identification and enzymatic characterization of the two types of PLC-δ1 genes were compared. Expression of mPLC-δ1-Lf was found to be tissue specific, whereas mPLC-δ1-Sf was widely distributed. The recombinant mPLC-δ1-Sf protein exhibited higher activity than recombinant mPLC-δ1-Lf protein. Although, the general catalytic and regulatory properties of mPLC-δ1-Lf are similar to those of PLC-δ1-Sf isozyme, the mPLC-δ1-Lf showed some distinct regulatory properties, such as tissue-specific expression and lipid binding specificity, particularly for phosphatidylserine.

  13. Different expression and subcellular localization of Phosphoinositide-specific Phospholipase C enzymes in differently polarized macrophages.

    PubMed

    Di Raimo, Tania; Leopizzi, Martina; Mangino, Giorgio; Rocca, Carlo Della; Businaro, Rita; Longo, Lucia; Lo Vasco, Vincenza Rita

    2016-12-01

    Macrophages' phenotypic and functional diversity depends on differentiating programs related to local environmental factors. Recent interest was deserved to the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. The expression panel of PLCs and the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), as well as in M1 and M2 macrophages to list the expressed isoforms and their subcellular localization. Furthermore, we investigated whether inflammatory stimulation modified the basal panel of PLCs' expression and subcellular localization. All PLC enzymes were detected within both M1 and M2 cells, but not in M0 cells. M0, as well as M1 and M2 cells own a specific panel of expression, different for both genes' mRNA expression and intracellular localization of PLC enzymes. The panel of PLC genes' expression and PLC proteins' presence slightly changes after inflammatory stimulation. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization.

  14. Involvement of phospholipases C and D in the defence responses of riboflavin-treated tobacco cells.

    PubMed

    Wang, Lianlian; Zhu, Xiaoping; Liu, Jinwei; Chu, Xiaojing; Jiao, Jiao; Liang, Yuancun

    2013-04-01

    Riboflavin is an activator of defence responses in plants that increases resistance against diseases caused by fungal, oomycete, bacterial and viral pathogens. However, the mechanisms driving defence activation by riboflavin are poorly understood. We investigated the signal transduction pathways of phospholipase C (PLC) and phospholipase D (PLD) in tobacco (Nicotiana tabacum) suspension cells using a pharmacological approach to confirm whether riboflavin-mediated activation of the defence response is dependent on both PLC and PLD. The expression patterns analysed by quantitative reverse transcription-polymerase chain reaction demonstrated that the tobacco PLC and PLD gene families were differentially expressed in riboflavin-treated tobacco cells. PLC and PLD expression accompanied defence responses including the expression of defence response genes (PAL, PR-1a and PR-1b), the production of hydrogen peroxide and the accumulation of the phytoalexin scopoletin in tobacco cells treated with riboflavin. These defence responses were significantly inhibited in the presence of the PLC inhibitor U73122 and the PLD inhibitor 1-butanol; however, inhibitor analogues had no effect. Moreover, treating tobacco cells with phosphatidic acid, a signalling molecule produced by phospholipase catalysis, induced the accumulation of the phytoalexin scopoletin and compensated for the suppressive effects of U73122 and 1-butanol on riboflavin-induced accumulation of the phytoalexin. These results offer pharmacological evidence that PLC and PLD play a role in riboflavin-induced defences of tobacco.

  15. Assay of phospholipases C and D in presence of other lipid hydrolases

    SciTech Connect

    Hostetler, K.Y.; Gardner, M.F.; Aldern, K.A.

    1991-01-01

    The activity of a phospholipase C or phospholipase D may be assessed by measuring the radioactivity or phosphate released into the aqueous phase of a lipid extract. However, in crude enzyme fractions, this type of analysis may not be possible due to formation of water-soluble metabolites by other enzymatic reactions, as demonstrated here with a crude lysosomal enzyme fraction. In such instances, analysis of both water-soluble and lipid-soluble metabolites, at various times of incubation, may still provide clear identification of phospholipases C or D, even when a variety of lipases and other hydrolases are present.

  16. Expression of Phosphoinositide-Specific Phospholipase C Isoforms in Native Endothelial Cells

    PubMed Central

    Béziau, Delphine M.; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R.; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

    2015-01-01

    Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the

  17. Direct activation of human phospholipase C by its well known inhibitor u73122.

    PubMed

    Klein, Ryan R; Bourdon, David M; Costales, Chester L; Wagner, Craig D; White, Wendy L; Williams, Jon D; Hicks, Stephanie N; Sondek, John; Thakker, Dhiren R

    2011-04-08

    Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCβ3 (hPLCβ3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCβ3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 μm. Activation of hPLCβ3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 μm) also activated hPLCγ1 (>10-fold) and hPLCβ2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.

  18. Phospholipase C-ε signaling mediates endothelial cell inflammation and barrier disruption in acute lung injury

    PubMed Central

    Bijli, Kaiser M.; Fazal, Fabeha; Slavin, Spencer A.; Leonard, Antony; Grose, Valerie; Alexander, William B.; Smrcka, Alan V.

    2016-01-01

    Phospholipase C-ε (PLC-ε) is a unique PLC isoform that can be regulated by multiple signaling inputs from both Ras family GTPases and heterotrimeric G proteins and has primary sites of expression in the heart and lung. Whereas the role of PLC-ε in cardiac function and pathology has been documented, its relevance in acute lung injury (ALI) is unclear. We used PLC-ε−/− mice to address the role of PLC-ε in regulating lung vascular inflammation and injury in an aerosolized bacterial LPS inhalation mouse model of ALI. PLC-ε−/− mice showed a marked decrease in LPS-induced proinflammatory mediators (ICAM-1, VCAM-1, TNF-α, IL-1β, IL-6, macrophage inflammatory protein 2, keratinocyte-derived cytokine, monocyte chemoattractant protein 1, and granulocyte-macrophage colony-stimulating factor), lung neutrophil infiltration and microvascular leakage, and loss of VE-cadherin compared with PLC-ε+/+ mice. These data identify PLC-ε as a critical determinant of proinflammatory and leaky phenotype of the lung. To test the possibility that PLC-ε activity in endothelial cells (EC) could contribute to ALI, we determined its role in EC inflammation and barrier disruption. RNAi knockdown of PLC-ε inhibited NF-κB activity in response to diverse proinflammatory stimuli, thrombin, LPS, TNF-α, and the nonreceptor agonist phorbol 13-myristate 12-acetate (phorbol esters) in EC. Depletion of PLC-ε also inhibited thrombin-induced expression of NF-κB target gene, VCAM-1. Importantly, PLC-ε knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and formation of actin stress fibers. These data identify PLC-ε as a novel regulator of EC inflammation and permeability and show a hitherto unknown role of PLC-ε in the pathogenesis of ALI. PMID:27371732

  19. A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility.

    PubMed

    Kashir, Junaid; Konstantinidis, Michalis; Jones, Celine; Lemmon, Bernadette; Lee, Hoi Chang; Hamer, Rebecca; Heindryckx, Bjorn; Deane, Charlotte M; De Sutter, Petra; Fissore, Rafael A; Parrington, John; Wells, Dagan; Coward, Kevin

    2012-01-01

    Male factor and idiopathic infertility contribute significantly to global infertility, with abnormal testicular gene expression considered to be a major cause. Certain types of male infertility are caused by failure of the sperm to activate the oocyte, a process normally regulated by calcium oscillations, thought to be induced by a sperm-specific phospholipase C, PLCzeta (PLCζ). Previously, we identified a point mutation in an infertile male resulting in the substitution of histidine for proline at position 398 of the protein sequence (PLCζ(H398P)), leading to abnormal PLCζ function and infertility. Here, using a combination of direct-sequencing and mini-sequencing of the PLCζ gene from the patient and his family, we report the identification of a second PLCζ mutation in the same patient resulting in a histidine to leucine substitution at position 233 (PLCζ(H233L)), which is predicted to disrupt local protein interactions in a manner similar to PLCζ(H398P) and was shown to exhibit abnormal calcium oscillatory ability following predictive 3D modelling and cRNA injection in mouse oocytes respectively. We show that PLCζ(H233L) and PLCζ(H398P) exist on distinct parental chromosomes, the former inherited from the patient's mother and the latter from his father. Neither mutation was detected utilizing custom-made single-nucleotide polymorphism assays in 100 fertile males and females, or 8 infertile males with characterized oocyte activation deficiency. Collectively, our findings provide further evidence regarding the importance of PLCζ at oocyte activation and forms of male infertility where this is deficient. Additionally, we show that the inheritance patterns underlying male infertility are more complex than previously thought and may involve maternal mechanisms.

  20. Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity

    PubMed Central

    Wu, Jun; Nilsson, Åke; Jönsson, Bo A. G.; Stenstad, Hanna; Agace, William; Cheng, Yajun; Duan, Rui-Dong

    2005-01-01

    Alkaline sphingomyelinase (alk-SMase) is a new member of the NPP (nucleotide pyrophosphatase/phosphodiesterase) family that hydrolyses SM (sphingomyelin) to generate ceramide in the intestinal tract. The enzyme may protect the intestinal mucosa from inflammation and tumorigenesis. PAF (platelet-activating factor) is a pro-inflammatory phospholipid involved in pathogenesis of inflammatory bowel diseases. We examined whether alk-SMase can hydrolyse and inactivate PAF. [3H]Octadecyl-labelled PAF was incubated with purified rat intestinal alk-SMase or recombinant human alk-SMase expressed in COS-7 cells. The hydrolytic products were assayed with TLC and MS. We found that alkSMase cleaved the phosphocholine head group from PAF and generated 1-O-alkyl-2-acetyl-sn-glycerol. Differing from the activity against SM, the activity against PAF was optimal at pH 7.5, inhibited by EDTA and stimulated by 0.1–0.25 mM Zn2+. The activity was abolished by site mutation of the predicted metal-binding sites that are conserved in all NPP members. Similar to the activity against SM, the activity against PAF was dependent on bile salt, particularly taurocholate and taurochenodeoxycholate. The Vmax for PAF hydrolysis was 374 μmol·h−1·(mg of protein)−1. The hydrolysis of PAF and SM could be inhibited by the presence of SM and PAF respectively, the inhibition of PAF hydrolysis by SM being stronger. The PAF-induced MAPK (mitogen-activated protein kinase) activation and IL-8 (interleukin 8) release in HT-29 cells, and chemotaxis in leucocytes were abolished by alk-SMase treatment. In conclusion, alk-SMase hydrolyses and inactivates PAF by a phospholipase C activity. The finding reveals a novel function, by which alk-SMase may counteract the development of intestinal inflammation and colon cancer. PMID:16255717

  1. Identification of the Elusive Mammalian Enzyme Phospatidylcholine-Specific Phospholipase C

    DTIC Science & Technology

    2015-01-01

    mammalian protein, phosphatidycholine- specific phospholipase C (PC-PLC) in the inflammatory processes involved in progression of rheumatoid arthritis (RA...serum, rheumatoid arthritis , transcriptome sequencing, HUVECs, U937 cells 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF...aims at identifying novel players that are critically involved in the progression of rheumatoid arthritis (RA). The identification of these factors

  2. Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi.

    PubMed Central

    Tsutsui, K; Minami, J; Matsushita, O; Katayama, S; Taniguchi, Y; Nakamura, S; Nishioka, M; Okabe, A

    1995-01-01

    The phylogenetic interrelationships between strains of 5 toxin types (A to E) of Clostridium perfringens were examined by analysis of differences in the nucleotide sequences of phospholipase C genes (plc genes) among 10 strains, including 3 strains for which the plc gene sequences have been previously reported. A plc gene was also cloned from a Clostridium novyi type A strain and sequenced to analyze the interspecies diversity of plc genes. Phylogenetic trees constructed by the neighbor-joining method revealed that the phylogeny of C. perfringens strains is not related to toxin typing, in agreement with the results of a comparative genome mapping study by Canard et al. (B. Canard, B. Saint-Joanis, and S. T. Cole, Mol. Microbiol. 6:1421-1429, 1992). Various C. perfringens phospholipase C enzymes were purified from cultures of Escherichia coli cells into which the encoding plc genes had been cloned. All of the enzymes showed the same specific activity. On the other hand, the level of plc transcripts differed greatly (up to 40-fold) from one C. perfringens strain to another. No significant difference in the nucleotide sequence of the plc promoter region was observed for any of the plc genes. These results suggest that the variation in phospholipase C activity among different strains is not due to mutation in the plc coding region but to that in an extragenic region. The evolution of C. perfringens phospholipase C is discussed on the basis of similarities and differences between clostridial plc genes. PMID:8522524

  3. Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi.

    PubMed

    Tsutsui, K; Minami, J; Matsushita, O; Katayama, S; Taniguchi, Y; Nakamura, S; Nishioka, M; Okabe, A

    1995-12-01

    The phylogenetic interrelationships between strains of 5 toxin types (A to E) of Clostridium perfringens were examined by analysis of differences in the nucleotide sequences of phospholipase C genes (plc genes) among 10 strains, including 3 strains for which the plc gene sequences have been previously reported. A plc gene was also cloned from a Clostridium novyi type A strain and sequenced to analyze the interspecies diversity of plc genes. Phylogenetic trees constructed by the neighbor-joining method revealed that the phylogeny of C. perfringens strains is not related to toxin typing, in agreement with the results of a comparative genome mapping study by Canard et al. (B. Canard, B. Saint-Joanis, and S. T. Cole, Mol. Microbiol. 6:1421-1429, 1992). Various C. perfringens phospholipase C enzymes were purified from cultures of Escherichia coli cells into which the encoding plc genes had been cloned. All of the enzymes showed the same specific activity. On the other hand, the level of plc transcripts differed greatly (up to 40-fold) from one C. perfringens strain to another. No significant difference in the nucleotide sequence of the plc promoter region was observed for any of the plc genes. These results suggest that the variation in phospholipase C activity among different strains is not due to mutation in the plc coding region but to that in an extragenic region. The evolution of C. perfringens phospholipase C is discussed on the basis of similarities and differences between clostridial plc genes.

  4. Phospholipase C Activity in Human Polymorphonuclear Leukocytes: Partial Characterization and Effect of Indomethacin

    DTIC Science & Technology

    1988-12-01

    phospholipase C activity alone, and in the presence of 0.5 mM and I mM indomethacin, is plotted according to Lineweaver and Burke as described previously...The data were plotted according to the method of Lineweaver and Burke (26). The values represent the mean + S.E.M. of values derived from neutrophils of 4 subjects. 18

  5. Modification of erythrocyte membranes by a purified phosphatidylinositol-specific phospholipase C (Staphylococcus aureus).

    PubMed Central

    Low, M G; Finean, J B

    1977-01-01

    A phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was purified by a three-step procedure. The specific activity of the purified enzyme was approx. 6000 times that of the culture supernatant, with an overall recovery of approx. 10%. Estimation of the molecular weight by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by gel filtration gave values of 33000 and 20000 respectively. A thiol group appears to be necessary for the activity of the enzyme. The purified enzyme had no detectable delta-haemolytic activity and was unable to hydrolyse S. aureus phospholipids. Phosphatidyl-inositol in erythrocyte 'ghosts' was readily hydrolysed by the purified phospholipase C. However, in contrast with our previous preliminary observations, phosphatidylinositol in intact erythrocytes was not significantly hydrolysed. These results suggest that at least 75-80% of the phosphatidylinositol is located at the inner leaflet of the membrane. PMID:849283

  6. A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.

    PubMed

    Shashidhar, M S; Volwerk, J J; Griffith, O H; Keana, J F

    1991-12-01

    A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.

  7. Identification of the Elusive Mammalian Enzyme Phosphatidylcholine-Specific Phospholipase C

    DTIC Science & Technology

    2014-07-01

    processes involved in progression of rheumatoid arthritis (RA). Thus, the main scopes of this proposal are: 1. to identify the PC-PLC gene and protein...of PC-PLC. 15. SUBJECT TERMS Phosphatidycholine-specific phospholipase C, lipopolisaccharide, oxidized lipoproteins, serum, rheumatoid arthritis ...present proposal aims at identifying novel players that are critically involved in the progression of rheumatoid arthritis (RA). The identification of

  8. Vascular smooth muscle cell spreading onto fibrinogen is regulated by calpains and phospholipase C.

    PubMed

    Paulhe, F; Bogyo, A; Chap, H; Perret, B; Racaud-Sultan, C

    2001-11-09

    Fibrinogen deposition and smooth muscle cell migration are important causes of atherosclerosis and angiogenesis. Involvement of calpains in vascular smooth muscle cell adhesion onto fibrinogen was investigated. Using calpain inhibitors, we showed that activation of calpains was required for smooth muscle cell spreading. An increase of (32)P-labeled phosphatidic acid and phosphatidylinositol-3,4-bisphosphate, respective products of phospholipase C and phosphoinositide 3-kinase activities, was measured in adherent cells. Addition of the calpain inhibitor calpeptin strongly decreased phosphatidic acid and phosphatidylinositol-3,4-bisphosphate. However, smooth muscle cell spreading was prevented by the phospholipase C inhibitor U-73122, but poorly modified by phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Moreover, PLC was found to act upstream of the PI 3-kinase IA isoform. Thus, our data provide the first evidence that calpains are required for smooth muscle cell spreading. Further, phospholipase C activation is pointed as a key step of cell-spreading regulation by calpains. Copyright 2001 Academic Press.

  9. The receptor tyrosine kinase MerTK activates phospholipase C γ2 during recognition of apoptotic thymocytes by murine macrophages

    PubMed Central

    Todt, Jill C.; Hu, Bin; Curtis, Jeffrey L.

    2008-01-01

    Apoptotic leukocytes must be cleared efficiently by macrophages (Mø). Apoptotic cell phagocytosis by Mø requires the receptor tyrosine kinase (RTK) MerTK (also known as c-Mer and Tyro12), the phosphatidylserine receptor (PS-R), and the classical protein kinase C (PKC) isoform βII, which translocates to Mø membrane and cytoskeletal fractions in a PS-R-dependent fashion. How these molecules cooperate to induce phagocytosis is unknown. Because the phosphatidylinositol-specific phospholipase (PI-PLC) PLC γ2 is downstream of RTKs in some cell types and can activate classical PKCs, we hypothesized that MerTK signals via PLC γ2. To test this hypothesis, we examined the interaction of MerTK and PLC γ2 in resident murine PMø and in the murine Mø cell line J774A.1 (J774) following exposure to apoptotic thymocytes. We found that, as with PMø, J774 phagocytosis of apoptotic thymocytes was inhibited by antibody against MerTK. Western blotting and immunoprecipitation showed that exposure to apoptotic cells produced three time-dependent changes in PMø and J774: (1) tyrosine phosphorylation of MerTK; (2) association of PLC γ2 with MerTK; and (3) tyrosine phosphorylation of PLC γ2. Phosphorylation of PLC γ2 and its association with MerTK was also induced by cross-linking MerTK using antibody. A PI-PLC appears to be required for phagocytosis of apoptotic cells because the PI-PLC inhibitor Et-18-OCH3 and the PLC inhibitor U73122, but not the inactive control U73343, blocked phagocytosis without impairing adhesion. On apoptotic cell adhesion to Mø, MerTK signals at least in part via PLC γ2. PMID:14704368

  10. Enhanced bradykinin-stimulated phospholipase C activity in murine embryonic stem cells lacking the G-protein alphaq-subunit.

    PubMed Central

    Ricupero, D A; Polgar, P; Taylor, L; Sowell, M O; Gao, Y; Bradwin, G; Mortensen, R M

    1997-01-01

    The gene coding for the G-protein alphaq subunit was interrupted by homologous recombination in murine embryonic stem cells (alphaq-null ES cells) as detected by Southern analysis and reverse-transcriptase PCR. The bradykinin (BK) B2 receptor was stably transfected into wild-type (WT) alphai-2-null and alphaq-null ES cells. The B2 receptor bound BK with high affinity and mobilized Ca2+. BK also activated phospholipase C (PLC), as determined by total inositol phosphate (IP) accumulation in a Bordetella pertussis toxin- and genistein-insensitive manner. In WT and alphai-2-null ES cells, BK increased IP levels approx. 4-fold above baseline. Most interestingly, in alphaq-null ES cells, BK increased IP accumulation approx. 9-fold above baseline. Re-expression of alphaq in alphaq-null ES cells resulted in normalization of the BK-stimulated IP accumulation (4-fold above baseline). These results suggest that the B2 receptor activates PLC through more than one member of the Gq family. Additionally, the absence of alphaq alters the kinetics of IP generation, which may reflect intrinsic characteristics of individual members of the Gq family or a decreased susceptibility to heterologous regulation in the alphaq-null ES cells, thus allowing for a more sustained generation of IP. PMID:9581559

  11. Cloning of a Phosphate-Regulated Hemolysin Gene (Phospholipase C) from Pseudomonas aeruginosa

    PubMed Central

    Vasil, Michael L.; Berka, Randy M.; Gray, Gregory L.; Nakai, Hiroshi

    1982-01-01

    Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa is a phosphate (Pi)-regulated extracellular protein which may be a significant virulence factor of this organism. The gene for this hemolytic enzyme was cloned on a 4.1-megadalton (Mdal) fragment from a BamHI digest of P. aeruginosa PAO1 genomic DNA and was inserted into the BamHI sites of the multicopy Escherichia coli(pBR322) and P. aeruginosa(pMW79) vectors. The E. coli and P. aeruginosa recombinant plasmids were designated pGV26 and pVB81, respectively. A restriction map of the 4.1-Mdal fragment from pGV26 was constructed, using double and single digestions with BamHI and EcoRI and several different restriction enzymes. Based on information from this map, a 2.4-Mdal BamHI/BglII fragment containing the gene for phospholipase C was subcloned to pBR322. The hybrid plasmids pGV26 and pVB81 direct the synthesis of enzymatically active phospholipase C, which is also hemolytic. The plasmid-directed synthesis of phospholipase C in E. coli or P. aeruginosa is not repressible by Pi as is the chromosomally directed synthesis in P. aeruginosa. Data are presented which suggest that the synthesis of phospholipase C from pGV26 and pVB81 is directed from the tetracycline resistance gene promoter. The level of enzyme activity produced by E. coli(pGV26) is slightly higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions. In contrast, the levels produced by P. aeruginosa(pVB81) are at least 600-fold higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions and approximately 20-fold higher than those produced by P. aeruginosa(pMW79) under derepressed conditions. The majority (85%) of the enzyme produced by E. coli(pGV26) remained cell associated, whereas >95% of the enzyme produced by P. aeruginosa(pVB81) was extracellular. Analysis of extracellular proteins from cultures of P. aeruginosa(pMW79) and P. aeruginosa(pVB81) by high-performance liquid chromotography and

  12. The phenotype of a phospholipase C (plc-1) mutant in a filamentous fungus, Neurospora crassa.

    PubMed

    Lew, Roger R; Giblon, Rachel E; Lorenti, Miranda S H

    2015-09-01

    In the filamentous fungus Neurospora crassa, phospholipase C may play a role in hyphal extension at the growing tips as part of a growth-sensing mechanism that activates calcium release from internal stores to mediate continued expansion of the hyphal tip. One candidate for a tip-localized phospholipase C is PLC-1. We characterized morphology and growth characteristics of a knockout mutant (KO plc-1) and a RIP mutated strain (RIP plc-1) (missense mutations and a nonsense mutation render the gene product non-functional). Growth and hyphal cytology of wildtype and KO plc-1 were similar, but the RIP plc-1 mutant grew slower and exhibited abnormal membrane structures at the hyphal tip, imaged using the fluorescence dye FM4-64. To test for causes of the slower growth of the RIP plc-1 mutant, we examined its physiological poise compared to wildtype and the KO plc-1 mutant. The electrical properties of all three strains and the electrogenic contribution of the plasma membrane H(+)-ATPase (identified by cyanide inhibition) were the same. Responses to high osmolarity were also similar. However, the RIP plc-1 mutant had a significantly lower turgor, a possible cause of its slower growth. While growth of all three strains was inhibited by the phospholipase C inhibitor 3-nitrocoumarin, the RIP plc-1 mutant did not exhibit hyphal bursting after addition of the inhibitor, observed in both wildtype and the KO plc-1 mutant. Although the plc-1 gene is not obligatory for tip growth, the phenotype of the RIP plc-1 mutant - abnormal tip cytology, lower turgor and resistance to inhibitor-induced hyphal bursting - suggest it does play a role in tip growth. The expression of a dysfunctional plc-1 gene may cause a shift to alternative mechanism(s) of growth sensing in hyphal extension.

  13. Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

    PubMed Central

    Stith, Bradley J.

    2015-01-01

    This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

  14. Inositol phosphosphingolipid phospholipase C1 regulates plasma membrane ATPase (Pma1) stability in Cryptococcus neoformans.

    PubMed

    Farnoud, Amir M; Mor, Visesato; Singh, Ashutosh; Del Poeta, Maurizio

    2014-11-03

    Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.

  15. Cyclin A2 modulates EMT via β-catenin and phospholipase C pathways.

    PubMed

    Cheung, Caroline T; Bendris, Nawal; Paul, Conception; Hamieh, Abdallah; Anouar, Youssef; Hahne, Michael; Blanchard, Jean-Marie; Lemmers, Bénédicte

    2015-08-01

    We have previously demonstrated that Cyclin A2 is involved in cytoskeletal dynamics, epithelial-mesenchymal transition (EMT) and metastasis. This phenotype was potentiated by activated oncogenic H-Ras. However, the mechanisms governing EMT in these cells have not yet been elucidated. Here, we dissected the pathways that are responsible for EMT in cells deficient for Cyclin A2. In Cyclin A2-depleted normal murine mammary gland (NMuMG) cells expressing RasV12, we found that β-catenin was liberated from the cell membrane and cell-cell junctions and underwent nuclear translocation and activation. Components of the canonical wingless (WNT) pathway, including WNT8b, WNT10a, WNT10b, frizzled 1 and 2 and TCF4 were upregulated at the messenger RNA and protein levels following Cyclin A2 depletion. However, suppression of the WNT pathway using the acetyltransferase porcupine inhibitor C59 did not reverse EMT whereas a dominant negative form of TCF4 as well as inhibition of phospholipase C using U73122 were able to do so. This suggests that a WNT-independent mechanism of β-catenin activation via phospholipase C is involved in the EMT induced by Cyclin A2 depletion. Our findings will broaden our knowledge on how Cyclin A2 contributes to EMT and metastasis.

  16. Substance P receptor desensitization requires receptor activation but not phospholipase C

    SciTech Connect

    Sugiya, Hiroshi; Putney, J.W. Jr. )

    1988-08-01

    Previous studies have shown that exposure of parotid acinar cells to substance P at 37{degree}C results in activation of phospholipase C, formation of ({sup 3}H)inositol 1,4,5-trisphosphate (IP{sub 3}), and persistent desensitization of the substance P response. In cells treated with antimycin in medium containing glucose, ATP was decreased to {approximately}20% of control values, IP{sub 3} formation was completely inhibited, but desensitization was unaffected. When cells were treated with antimycin in the absence of glucose, cellular ATP was decreased to {approximately}5% of control values, and both IP{sub 3} formation and desensitization were blocked. A series of substance P-related peptides increased the formation of ({sup 3}H)IP{sub 3} and induced desensitization of the substance P response with a similar rank order of potencies. The substance P antagonist, (D-Pro{sup 2}, D-Try{sup 7,9})-substance P, inhibited substance P-induced IP{sub 3} formation and desensitization but did not induce desensitization. These results suggest that the desensitization of substance P-induced IP{sub 3} formation requires agonist activation of a P-type substance P receptor, and that one or more cellular ATP-dependent processes are required for this reaction. However, activation of phospholipase C and the generation of inositol phosphates does not seem to be a prerequisite for desensitization.

  17. Grb2 negatively regulates epidermal growth factor-induced phospholipase C-gamma1 activity through the direct interaction with tyrosine-phosphorylated phospholipase C-gamma1.

    PubMed

    Choi, Jang Hyun; Hong, Won-Pyo; Yun, Sanguk; Kim, Hyeon Soo; Lee, Jong-Ryul; Park, Jong Bae; Bae, Yun Soo; Ryu, Sung Ho; Suh, Pann-Ghill

    2005-10-01

    Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation. Upon the stimulation of growth factors and hormones, PLC-gamma1 is rapidly phosphorylated at three known sites; Tyr771, Tyr783 and Tyr1254 and its enzymatic activity is up-regulated. In this study, we demonstrate for the first time that Grb2, an adaptor protein, specifically interacts with tyrosine-phosphorylated PLC-gamma1 at Tyr783. The association of Grb2 with PLC-gamma1 was induced by the treatment with epidermal growth factor (EGF). Replacement of Tyr783 with Phe completely blocked EGF-induced interaction of PLC-gamma1 with Grb2, indicating that tyrosine phosphorylation of PLC-gamma1 at Tyr783 is essential for the interaction with Grb2. Interestingly, the depletion of Grb2 from HEK-293 cells by RNA interference significantly enhanced increased EGF-induced PLC-gamma1 enzymatic activity and mobilization of the intracellular Ca2+, while it did not affect EGF-induced tyrosine phosphorylation of PLC-gamma1. Furthermore, overexpression of Grb2 inhibited PLC-gamma1 enzymatic activity. Taken together, these results suggest Grb2, in addition to its key function in signaling through Ras, may have a negatively regulatory role on EGF-induced PLC-gamma1 activation.

  18. Development of a highly efficient oil degumming process using a novel phosphatidylinositol-specific phospholipase C enzyme.

    PubMed

    Cerminati, Sebastián; Eberhardt, Florencia; Elena, Claudia E; Peirú, Salvador; Castelli, María E; Menzella, Hugo G

    2017-06-01

    Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14 g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

  19. Prostacyclin receptor-independent inhibition of phospholipase C activity by non-prostanoid prostacyclin mimetics

    PubMed Central

    Chow, Kevin B S; Wong, Yung H; Wise, Helen

    2001-01-01

    Chinese hamster ovary (CHO) cells were transiently transfected with the mouse prostacyclin (mIP) receptor to examine IP agonist-mediated stimulation of [3H]-cyclic AMP and [3H]-inositol phosphate production.The prostacyclin analogues, cicaprost, iloprost, carbacyclin and prostaglandin E1, stimulated adenylyl cyclase activity with EC50 values of 5, 6, 25 and 95 nM, respectively. These IP agonists also stimulated the phospholipase C pathway with 10 – 40 fold lower potency than stimulation of adenylyl cyclase.The non-prostanoid prostacyclin mimetics, octimibate, BMY 42393 and BMY 45778, also stimulated adenylyl cyclase activity, with EC50 values of 219, 166 and 398 nM, respectively, but failed to stimulate [3H]-inositol phosphate production.Octimibate, BMY 42393 and BMY 45778 inhibited iloprost-stimulated [3H]-inositol phosphate production in a non-competitive manner.Activation of the endogenously-expressed P2 purinergic receptor by ATP led to an increase in [3H]-inositol phosphate production which was inhibited by the non-prostanoid prostacyclin mimetics in non-transfected CHO cells. Prostacyclin analogues and other prostanoid receptor ligands failed to inhibit ATP-stimulated [3H]-inositol phosphate production.A comparison between the IP receptor-specific non-prostanoid ONO-1310 and the structurally-related EP3 receptor-specific agonist ONO-AP-324, indicated that the inhibitory effect of non-prostanoids was specific for those compounds known to activate IP receptors.The non-prostanoid prostacyclin mimetics also inhibited phospholipase C activity when stimulated by constitutively-active mutant GαqRC, Gα14RC and Gα16QL transiently expressed in CHO cells. These drugs did not inhibit adenylyl cyclase activity when stimulated by the constitutively-active mutant GαsQL.These results suggest that non-prostanoid prostacyclin mimetics can specifically inhibit [3H]-inositol phosphate production by targeting Gq/11 and/or phospholipase C in CHO cells, and

  20. On-Tissue Phospholipase C Digestion for Enhanced MALDI-MS Imaging of Neutral Glycosphingolipids.

    PubMed

    Vens-Cappell, Simeon; Kouzel, Ivan U; Kettling, Hans; Soltwisch, Jens; Bauwens, Andreas; Porubsky, Stefan; Müthing, Johannes; Dreisewerd, Klaus

    2016-06-07

    Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to simultaneously visualize the lateral distribution of different lipid classes in tissue sections, but the applicability of the method to real-life samples is often limited by ion suppression effects. In particular, the presence of abundant phosphatidylcholines (PCs) can reduce the ion yields for all other lipid species in positive ion mode measurements. Here, we used on-tissue treatment with buffer-free phospholipase C (PLC) to near-quantitatively degrade PCs in fresh-frozen tissue sections. The ion signal intensities of mono-, di-, and oligohexosylceramides were enhanced by up to 10-fold. In addition, visualization of Shiga toxin receptor globotriaosylceramide (Gb3Cer) in the kidneys of wild-type and α-galactosidase A-knockout (Fabry) mice was possible at about ten micrometer resolution. Importantly, the PLC treatment did not decrease the high lateral resolution of the MS imaging analysis.

  1. Mutations in phospholipase C epsilon 1 are not sufficient to cause diffuse mesangial sclerosis.

    PubMed

    Gilbert, Rodney D; Turner, Claire L S; Gibson, Jane; Bass, Paul S; Haq, Mushfequr R; Cross, Esta; Bunyan, David J; Collins, Andrew R; Tapper, William J; Needell, Juliet C; Dell, Beverley; Morton, Newton E; Temple, I Karen; Robinson, David O

    2009-02-01

    Diffuse mesangial sclerosis occurs as an isolated abnormality or as a part of a syndrome. Recently, mutations in phospholipase C epsilon 1 (PLCE1) were found to cause a nonsyndromic, autosomal recessive form of this disease. Here we describe three children from one consanguineous kindred of Pakistani origin with diffuse mesangial sclerosis who presented with congenital or infantile nephrotic syndrome. Homozygous mutations in PLCE1 (also known as KIAA1516, PLCE, or NPHS3) were identified following genome-wide mapping of single-nucleotide polymorphisms. All affected children were homozygous for a four-basepair deletion in exon 3, which created a premature translational stop codon. Analysis of the asymptomatic father of two of the children revealed that he was also homozygous for the same mutation. We conclude this nonpenetrance may be due to compensatory mutations at a second locus and that mutation within PLCE1 is not always sufficient to cause diffuse mesangial sclerosis.

  2. An autoinhibitory helix in the C-terminal region of phospholipase C-[beta] mediates G[alpaha subscript q] activation

    SciTech Connect

    Lyon, Angeline M.; Tesmer, Valerie M.; Dhamsania, Vishan D.; Thal, David M.; Gutierrez, Joanne; Chowdhury, Shoaib; Suddala, Krishna C.; Northup, John K.; Tesmer, John J.G.

    2012-03-16

    The enzyme phospholipase C-{beta} (PLC{beta}) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the G{sub q} family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLC{beta} (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLC{beta}3 considerably increase basal activity and diminish stimulation by G{alpha}{sub q}. G{alpha}{sub q} binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLC{beta}.

  3. In vivo Detection of Phospholipase C by Enzyme-Activated Near-infrared Probes

    PubMed Central

    Mawn, Theresa M.; Popov, Anatoliy V.; Beardsley, Nancy J.; Stefflova, Klara; Milkevitch, Matthew; Zheng, Gang; Delikatny, E. James

    2011-01-01

    In this paper the characterization of the first near-infrared (NIR) phospholipase-activated molecular beacon is reported and its utility for in vivo cancer imaging is demonstrated. The probe consists of three elements: a phospholipid (PL) backbone to which the NIR fluorophore, pyropheophorbide a (Pyro), and the NIR Black Hole Quencher 3 (BHQ) were conjugated. Due to the close proximity of BHQ to Pyro, the Pyro-PtdEtn-BHQ probe is self-quenched until enzyme hydrolysis releases the fluorophore. The Pyro-PtdEtn-BHQ probe is highly specific to one isoform of phospholipase C, phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine. Incubation of Pyro-PtdEtn-BHQ in vitro with PC-PLC demonstrated a 150-fold increase in fluorescence that could be inhibited by the specific PC-PLC inhibitor tricyclodecan-9-yl xanthogenate (D609) with an IC50 of 34±8 µM. Since elevations in phosphocholine have been consistently observed by magnetic resonance spectroscopy in a wide array of cancer cells and solid tumors, we assessed the utility of Pyro-PtdEtn-BHQ as a probe for targeted tumor imaging. Injection of Pyro-PtdEtn-BHQ into mice bearing DU145 human prostate tumor xenografts followed by in vivo NIR imaging resulted in a 4-fold increase in tumor radiance over background and a 2 fold increase in the tumor:muscle ratio. Tumor fluorescence enhancement was inhibited with administration of D609. The ability to image PC-PLC activity in vivo provides a unique and sensitive method of monitoring one of the critical phospholipase signaling pathways activated in cancer, as well as the phospholipase activities that are altered in response to cancer treatment. PMID:22034913

  4. C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C.

    PubMed

    Rupwate, Sunny D; Rajasekharan, Ram

    2012-02-01

    Phosphoinositide-specific phospholipase C (PLC) is involved in Ca²⁺ mediated signalling events that lead to altered cellular status. Using various sequence-analysis methods, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca²⁺ binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca²⁺ signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca²⁺ binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. Overall, these data suggest that the C2 domain of PLC plays a vital role in calcium signalling.

  5. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase

    PubMed Central

    Ruisanchez, Éva; Dancs, Péter; Kerék, Margit; Németh, Tamás; Faragó, Bernadett; Balogh, Andrea; Patil, Renukadevi; Jennings, Brett L.; Liliom, Károly; Malik, Kafait U.; Smrcka, Alan V.; Tigyi, Gabor; Benyó, Zoltán

    2014-01-01

    Lysophosphatidic acid (LPA) has been implicated as a mediator of several cardiovascular functions, but its potential involvement in the control of vascular tone is obscure. Here, we show that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1–3 receptors) relax intact mouse thoracic aorta with similar Emax values (53.9 and 51.9% of phenylephrine-induced precontraction), although the EC50 of LPA- and VPC31143-induced vasorelaxations were different (400 vs. 15 nM, respectively). Mechanical removal of the endothelium or genetic deletion of endothelial nitric oxide synthase (eNOS) not only diminished vasorelaxation by LPA or VPC31143 but converted it to vasoconstriction. Freshly isolated mouse aortic endothelial cells expressed LPA1, LPA2, LPA4 and LPA5 transcripts. The LPA1,3 antagonist Ki16425, the LPA1 antagonist AM095, and the genetic deletion of LPA1, but not that of LPA2, abolished LPA-induced vasorelaxation. Inhibition of the phosphoinositide 3 kinase–protein kinase B/Akt pathway by wortmannin or MK-2206 failed to influence the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edelfosine, but not genetic deletion of PLCε, abolished LPA-induced vasorelaxation and indicated that a PLC enzyme, other than PLCε, mediates the response. In summary, the present study identifies LPA as an endothelium-dependent vasodilator substance acting via LPA1, PLC, and eNOS.—Ruisanchez, É., Dancs, P., Kerék, M., Németh, T., Faragó, B., Balogh, A., Patil, R., Jennings, B. L., Liliom, K., Malik, K. U., Smrcka, A. V., Tigyi, G., Benyó, Z. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase. PMID:24249637

  6. Brain-derived neurotrophic factor enhances cholinergic contraction of longitudinal muscle of rabbit intestine via activation of phospholipase C.

    PubMed

    Al-Qudah, M; Anderson, C D; Mahavadi, S; Bradley, Z L; Akbarali, H I; Murthy, K S; Grider, J R

    2014-02-15

    Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of proteins best known for its role in neuronal survival, differentiation, migration, and synaptic plasticity in central and peripheral neurons. BDNF is also widely expressed in nonneuronal tissues including the gastrointestinal tract. The role of BDNF in intestinal smooth muscle contractility is not well defined. The aim of this study was to identify the role of BDNF in carbachol (CCh)- and substance P (SP)-induced contraction of intestinal longitudinal smooth muscle. BDNF, selective tropomyosin-related kinase B (TrkB) receptor agonists, and pharmacological inhibitors of signaling pathways were examined for their effects on contraction of rabbit intestinal longitudinal muscle strips induced by CCh and SP. BDNF activation of intracellular signaling pathways was examined by Western blot in homogenates of muscle strips and isolated muscle cells. One-hour preincubation with BDNF enhanced intestinal muscle contraction induced by CCh but not by SP. The selective synthetic TrkB agonists LM 22A4 and 7,8-dihydroxyflavone produced similar effects to BDNF. The Trk antagonist K-252a, a TrkB antibody but not p75NTR antibody, blocked the effect of BDNF. The enhancement of CCh-induced contraction by BDNF was blocked by the phospholipase C (PLC) antagonist U73122, but not by ERK1/2 or Akt antagonists. Direct measurement in muscle strips and isolated muscle cells showed that BDNF caused phosphorylation of TrkB receptors and PLC-γ, but not ERK1/2 or Akt. We conclude that exogenous BDNF augments the CCh-induced contraction of longitudinal muscle from rabbit intestine by activating TrkB receptors and subsequent PLC activation.

  7. Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

    PubMed Central

    Kadamur, Ganesh

    2016-01-01

    Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. PMID:27002154

  8. Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

    PubMed

    Kadamur, Ganesh; Ross, Elliott M

    2016-05-20

    Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. A Cell-Permeable Phospholipase C[gamma]1-Binding Peptide Transduces Neurons and Impairs Long-Term Spatial Memory

    ERIC Educational Resources Information Center

    Blum, Sonja; Dash, Pramod K.

    2004-01-01

    Growth factor-mediated signaling has emerged as an essential component of memory formation. In this study, we used a phospholipase C gamma 1 (PLC[gamma]1) binding, cell-penetrating peptide to sequester PLC[gamma]1 away from its target, the phosphotyrosine residues within the activated growth factor receptor. Peptides appear to transduce neurons…

  10. A Cell-Permeable Phospholipase C[gamma]1-Binding Peptide Transduces Neurons and Impairs Long-Term Spatial Memory

    ERIC Educational Resources Information Center

    Blum, Sonja; Dash, Pramod K.

    2004-01-01

    Growth factor-mediated signaling has emerged as an essential component of memory formation. In this study, we used a phospholipase C gamma 1 (PLC[gamma]1) binding, cell-penetrating peptide to sequester PLC[gamma]1 away from its target, the phosphotyrosine residues within the activated growth factor receptor. Peptides appear to transduce neurons…

  11. Differential coupling of the human P2Y11 receptor to phospholipase C and adenylyl cyclase

    PubMed Central

    Qi, Ai-Dong; Kennedy, Charles; Harden, T Kendall; Nicholas, Robert A

    2001-01-01

    The human P2Y11 (hP2Y11) receptor was stably expressed in two cell lines, 1321N1 human astrocytoma cells (1321N1-hP2Y11) and Chinese hamster ovary cells (CHO-hP2Y11), and its coupling to phospholipase C and adenylyl cyclase was assessed. In 1321N1-hP2Y11 cells, ATP promoted inositol phosphate (IP) accumulation with low μM potency (EC50=8.5±0.1 μM), whereas it was 15 fold less potent (130±10 μM) in evoking cyclic AMP production. In CHO-hP2Y11 cells, ATP promoted IP accumulation with slightly higher potency (EC50=3.6±1.3 μM) than in 1321N1-hP2Y11 cells, but it was still 15 fold less potent in promoting cyclic AMP accumulation (EC50=62.4±15.6 μM) than for IP accumulation. Comparable differences in potencies for promoting the two second messenger responses were observed with other adenosine nucleotide analogues. In 1321N1-hP2Y11 and CHO-hP2Y11 cells, down regulation of PKC by chronic treatment with phorbol ester decreased ATP-promoted cyclic AMP accumulation by 60 – 80% (P<0.001) with no change in its potency. Likewise, chelation of intracellular Ca2+ decreased ATP-promoted cyclic AMP accumulation by ∼45% in 1321N1-hP2Y11 cells, whereas chelation had no effect on either the efficacy or potency of ATP in CHO-hP2Y11 cells. We conclude that coupling of hP2Y11 receptors to adenylyl cyclase in these cell lines is much weaker than coupling to phospholipase C, and that activation of PKC and intracellular Ca2+ mobilization as consequences of inositol lipid hydrolysis potentiates the capacity of ATP to increase cyclic AMP accumulation in both 1321N1-hP2Y11 and CHO-hP2Y11 cells. PMID:11156592

  12. Revisiting the role of phospholipases C in virulence and the lifecycle of Mycobacterium tuberculosis

    PubMed Central

    Le Chevalier, Fabien; Cascioferro, Alessandro; Frigui, Wafa; Pawlik, Alexandre; Boritsch, Eva C.; Bottai, Daria; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

    2015-01-01

    Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis. PMID:26603639

  13. Inhibition of phosphatidylcholine-specific phospholipase C prevents bone marrow stromal cell senescence in vitro.

    PubMed

    Sun, Chunhui; Wang, Nan; Huang, Jie; Xin, Jie; Peng, Fen; Ren, Yinshi; Zhang, Shangli; Miao, Junying

    2009-10-01

    Bone marrow stromal cells (BMSCs) can proliferate in vitro and can be transplanted for treating many kinds of diseases. However, BMSCs become senescent with long-term culture, which inhibits their application. To understand the mechanism underlying the senescence, we investigated the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and levels of integrin beta4, caveolin-1 and ROS with BMSC senescence. The activity of PC-PLC and levels of integrin beta4, caveolin-1 and ROS increased greatly during cell senescence. Selective inhibition of increased PC-PLC activity with D609 significantly decreased the number of senescence-associated beta galactosidase positive cells in BMSCs. Furthermore, D609 restored proliferation of BMSCs and their differentiation into adipocytes. Moreover, D609 suppressed the elevated levels of integrin beta4, caveolin-1 and ROS. The data suggest that PC-PLC is involved in senescence of BMSCs, and its function is associated with integrin beta4, caveolin-1 and ROS.

  14. The potential role of postsynaptic phospholipase C activity in synaptic facilitation and behavioral sensitization in Aplysia.

    PubMed

    Fulton, Daniel; Condro, Michael C; Pearce, Kaycey; Glanzman, David L

    2008-07-01

    Previous findings indicate that synaptic facilitation, a cellular mechanism underlying sensitization of the siphon withdrawal response (SWR) in Aplysia, depends on a cascade of postsynaptic events, including activation of inositol triphosphate (IP3) receptors and release of Ca2+ from postsynaptic intracellular stores. These findings suggest that phospholipase C (PLC), the enzyme that catalyzes IP3 formation, may play an important role in postsynaptic signaling during facilitation and learning in Aplysia. Using the PLC inhibitor U73122, we found that PLC activity is required for synaptic facilitation following a 10-min treatment with 5-HT, as measured at 20 min after 5-HT washout. Prior work has indicated that facilitation at this time is supported primarily by postsynaptic processes. To determine whether postsynaptic PLC activity is involved in 5-HT-mediated facilitatory actions, we examined the effect of U73122 on enhancement of the response of motor neurons isolated in cell culture to glutamate, the sensory neuron transmitter. A 10-min application of 5-HT induced persistent (>40 min) enhancement of glutamate-evoked potentials (Glu-EPs) recorded from isolated motor neurons, and this enhancement was blocked by U73122. Finally, we showed that injecting U73122 into intact animals before behavioral training impaired intermediate-term sensitization, indicating that PLC activity contributes to this form of nonassociative learning.

  15. Multiple receptors coupled to phospholipase C gate long-term depression in visual cortex.

    PubMed

    Choi, Se-Young; Chang, Jeff; Jiang, Bin; Seol, Geun-Hee; Min, Sun-Seek; Han, Jung-Soo; Shin, Hee-Sup; Gallagher, Michela; Kirkwood, Alfredo

    2005-12-07

    Long-term depression (LTD) in sensory cortices depends on the activation of NMDA receptors. Here, we report that in visual cortical slices, the induction of LTD (but not long-term potentiation) also requires the activation of receptors coupled to the phospholipase C (PLC) pathway. Using immunolesions in combination with agonists and antagonists, we selectively manipulated the activation of alpha1 adrenergic, M1 muscarinic, and mGluR5 glutamatergic receptors. Inactivation of these PLC-coupled receptors prevents the induction of LTD, but only when the three receptors were inactivated together. LTD is fully restored by activating any one of them or by supplying intracellular D-myo-inositol-1,4,5-triphosphate (IP3). LTD was also impaired by intracellular application of PLC or IP3 receptor blockers, and it was absent in mice lacking PLCbeta1, the predominant PLC isoform in the forebrain. We propose that visual cortical LTD requires a minimum of PLC activity that can be supplied independently by at least three neurotransmitter systems. This essential requirement places PLC-linked receptors in a unique position to control the induction of LTD and provides a mechanism for gating visual cortical plasticity via extra-retinal inputs in the intact organism.

  16. Kinetic Analysis of Phospholipase C from Catharanthus roseus Transformed Roots Using Different Assays1

    PubMed Central

    Hernández-Sotomayor, S.M. Teresa; De Los Santos-Briones, César; Muñoz-Sánchez, J. Armando; Loyola-Vargas, Victor M.

    1999-01-01

    The properties of phospholipase C (PLC) partially purified from Catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles, and phospholipid monolayers spread at an air-water interface. Using [33P]phosphatidylinositol 4,5-bisphosphate (PIP2) of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphate and its subsequent dissolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure. The optimum surface pressure for PIP2 hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP2 vesicles followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determine whether PLC activity was dependent on both bulk PIP2 and PIP2 surface concentrations in the micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol fraction, and the equilibrium dissociation constant of PLC for the lipid was 45.5 μm. These findings will add to our understanding of the mechanisms of regulation of plant PLC. PMID:10444091

  17. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    SciTech Connect

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).

  18. Molecular cloning and characterization of a novel phospholipase C, PLC-eta.

    PubMed

    Hwang, Jong-Ik; Oh, Yong-Seok; Shin, Kum-Joo; Kim, Hyun; Ryu, Sung Ho; Suh, Pann-Ghill

    2005-07-01

    PLC (phospholipase C) plays an important role in intracellular signal transduction by hydrolysing phosphatidylinositol 4,5-bisphosphate, a membrane phospholipid. To date, 12 members of the mammalian PLC isoforms have been identified and classified into five isotypes beta, gamma, delta, epsilon and zeta, which are regulated by distinct mechanisms. In the present study, we describe the identification of a novel PLC isoform in the brains of human and mouse, named PLC-eta, which contains the conserved pleckstrin homology domain, X and Y domains for catalytic activity and the C2 domain. The first identified gene encoded 1002 (human) or 1003 (mouse) amino acids with an estimated molecular mass of 115 kDa. The purified recombinant PLC-eta exhibited Ca2+-dependent catalytic activity on phosphatidylinositol 4,5-bisphosphate. Furthermore, molecular biological analysis revealed that the PLC-eta gene was transcribed to several splicing variants. Although some transcripts were detected in most of the tissues we examined, the transcript encoding 115 kDa was restricted to the brain and lung. In addition, the expression of the 115 kDa protein was defined in only nerve tissues such as the brain and spinal cord. In situ hybridization analysis with brain revealed that PLC-eta was abundantly expressed in various regions including cerebral cortex, hippocampus, zona incerta and cerebellar Purkinje cell layer, which are neuronal cell-enriched regions. These results suggest that PLC-eta may perform fundamental roles in the brain.

  19. Cbl competitively inhibits epidermal growth factor-induced activation of phospholipase C-gamma1.

    PubMed

    Choi, Jang Hyun; Bae, Sun Sik; Park, Jong Bae; Ha, Sang Hoon; Song, Hebok; Kim, Jae-Ho; Cocco, Lucio; Ryu, Sung Ho; Suh, Pann-Ghill

    2003-04-30

    Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation through its two Src homology (SH) 2 domains and its single SH3 domain, which interact with signaling molecules in response to various growth factors and hormones. However, the role of the SH domains in the growth factor-induced regulation of PLC-gamma1 is unclear. By peptide-mass fingerprinting analysis we have identified Cbl as a binding protein for the SH3 domain of PLC-gamma1 from rat pheochromatocyte PC12 cells. Association of Cbl with PLC-gamma1 was induced by epidermal growth factor (EGF) but not by nerve growth factor (NGF). Upon EGF stimulation, both Cbl and PLC-gamma1 were recruited to the activated EGF receptor through their SH2 domains. Mutation of the SH2 domains of either Cbl or PLC-gamma1 abrogated the EGF-induced interaction of PLC-gamma1 with Cbl, indicating that SH2-mediated translocation is essential for the association of PLC-gamma1 and Cbl. Overexpression of Cbl attenuated EGF-induced tyrosine phosphorylation and the subsequent activation of PLC-gamma1 by interfering competitively with the interaction between PLC-gamma1 and EGFR. Taken together, these results provide the first indications that Cbl may be a negative regulator of intracellular signaling following EGF-induced PLC-gamma1 activation.

  20. Differential subcellular distribution of four phospholipase C isoforms and secretion of GPI-PLC activity.

    PubMed

    Staudt, Emanuel; Ramasamy, Pathmanaban; Plattner, Helmut; Simon, Martin

    2016-12-01

    Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP3. In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface.

  1. Inhibition and Activation by CD244 Depends on CD2 and Phospholipase C-γ1*

    PubMed Central

    Clarkson, Nicholas G.; Brown, Marion H.

    2009-01-01

    Regulation by the NK and T cell surface receptor CD244 in mice and humans depends both on engagement at the cell surface by CD48 and intracellular interactions with SAP and EAT-2. Relevance to human disease by manipulating CD244 in mouse models is complicated by rodent CD2 also binding CD48. We distinguish between contributions of mouse CD244 and CD2 on engagement of CD48 in a mouse T cell hybridoma. CD2 and CD244 both contribute positively to the immune response as mutation of proline-rich motifs or tyrosine motifs in the tails of CD2 and CD244, respectively, result in a decrease in antigen-specific interleukin-2 production. Inhibitory effects of mouse CD244 are accounted for by competition with CD2 at the cell surface for CD48. In humans CD2 and CD244 are engaged separately at the cell surface but biochemical data suggest a potential conserved intracellular link between the two receptors through FYN kinase. We identify a novel signaling mechanism for CD244 through its potential to recruit phospholipase C-γ1 via the conserved phosphorylated tyrosine motif in the tail of the adaptor protein EAT-2, which we show is important for function. PMID:19586919

  2. Mycobacterium tuberculosis expressing phospholipase C subverts PGE2 synthesis and induces necrosis in alveolar macrophages

    PubMed Central

    2014-01-01

    Background Phospholipases C (PLCs) are virulence factors found in several bacteria. In Mycobacterium tuberculosis (Mtb) they exhibit cytotoxic effects on macrophages, but the mechanisms involved in PLC-induced cell death are not fully understood. It has been reported that induction of cell necrosis by virulent Mtb is coordinated by subversion of PGE2, an essential factor in cell membrane protection. Results Using two Mtb clinical isolates carrying genetic variations in PLC genes, we show that the isolate 97-1505, which bears plcA and plcB genes, is more resistant to alveolar macrophage microbicidal activity than the isolate 97-1200, which has all PLC genes deleted. The isolate 97-1505 also induced higher rates of alveolar macrophage necrosis, and likewise inhibited COX-2 expression and PGE2 production. To address the direct effect of mycobacterial PLC on cell necrosis and PGE2 inhibition, both isolates were treated with PLC inhibitors prior to macrophage infection. Interestingly, inhibition of PLCs affected the ability of the isolate 97-1505 to induce necrosis, leading to cell death rates similar to those induced by the isolate 97-1200. Finally, PGE2 production by Mtb 97-1505-infected macrophages was restored to levels similar to those produced by 97-1200-infected cells. Conclusions Mycobacterium tuberculosis bearing PLCs genes induces alveolar macrophage necrosis, which is associated to subversion of PGE2 production. PMID:24886263

  3. Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C

    PubMed Central

    Seo, Jong Bae; Jung, Seung-Ryoung; Huang, Weigang; Zhang, Qisheng; Koh, Duk-Su

    2015-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically. PMID:26658739

  4. Mycobacterium tuberculosis expressing phospholipase C subverts PGE2 synthesis and induces necrosis in alveolar macrophages.

    PubMed

    Assis, Patricia A; Espíndola, Milena S; Paula-Silva, Francisco W G; Rios, Wendy M; Pereira, Priscilla A T; Leão, Sylvia C; Silva, Célio L; Faccioli, Lúcia H

    2014-05-19

    Phospholipases C (PLCs) are virulence factors found in several bacteria. In Mycobacterium tuberculosis (Mtb) they exhibit cytotoxic effects on macrophages, but the mechanisms involved in PLC-induced cell death are not fully understood. It has been reported that induction of cell necrosis by virulent Mtb is coordinated by subversion of PGE2, an essential factor in cell membrane protection. Using two Mtb clinical isolates carrying genetic variations in PLC genes, we show that the isolate 97-1505, which bears plcA and plcB genes, is more resistant to alveolar macrophage microbicidal activity than the isolate 97-1200, which has all PLC genes deleted. The isolate 97-1505 also induced higher rates of alveolar macrophage necrosis, and likewise inhibited COX-2 expression and PGE2 production. To address the direct effect of mycobacterial PLC on cell necrosis and PGE2 inhibition, both isolates were treated with PLC inhibitors prior to macrophage infection. Interestingly, inhibition of PLCs affected the ability of the isolate 97-1505 to induce necrosis, leading to cell death rates similar to those induced by the isolate 97-1200. Finally, PGE2 production by Mtb 97-1505-infected macrophages was restored to levels similar to those produced by 97-1200-infected cells. Mycobacterium tuberculosis bearing PLCs genes induces alveolar macrophage necrosis, which is associated to subversion of PGE2 production.

  5. Mechanism of phosphorylation-induced activation of phospholipase C-gamma isozymes.

    PubMed

    Gresset, Aurelie; Hicks, Stephanie N; Harden, T Kendall; Sondek, John

    2010-11-12

    The lipase activity of most phospholipases C (PLCs) is basally repressed by a highly degenerate and mostly disordered X/Y linker inserted within the catalytic domain. Release of this auto-inhibition is driven by electrostatic repulsion between the plasma membrane and the electronegative X/Y linker. In contrast, PLC-γ isozymes (PLC-γ1 and -γ2) are structurally distinct from other PLCs because multiple domains are present in their X/Y linker. Moreover, although many tyrosine kinases directly phosphorylate PLC-γ isozymes to enhance their lipase activity, the underlying molecular mechanism of this activation remains unclear. Here we define the mechanism for the unique regulation of PLC-γ isozymes by their X/Y linker. Specifically, we identify the C-terminal SH2 domain within the X/Y linker as the critical determinant for auto-inhibition. Tyrosine phosphorylation of the X/Y linker mediates high affinity intramolecular interaction with the C-terminal SH2 domain that is coupled to a large conformational rearrangement and release of auto-inhibition. Consequently, PLC-γ isozymes link phosphorylation to phospholipase activation by elaborating upon primordial regulatory mechanisms found in other PLCs.

  6. Identification of a novel class of mammalian phosphoinositol-specific phospholipase C enzymes.

    PubMed

    Stewart, Alan J; Mukherjee, Joy; Roberts, Scott J; Lester, Douglas; Farquharson, Colin

    2005-01-01

    Phosphoinositol (PhoIns)-specific phospholipase C enzymes (PLCs) are central to the inositol lipid signaling pathways and contribute to intracellular Ca2+ release and protein kinase C activation. Five distinct classes of PhoIns-specific PLCs are known to exist in mammals, which are activated by membrane receptor-mediated events. Here we have identified a sixth class of PhoIns-specific PLC with a novel domain structure, which we have termed PLC-eta. Two putative PLC-eta enzymes were identified in humans and in mice. Sequence analysis revealed that residues implicated in substrate binding and catalysis from other PhoIns-specific PLCs are conserved in the novel enzymes. PLC-eta enzymes are most closely related to the PLC-delta class and share a close evolutionary relationship with other PLC isozymes. EST analysis and RT-PCR data suggest that PLC-eta enzymes are expressed in several cell types and, by analogy with other mammalian PhoIns-specific PLCs, are likely to be involved in signal transduction pathways.

  7. Phospholipase C-delta1 and oxytocin receptor signalling: evidence of its role as an effector.

    PubMed

    Park, E S; Won, J H; Han, K J; Suh, P G; Ryu, S H; Lee, H S; Yun, H Y; Kwon, N S; Baek, K J

    1998-04-01

    Although the oxytocin receptor modulates intracellular Ca2+ ion levels in myometrium, the identities of signal molecules have not been clearly clarified. Our previous studies on oxytocin receptor signalling demonstrated that 80 kDa Ghalpha is a signal mediator [Baek, Kwon, Lee, Kim, Muralidhar and Im (1996) Biochem. J. 315, 739-744]. To elucidate the effector in the oxytocin receptor signalling pathway, we evaluated the oxytocin-mediated activation of phospholipase C (PLC) by using solubilized membranes from human myometrium and a three-component preparation containing the oxytocin receptor-Ghalpha-PLC-delta1 complex. PLC-delta1 activity in the three-component preparation, as well as PLC activity in solubilized membranes, was increased by oxytocin in the presence of Ca2+ and activated Ghalpha (GTP-bound Ghalpha). Furthermore the stimulated PLC-delta1 activity resulting from activation of Ghalpha via the oxytocin receptor was significantly attenuated by the selective oxytocin antagonist desGly-NH2d(CH2)5[Tyr(Me)2,Thr4]ornithine vasotocin or GDP. Consistent with these observations, co-immunoprecipitation and co-immunoadsorption of PLC-delta1 in the three-component preparation by anti-Gh7alpha antibody resulted in the PLC-delta1 being tightly coupled to activated Ghalpha on stimulation of the oxytocin receptor. These results indicate that PLC-delta1 is the effector for Ghalpha-mediated oxytocin receptor signalling.

  8. Phospholipase C beta 4 in the medial septum controls cholinergic theta oscillations and anxiety behaviors.

    PubMed

    Shin, Jonghan; Gireesh, Gangadharan; Kim, Seong-Wook; Kim, Duk-Soo; Lee, Sukyung; Kim, Yeon-Soo; Watanabe, Masahiko; Shin, Hee-Sup

    2009-12-09

    Anxiety is among the most prevalent and costly diseases of the CNS, but its underlying mechanisms are not fully understood. Although attenuated theta rhythms have been observed in human subjects with increased anxiety, no study has been done on the possible physiological link between these two manifestations. We found that the mutant mouse for phospholipase C beta 4 (PLC-beta 4(-/-)) showed attenuated theta rhythm and increased anxiety, presenting the first animal model for the human condition. PLC-beta 4 is abundantly expressed in the medial septum, a region implicated in anxiety behavior. RNA interference-mediated PLC-beta 4 knockdown in the medial septum produced a phenotype similar to that of PLC-beta 4(-/-) mice. Furthermore, increasing cholinergic signaling by administering an acetylcholinesterase inhibitor cured the anomalies in both cholinergic theta rhythm and anxiety behavior observed in PLC-beta 4(-/-) mice. These findings suggest that (1) PLC-beta 4 in the medial septum is involved in controlling cholinergic theta oscillation and (2) cholinergic theta rhythm plays a critical role in suppressing anxiety. We propose that defining the cholinergic theta rhythm profile may provide guidance in subtyping anxiety disorders in humans for more effective diagnosis and treatments.

  9. Retinal phospholipase C from squid is a regulator of Gq alpha GTPase activity.

    PubMed

    Mayeenuddin, L H; Bamsey, C; Mitchell, J

    2001-09-01

    The phospholipase C (PLC) pathway is the major signaling mechanism of photoactivation in invertebrate photoreceptors. Here we report the cloning of a cDNA encoding a 140-kDa retinal PLC that is uniquely expressed in squid photoreceptors. This cDNA encodes a protein with multiple distinct modular domains: PH, X and Y catalytic, and C2 domains, as well as G- and P-box motifs and two GTP/ATP binding motifs. The PLC was stimulated by activated squid Gq alpha but not by squid Gq beta gamma or mammalian beta gamma subunits. The PLC was inhibited by monophosphate, diphosphate and triphosphate nucleotides but not cyclic nucleosides. We also tested the ability of PLC-140 to regulate the GTPase activity of Gq alpha in the rhabdomeric membranes. Depletion of PLC-140 from the rhabdomeric membranes decreased the GTP hydrolysis but not GTP gamma S binding to the membranes. Reconstitution of purified PLC-140 with membranes accelerated Gq alpha GTPase activity by fivefold at a concentration of 2.5 microM. Our data suggest that PLC-140 plays an important role in both the activation and inactivation pathways of invertebrate visual transduction.

  10. Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C.

    PubMed

    Seo, Jong Bae; Jung, Seung-Ryoung; Huang, Weigang; Zhang, Qisheng; Koh, Duk-Su

    2015-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically.

  11. Purification of the trypanosome phospholipase C which cleaves the variant surface glycoprotein

    SciTech Connect

    Hereld, D.; Hart, G.W.; Englund, P.T.

    1986-05-01

    The surface coat of Trypanosoma brucei is composed of many copies of the Variant Surface Glycoprotein (VSG). This protein is tethered to the cell membrane by a glycolipid moiety which contains dimyristylphosphatidylinositol. Following cell lysis, an endogenous, membrane-bound phospholipase C cleaves the glycolipid and releases the VSG in soluble form. The authors have purified a lipase which they believe is responsible for VSG release. This enzyme, designated VSG lipase, is assayed by measuring release of butanol-soluble /sup 3/H from VSG labeled with (/sup 3/H)myristate. The purification involves detergent extraction of trypanosome membranes, ammonium sulfate fractionation, hydrophobic chromatography, and cation exchange chromatography. The enzyme is purified roughly 2500 fold and is nearly homogeneous. Based on SDS-PAGE, it has an apparent subunit molecular weight of 37,000 daltons. This polypeptide co-fractionates with the activity during several fractionation procedures. The enzyme has an apparent s/sub 20,w/ of 3.8 S. The purified VSG lipase is active in the presence of EDTA; its activity is inhibited by organomercurials and stimulated by dithiothreitol. The purified enzyme releases dimyristylglycerol from VSG.

  12. Propofol Anesthesia Is Reduced in Phospholipase C-Related Inactive Protein Type-1 Knockout Mice.

    PubMed

    Nikaido, Yoshikazu; Furukawa, Tomonori; Shimoyama, Shuji; Yamada, Junko; Migita, Keisuke; Koga, Kohei; Kushikata, Tetsuya; Hirota, Kazuyoshi; Kanematsu, Takashi; Hirata, Masato; Ueno, Shinya

    2017-06-01

    The GABA type A receptor (GABAA-R) is a major target of intravenous anesthetics. Phospholipase C-related inactive protein type-1 (PRIP-1) is important in GABAA-R phosphorylation and membrane trafficking. In this study, we investigated the role of PRIP-1 in general anesthetic action. The anesthetic effects of propofol, etomidate, and pentobarbital were evaluated in wild-type and PRIP-1 knockout (PRIP-1 KO) mice by measuring the latency and duration of loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR). The effect of pretreatment with okadaic acid (OA), a protein phosphatase 1/2A inhibitor, on propofol- and etomidate-induced LORR was also examined. PRIP-1 deficiency provided the reduction of LORR and LTWR induced by propofol but not by etomidate or pentobarbital, indicating that PRIP-1 could determine the potency of the anesthetic action of propofol. Pretreatment with OA recovered the anesthetic potency induced by propofol in PRIP-1 KO mice. OA injection enhanced phosphorylation of cortical the GABAA-R β3 subunit in PRIP-1 KO mice. These results suggest that PRIP-1-mediated GABAA-R β3 subunit phosphorylation might be involved in the general anesthetic action induced by propofol but not by etomidate or pentobarbital. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Membrane-induced Allosteric Control of Phospholipase C-β Isozymes*

    PubMed Central

    Charpentier, Thomas H.; Waldo, Gary L.; Barrett, Matthew O.; Huang, Weigang; Zhang, Qisheng; Harden, T. Kendall; Sondek, John

    2014-01-01

    All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-β (PLC-β) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-β isozymes are autoinhibited, and several proteins, including Gαq, Gβγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP2 that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gαq or Gβ1γ2 did not activate purified PLC-β3 under these conditions despite their robust capacity to activate PLC-β3 at membranes. In addition, mutants of PLC-β3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP2 in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-β isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-β isozymes. PMID:25193662

  14. Mechanism of Phosphorylation-induced Activation of Phospholipase C-γ Isozymes*♦

    PubMed Central

    Gresset, Aurelie; Hicks, Stephanie N.; Harden, T. Kendall; Sondek, John

    2010-01-01

    The lipase activity of most phospholipases C (PLCs) is basally repressed by a highly degenerate and mostly disordered X/Y linker inserted within the catalytic domain. Release of this auto-inhibition is driven by electrostatic repulsion between the plasma membrane and the electronegative X/Y linker. In contrast, PLC-γ isozymes (PLC-γ1 and -γ2) are structurally distinct from other PLCs because multiple domains are present in their X/Y linker. Moreover, although many tyrosine kinases directly phosphorylate PLC-γ isozymes to enhance their lipase activity, the underlying molecular mechanism of this activation remains unclear. Here we define the mechanism for the unique regulation of PLC-γ isozymes by their X/Y linker. Specifically, we identify the C-terminal SH2 domain within the X/Y linker as the critical determinant for auto-inhibition. Tyrosine phosphorylation of the X/Y linker mediates high affinity intramolecular interaction with the C-terminal SH2 domain that is coupled to a large conformational rearrangement and release of auto-inhibition. Consequently, PLC-γ isozymes link phosphorylation to phospholipase activation by elaborating upon primordial regulatory mechanisms found in other PLCs. PMID:20807769

  15. Effect of parathyroid hormone on healing in osteoporotic fractures via a phospholipase C-independent pathway

    PubMed Central

    Li, Wei-Long; Yu, Xiao; Huang, Zhi-Ping

    2017-01-01

    Objective This study was performed to investigate the effect of parathyroid hormone (PTH) on healing in osteoporotic fractures via a phospholipase C (PLC)-independent pathway and explore the mechanism of PTH-mediated bone formation. Methods Ninety-six 12-week-old C57BL/6J female mice underwent bilateral ovariectomy. One month later, the lower third of the femur was fractured and the mice were treated using saline, PTH(1-28), PTH(1-34), zoledronic acid (ZA), PTH(1-28)+ZA, and PTH(1-34)+ZA. The mice were killed at weeks 2 and 4 in each group. Biomechanical testing and micro-computed tomography were performed. Results The formation and strength of the callus increased in all but the saline group. The mice treated with PTH(1-34) showed a significantly higher ultimate bending force, bending rigidity, bone mineral density, percent bone volume, and trabecular thickness than those treated with PTH(1-28). The PTH(1-34)+ZA group demonstrated the greatest improvements in the ultimate bending force, bending rigidity, bone mineral density, and relative bone volume. Conclusions PTH can promote fracture healing and callus hardness in ovariectomized mice by increasing callus formation and reconstructing trabecular bone via a PLC-independent pathway. PTH combined with ZA has a cumulative effect on the healing of fractures in ovariectomized mice. PMID:28534698

  16. Infrared neural stimulation induces intracellular Ca(2+) release mediated by phospholipase C.

    PubMed

    Moreau, David; Lefort, Claire; Pas, Jolien; Bardet, Sylvia M; Leveque, Philippe; O'Connor, Rodney P

    2017-07-12

    The influence of infrared laser pulses on intracellular Ca(2+) signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo-4 was used to measure Ca(2+) in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo-4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca(2+) transient with similar dose-response characteristics in hippocampal neurons and glioblastoma cells (half-maximal effective energy density EC50 of around 58 J.cm(-2) ). For both type of cells, the source of the infrared-induced Ca(2+) transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP3 -induced Ca(2+) release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP3 cell signaling cascade has many potential applications including the development of optoceutical therapeutics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Simple purification and functional reconstitution of octopus photoreceptor Gq, which couples rhodopsin to phospholipase C.

    PubMed

    Kikkawa, S; Tominaga, K; Nakagawa, M; Iwasa, T; Tsuda, M

    1996-12-10

    In invertebrate photoreceptors, illuminated rhodopsin activates multiple G proteins, which are assumed to initiate multiple phototransduction cascades. In this paper, we focused on one of the phototransduction cascades, which utilizes rhodopsin, a Gq-like G protein, and phospholipase C (PLC). A Gq-like G protein from octopus photoreceptors was successfully purified to apparent homogeneity as an active form by simple two-step chromatography. The purified G protein had an alpha beta gamma-trimeric structure consisting of 44-kDa alpha, 37-kDa beta, and 9-kDa gamma subunits. The 44-kDa alpha subunit was assigned to the Gq class by western blot with antiserum against mammalian Gq alpha and by partial amino acid sequencing of its proteolytic fragments. Light-dependent binding of GTP gamma S was observed when the purified octopus Gq was reconstituted with octopus rhodopsin that had been integrated into phospholipid vesicles. Octopus Gq activated PLC beta 1 purified from bovine brain dose-dependently in the presence of A1F4-. Finally, light- and GTP-dependent activation of PLC beta 1 was observed in a reconstitution system consisting of octopus rhodopsin, Gq, and bovine PLC beta 1.

  18. Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development

    SciTech Connect

    Sone, Yoshie; Ito, Masahiko; Shirakawa, Hideki; Shikano, Tomohide; Takeuchi, Hiroyuki; Kinoshita, Katsuyuki; Miyazaki, Shunichi . E-mail: shunm@research.twmu.ac.jp

    2005-05-13

    Phospholipase C-zeta (PLC{zeta}), a strong candidate of the egg-activating sperm factor, causes intracellular Ca{sup 2+} oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLC{zeta}. Changes in the localization of expressed PLC{zeta} were investigated by tagging with a fluorescent protein. PLC{zeta} began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLC{zeta} in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated 1-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLC{zeta} was recognized in every embryo up to blastocyst. Thus, PLC{zeta} exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca{sup 2+} oscillations in early embryogenesis.

  19. Mechanosensitivity of human osteosarcoma cells and phospholipase C {beta}2 expression

    SciTech Connect

    Hoberg, M. . E-mail: Maik.Hoberg@med.uni-tuebingen.de; Gratz, H.-H.; Noll, M.; Jones, D.B.

    2005-07-22

    Bone adapts to mechanical load by osteosynthesis, suggesting that osteoblasts might respond to mechanical stimuli. We therefore investigated cell proliferation and phospholipase C (PLC) expression in osteoblasts. One Hertz uniaxial stretching at 4000 {mu}strains significantly increased the proliferation rates of human osteoblast-like osteosarcoma cell line MG-63 and primary human osteoblasts. However, U-2/OS, SaOS-2, OST, and MNNG/HOS cells showed no significant changes in proliferation rate. We investigated the expression pattern of different isoforms of PLC in these cell lines. We were able to detect PLC {beta}1, {beta}3, {gamma}1, {gamma}2, and {delta}1 in all cells, but PLC {beta}2 was only detectable in the mechanosensitive cells. We therefore investigated the possible role of PLC {beta}2 in mechanotransduction. Inducible antisense expression for 24 h inhibited the translation of PLC {beta}1 in U-2/OS cells by 35% and PLC {beta}2 in MG-63 by 29%. Fluid shear flow experiments with MG-63 lacking PLC {beta}2 revealed a significantly higher level of cells losing attachment to coverslips and a significantly lower number of cells increasing intracellular free calcium.

  20. Characterization of phospholipases C beta and gamma and their possible roles in Chaetopterus egg activation.

    PubMed

    Yin, Xunqin; Eckberg, William R

    2009-05-01

    Intracellular calcium release from the endoplasmic reticulum is a hallmark at egg activation of both vertebrates and invertebrates. This fertilization-associated calcium release results from generation of the second messenger inositol 1,4,5-trisphosphate (IP(3)) by one or more phospholipases C (PLC). We characterized Chaetopterus PLCbeta and gamma by reverse transcription/degenerate oligonucleotide primed PCR and rapid amplification of cDNA end PCR. Phylogenetic analyses suggested that the deduced PLCbeta protein shared the greatest homology with mammalian PLCbeta4; the deduced PLCgamma protein shared the greatest homology with starfish PLCgamma and diverged from mammalian PLCgamma before mammalian the PLCgamma1 and gamma2 isoforms diverged. Western blot analyses with specific anti-PLCbeta and gamma antibodies, respectively, revealed that 135 and 150 kDa proteins were expressed in eggs. The general PLC antagonist U-73122 blocked fertilization-induced egg activation; however, the inactive analog, U-73343, had no effect on egg activation. We further tested whether egg activation was G protein-PLCbeta and/or protein tyrosine kinase-PLCgamma dependent. Cholera and pertussis toxins, well-known effectors of G proteins, had no effect on egg activation; while two antagonists of PTK, genistein and tyrphostin B42, inhibited both fertilization-induced and artificial egg activation. Taken together, our studies suggested that PLC activity from eggs contributes to Chaetopterus egg activation and PLCgamma might play an important role during this biological process.

  1. Revisiting the role of phospholipases C in virulence and the lifecycle of Mycobacterium tuberculosis.

    PubMed

    Le Chevalier, Fabien; Cascioferro, Alessandro; Frigui, Wafa; Pawlik, Alexandre; Boritsch, Eva C; Bottai, Daria; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

    2015-11-25

    Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis.

  2. A lobster phospholipase C-beta that associates with G-proteins in response to odorants.

    PubMed

    Xu, F; McClintock, T S

    1999-06-15

    A cDNA clone encoding a protein of 1116 amino acids with significant homology to beta-isoforms of phospholipase C was isolated from lobster olfactory organ cDNA libraries and named lobPLCbeta. This cDNA hybridized predominantly to a 9 kb transcript in RNA from olfactory organ, pereiopod, brain, and eye-eyestalk and to several smaller minor transcripts only in eye-eyestalk. An antiserum raised to the C terminus of lobPLCbeta detected immunoreactivity in a single 130 kDa band in olfactory aesthetasc hairs, olfactory organ, pereiopod, dactyl, and brain. In eye-eyestalk this 130 kDa band was abundant, and minor bands of 100, 79, and 57 kDa also were detected. In cross sections of the aesthetasc hairs, immunoreactivity was detected in the outer dendritic segments of the olfactory receptor neurons, the site of olfactory transduction. A complex odorant caused lobPLCbeta immunoreactivity to increase in membrane fractions and decrease in soluble fractions of homogenates of aesthetasc hairs. The odorant also increased the amount of lobPLCbeta in immunoprecipitates of Galphaq and Gbeta from homogenates of aesthetasc hairs. These results support the conclusion that lobPLCbeta mediates olfactory transduction.

  3. Membrane translocation of protein kinase Ctheta during T lymphocyte activation requires phospholipase C-gamma-generated diacylglycerol.

    PubMed

    Díaz-Flores, Ernesto; Siliceo, María; Martínez-A, Carlos; Mérida, Isabel

    2003-08-01

    Protein kinase C (PKC) is the only PKC isoform recruited to the immunological synapse after T cell receptor stimulation, suggesting that its activation mechanism differs from that of the other isoforms. Previous studies have suggested that this selective PKC recruitment may operate via a Vav-regulated, cytoskeletal-dependent mechanism, independent of the classical phospholipase C/diacylglycerol pathway. Here, we demonstrate that, together with tyrosine phosphorylation of PKC in the regulatory domain, binding of phospholipase C-dependent diacylglycerol is required for PKC recruitment to the T cell synapse. In addition, we demonstrate that diacylglycerol kinase alpha-dependent diacylglycerol phosphorylation provides the negative signal required for PKC inactivation, ensuring fine control of the T cell activation response.

  4. Endothelin stimulates phospholipase C, Na+/H+ exchange, c-fos expression, and mitogenesis in rat mesangial cells.

    PubMed Central

    Simonson, M S; Wann, S; Mené, P; Dubyak, G R; Kester, M; Nakazato, Y; Sedor, J R; Dunn, M J

    1989-01-01

    A recently described peptide hormone, endothelin, is a potent vasoconstrictor, but it is unclear whether endothelin has other biological actions. These experiments extend the range of biological actions of endothelin to stimulation of mitogenesis. Endothelin at low concentrations (0.1-10 nM) induced mitogenesis by quiescent rat glomerular mesangial cells in culture. Mitogenesis induced by endothelin was accompanied by activation of phospholipase C with increased inositol phosphate turnover and increments of intracellular [Ca2+]. Endothelin also activated Na+/H+ exchange, causing cytosolic alkalinization, and enhanced transcription of the c-fos protooncogene, additional biochemical signals closely linked to proliferation. In addition to being a vasoconstrictor, endothelin thus also functions as a mitogen, presumably through activation of phospholipase C. Images PMID:2536405

  5. Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity.

    PubMed

    Siqueira, Flávia F; Almeida, Marcelle O; Barroca, Tatiana M; Horta, Carolina C R; Carmo, Anderson O; Silva, Rodrigo O S; Pires, Prhiscylla S; Lobato, Francisco C F; Kalapothakis, Evanguedes

    2012-10-12

    Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Role of Inositol Phosphosphingolipid Phospholipase C1, the Yeast Homolog of Neutral Sphingomyelinases in DNA Damage Response and Diseases.

    PubMed

    Tripathi, Kaushlendra

    2015-01-01

    Sphingolipids play a very crucial role in many diseases and are well-known as signaling mediators in many pathways. Sphingolipids are produced during the de novo process in the ER (endoplasmic reticulum) from the nonsphingolipid precursor and comprise both structural and bioactive lipids. Ceramide is the central core of the sphingolipid pathway, and its production has been observed following various treatments that can induce several different cellular effects including growth arrest, DNA damage, apoptosis, differentiation, and senescence. Ceramides are generally produced through the sphingomyelin hydrolysis and catalyzed by the enzyme sphingomyelinase (SMase) in mammals. Presently, there are many known SMases and they are categorized into three groups acid SMases (aSMases), alkaline SMases (alk-SMASES), and neutral SMases (nSMases). The yeast homolog of mammalians neutral SMases is inositol phosphosphingolipid phospholipase C. Yeasts generally have inositol phosphosphingolipids instead of sphingomyelin, which may act as a homolog of mammalian sphingomyelin. In this review, we shall explain the structure and function of inositol phosphosphingolipid phospholipase C1, its localization inside the cells, mechanisms, and its roles in various cell responses during replication stresses and diseases. This review will also give a new basis for our understanding for the mechanisms and nature of the inositol phosphosphingolipid phospholipase C1/nSMase.

  7. Inhibition of phosphatidylcholine-specific phospholipase C downregulates HER2 overexpression on plasma membrane of breast cancer cells

    PubMed Central

    2010-01-01

    Introduction Overexpression on plasma membrane of human epidermal growth factor receptor 2 (HER2) is reported in 25% to 30% of breast cancers. Heterodimer formation with cognate members of the epidermal growth factor receptor (EGFR) family, such as HER3 and EGFR, activates abnormal cell-signalling cascades responsible for tumorigenesis and further transcriptional HER2 gene upregulation. Targeting the molecular mechanisms controlling HER2 overexpression and recycling may effectively deactivate this feedback-amplification loop. We recently showed that inactivation of phosphatidylcholine-specific phospholipase C (PC-PLC) may exert a pivotal role in selectively modulating the expression on the membrane of specific receptors or proteins relevant to cell function. In the present study, we investigated the capability of PC-PLC inhibition to target the molecular mechanisms controlling HER2 overexpression on the membrane of breast cancer cells by altering the rates of its endocytosis and lysosomal degradation. Methods Localization on the membrane and interaction of PC-PLC with HER2, EGFR, and HER3 were investigated on HER2-overexpressing and HER2-low breast cancer cell lines, by using confocal laser scanning microscopy, flow cytometry, cell-surface biotinylation, isolation of lipid rafts, and immunoprecipitation experiments. The effects of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609) on HER2 expression on the membrane and on the levels of overall HER2, HER2-HER3, and HER2-EGFR contents were monitored in the HER2-overexpressing SKBr3 cells, after either transient or continuous receptor engagement with anti-HER2 monoclonal antibodies, including trastuzumab. Changes of HER2 expression and cell proliferation were examined in SKBr3, BT-474, and MDA-MB-453 cells continuously exposed to D609 alone or combined with trastuzumab. Results PC-PLC selectively accumulates on the plasma membrane of HER2-overexpressing cells, where it colocalizes and associates with

  8. Phospholipase C Epsilon (PLCε) Induced TRPC6 Activation: A Common but Redundant Mechanism in Primary Podocytes

    PubMed Central

    Kalwa, Hermann; Storch, Ursula; Demleitner, Jana; Fiedler, Susanne; Mayer, Tim; Kannler, Martina; Fahlbusch, Meike; Barth, Holger; Smrcka, Alan; Hildebrandt, Friedhelm; Gudermann, Thomas; Dietrich, Alexander

    2016-01-01

    In eukaryotic cells, activation of phospholipase C (PLC)-coupled membrane receptors by hormones leads to an increase in the intracellular Ca2+ concentration [Ca2+]i. Catalytic activity of PLCs results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which opens DAG-sensitive classical transient receptor channels 3, 6, and 7 (TRPC3/6/7), initiating Ca2+ influx from the extracellular space. Patients with focal segmental glomerulosclerosis (FSGS) express gain-of-function mutants of TRPC6, while others carry loss-of-function mutants of PLCε, raising the intriguing possibility that both proteins interact and might work in the same signalling pathway. While TRPC6 activation by PLCβ and PLCγ isozymes was extensively studied, the role of PLCε in TRPC6 activation remains elusive. TRPC6 was co-immunoprecipitated with PLCε in a heterologous overexpression system in HEK293 cells as well as in freshly isolated murine podocytes. Receptor-operated TRPC6 currents in HEK293 cells expressing TRPC6 were reduced by a specific PLCε siRNA and by a PLCε loss-of-function mutant isolated from a patient with FSGS. PLCε-induced TRPC6 activation was also identified in murine embryonic fibroblasts (MEFs) lacking Gαq/11 proteins. Further analysis of the signal transduction pathway revealed a Gα12/13 Rho-GEF activation which induced Rho-mediated PLCε stimulation. Therefore, we identified a new pathway for TRPC6 activation by PLCε. PLCε-/- podocytes however, were undistinguishable from WT podocytes in their angiotensin II-induced formation of actin stress fibers and their GTPγS-induced TRPC6 activation, pointing to a redundant role of PLCε-mediated TRPC6 activation at least in podocytes. PMID:25521631

  9. Iron-Regulated Phospholipase C Activity Contributes to the Cytolytic Activity and Virulence of Acinetobacter baumannii

    PubMed Central

    Fiester, Steven E.; Schmidt, Robert E.; Beckett, Amber C.; Ticak, Tomislav; Carrier, Mary V.; Ghosh, Rajarshi; Ohneck, Emily J.; Metz, Maeva L.; Sellin Jeffries, Marlo K.; Actis, Luis A.

    2016-01-01

    Acinetobacter baumannii is an opportunistic Gram-negative pathogen that causes a wide range of infections including pneumonia, septicemia, necrotizing fasciitis and severe wound and urinary tract infections. Analysis of A. baumannii representative strains grown in Chelex 100-treated medium for hemolytic activity demonstrated that this pathogen is increasingly hemolytic to sheep, human and horse erythrocytes, which interestingly contain increasing amounts of phosphatidylcholine in their membranes. Bioinformatic, genetic and functional analyses of 19 A. baumannii isolates showed that the genomes of each strain contained two phosphatidylcholine-specific phospholipase C (PC-PLC) genes, which were named plc1 and plc2. Accordingly, all of these strains were significantly hemolytic to horse erythrocytes and their culture supernatants tested positive for PC-PLC activity. Further analyses showed that the transcriptional expression of plc1 and plc2 and the production of phospholipase and thus hemolytic activity increased when bacteria were cultured under iron-chelation as compared to iron-rich conditions. Testing of the A. baumannii ATCC 19606T plc1::aph-FRT and plc2::aph isogenic insertion derivatives showed that these mutants had a significantly reduced PC-PLC activity as compared to the parental strain, while testing of plc1::ermAM/plc2::aph demonstrated that this double PC-PLC isogenic mutant expressed significantly reduced cytolytic and hemolytic activity. Interestingly, only plc1 was shown to contribute significantly to A. baumannii virulence using the Galleria mellonella infection model. Taken together, our data demonstrate that both PLC1 and PLC2, which have diverged from a common ancestor, play a concerted role in hemolytic and cytolytic activities; although PLC1 seems to play a more critical role in the virulence of A. baumannii when tested in an invertebrate model. These activities would provide access to intracellular iron stores this pathogen could use during

  10. M1-muscarinic receptors promote fear memory consolidation via phospholipase C and the M-current.

    PubMed

    Young, Matthew B; Thomas, Steven A

    2014-01-29

    Neuromodulators released during and after a fearful experience promote the consolidation of long-term memory for that experience. Because overconsolidation may contribute to the recurrent and intrusive memories of post-traumatic stress disorder, neuromodulatory receptors provide a potential pharmacological target for prevention. Stimulation of muscarinic receptors promotes memory consolidation in several conditioning paradigms, an effect primarily associated with the M1 receptor (M1R). However, neither inhibiting nor genetically disrupting M1R impairs the consolidation of cued fear memory. Using the M1R agonist cevimeline and antagonist telenzepine, as well as M1R knock-out mice, we show here that M1R, along with β2-adrenergic (β2AR) and D5-dopaminergic (D5R) receptors, regulates the consolidation of cued fear memory by redundantly activating phospholipase C (PLC) in the basolateral amygdala (BLA). We also demonstrate that fear memory consolidation in the BLA is mediated in part by neuromodulatory inhibition of the M-current, which is conducted by KCNQ channels and is known to be inhibited by muscarinic receptors. Manipulating the M-current by administering the KCNQ channel blocker XE991 or the KCNQ channel opener retigabine reverses the effects on consolidation caused by manipulating β2AR, D5R, M1R, and PLC. Finally, we show that cAMP and protein kinase A (cAMP/PKA) signaling relevant to this stage of consolidation is upstream of these neuromodulators and PLC, suggesting an important presynaptic role for cAMP/PKA in consolidation. These results support the idea that neuromodulatory regulation of ion channel activity and neuronal excitability is a critical mechanism for promoting consolidation well after acquisition has occurred.

  11. Cholesterol regulates HERG K+ channel activation by increasing phospholipase C β1 expression.

    PubMed

    Chun, Yoon Sun; Oh, Hyun Geun; Park, Myoung Kyu; Cho, Hana; Chung, Sungkwon

    2013-01-01

    Human ether-a-go-go-related gene (HERG) K(+) channel underlies the rapidly activating delayed rectifier K(+) conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of β1 and β3 isoforms of PLC (PLCβ1, PLCβ3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCβ1 expression, we conclude that increased PLCβ1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels.

  12. Filamin and Phospholipase C-ε Are Required for Calcium Signaling in the Caenorhabditis elegans Spermatheca

    PubMed Central

    Kovacevic, Ismar; Orozco, Jose M.; Cram, Erin J.

    2013-01-01

    The Caenorhabditis elegans spermatheca is a myoepithelial tube that stores sperm and undergoes cycles of stretching and constriction as oocytes enter, are fertilized, and exit into the uterus. FLN-1/filamin, a stretch-sensitive structural and signaling scaffold, and PLC-1/phospholipase C-ε, an enzyme that generates the second messenger IP3, are required for embryos to exit normally after fertilization. Using GCaMP, a genetically encoded calcium indicator, we show that entry of an oocyte into the spermatheca initiates a distinctive series of IP3-dependent calcium oscillations that propagate across the tissue via gap junctions and lead to constriction of the spermatheca. PLC-1 is required for the calcium release mechanism triggered by oocyte entry, and FLN-1 is required for timely initiation of the calcium oscillations. INX-12, a gap junction subunit, coordinates propagation of the calcium transients across the spermatheca. Gain-of-function mutations in ITR-1/IP3R, an IP3-dependent calcium channel, and loss-of-function mutations in LFE-2, a negative regulator of IP3 signaling, increase calcium release and suppress the exit defect in filamin-deficient animals. We further demonstrate that a regulatory cassette consisting of MEL-11/myosin phosphatase and NMY-1/non-muscle myosin is required for coordinated contraction of the spermatheca. In summary, this study answers long-standing questions concerning calcium signaling dynamics in the C. elegans spermatheca and suggests FLN-1 is needed in response to oocyte entry to trigger calcium release and coordinated contraction of the spermathecal tissue. PMID:23671426

  13. Costimulation of AMPA and metabotropic glutamate receptors underlies phospholipase C activation by glutamate in hippocampus.

    PubMed

    Kim, Hye-Hyun; Lee, Kyu-Hee; Lee, Doyun; Han, Young-Eun; Lee, Suk-Ho; Sohn, Jong-Woo; Ho, Won-Kyung

    2015-04-22

    Glutamate, a major neurotransmitter in the brain, activates ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs, respectively). The two types of glutamate receptors interact with each other, as exemplified by the modulation of iGluRs by mGluRs. However, the other way of interaction (i.e., modulation of mGluRs by iGluRs) has not received much attention. In this study, we found that group I mGluR-specific agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) alone is not sufficient to activate phospholipase C (PLC) in rat hippocampus, while glutamate robustly activates PLC. These results suggested that additional mechanisms provided by iGluRs are involved in group I mGluR-mediated PLC activation. A series of experiments demonstrated that glutamate-induced PLC activation is mediated by mGluR5 and is facilitated by local Ca(2+) signals that are induced by AMPA-mediated depolarization and L-type Ca(2+) channel activation. Finally, we found that PLC and L-type Ca(2+) channels are involved in hippocampal mGluR-dependent long-term depression (mGluR-LTD) induced by paired-pulse low-frequency stimulation, but not in DHPG-induced chemical LTD. Together, we propose that AMPA receptors initiate Ca(2+) influx via the L-type Ca(2+) channels that facilitate mGluR5-PLC signaling cascades, which underlie mGluR-LTD in rat hippocampus.

  14. Phospholipase C-beta3 mediates the thrombin-induced Ca2+ response in glial cells.

    PubMed

    Hwang, Jong-Ik; Shin, Kum-Joo; Oh, Yong-Seok; Choi, Jung-Woong; Lee, Zee-Won; Kim, Daesoo; Ha, Kwon-Soo; Shin, Hee-Sup; Ryu, Sung Ho; Suh, Pann-Ghill

    2005-06-30

    Phospholipase C-beta (PLC-beta) hydrolyses phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5-trisphosphate in response to activation of various G protein-coupled receptors (GPCRs). Using glial cells from knock-out mice lacking either PLC-beta1 [PLC-beta1 (-/-)] or PLC-beta3 [PLC-beta3 (-/-)], we examined which isotype of PLC-beta participated in the cellular signaling events triggered by thrombin. Generation of inositol phosphates (IPs) was enhanced by thrombin in PLC-beta1 (-/-) cells, but was negligible in PLC-beta3 (-/-) cells. Expression of PLC-beta3 in PLC-beta3 (-/-) cells resulted in an increase in pertussis toxin (PTx)-sensitive IPs in response to thrombin as well as to PAR1-specific peptide, while expression of PLC-beta1 in PLC-beta1 (-/-) cells did not have any effect on IP generation. The thrombin-induced [Ca2+]i increase was delayed and attenuated in PLC-beta3 (-/-) cells, but normal in PLC-beta1 (-/-) cells. Pertussis toxin evoked a delayed [Ca2+]i increase in PLC-beta3 (-/-) cells as well as in PLC-beta1 (-/-) cells. These results suggest that activation of PLC-beta3 by pertussis toxin-sensitive G proteins is responsible for the transient [Ca2+]i increase in response to thrombin, whereas the delayed [Ca2+]i increase may be due to activation of some other PLC, such as PLC-beta4, acting via PTx-insensitive G proteins.

  15. Developmental increase in hippocampal endocannabinoid mobilization: role of metabotropic glutamate receptor subtype 5 and phospholipase C.

    PubMed

    Liang, Shu-Ling; Alger, Bradley E; McCarthy, Margaret M

    2014-11-15

    Endocannabinoids (eCBs) released from postsynaptic neurons mediate retrograde suppression of neurotransmitter release at central synapses. eCBs are crucial for establishing proper synaptic connectivity in the developing nervous system. Mobilization of eCBs is driven either by a rise in intracellular Ca(2+) (depolarization-induced suppression of inhibition, DSI) or postsynaptic G protein-coupled receptors (GPCRs) that activate phospholipase C beta (PLCβ). To determine whether eCB mobilization changes between neonatal and juvenile ages, we used whole cell voltage-clamp recordings of CA1 neurons from rat hippocampal slices at postnatal days 1-18 (neonatal) and 19-43 (juvenile), because many neurophysiological parameters change dramatically between approximately postnatal days 18-20. We found that DSI was slightly greater in juveniles than in neonates, while eCB mobilization stimulated by GPCRs was unchanged. However, when DSI was elicited during GPCR activation, its increase was much greater in juveniles, suggesting that eCB mobilization caused by the synergy between the Ca(2+) and GPCR pathways is developmentally upregulated. Western blotting revealed significant increases in both metabotropic type glutamate receptor 5 (mGluR5) and PLCβ1 proteins in juveniles compared with neonates. Responses to pharmacological activation or inhibition of PLC implied that eCB upregulation is associated with a functional increase in PLC activity. We conclude that synergistic eCB mobilization in hippocampal CA1 neurons is greater in juveniles than in neonates, and that this may result from increases in the mGluR5-PLCβ1 eCB pathway. The data enhance our understanding of the developmental regulation of the eCB system and may provide insight into diseases caused by improper cortical wiring, or the impact of cannabis exposure during development. Copyright © 2014 the American Physiological Society.

  16. Growth hormone activates phospholipase C in proximal tubular basolateral membranes from canine kidney

    SciTech Connect

    Rogers, S.A.; Hammerman, M.R. )

    1989-08-01

    To delineate pathways for signal transduction by growth hormone (GH) in proximal tubule, the authors incubated basolateral membranes isolated from canine kidney with human growth hormone (hGH) or human prolactin (hPrl) and measured levels of inositol trisphosphate (InsP{sub 3}) in suspensions and of diacylglycerol extractable from the membranes. Incubation with hGH, but not hPrl, increased levels of InsP{sub 3} and diacylglycerol in a concentration-dependent manner. Half-maximal effects occurred between 0.1 and 1 nM hGH. Increased levels of InsP{sub 3} were measured after as little as 5 sec of incubation with 1 nM hGH, and increase was maximal after 15 sec. Increases were no longer detectable after 60 sec because of dephosphorylation of InsP{sub 3} in membrane suspensions. hGH did not affect rates of dephosphorylation. hGH-stimulated increases in InsP{sub 3} were detectable in membranes suspended in 0, 0.1, and 0.2 {mu}M calcium but not in 0.3 or 1.0 {mu}M calcium. {sup 125}I-labeled hGH-receptor complexes with M{sub r} values of 66,000 and 140,000 were identified in isolated basolateral membranes. The findings establish that GH activates phospholipase C in isolated canine renal proximal tubular basolateral membranes, potentially after binding to a specific receptor. This process could mediate signal transmission by GH across the plasma membrane of the proximal tubular cell and elsewhere.

  17. The sperm phospholipase C-ζ and Ca2+ signalling at fertilization in mammals.

    PubMed

    Swann, Karl; Lai, F Anthony

    2016-02-01

    A series of intracellular oscillations in the free cytosolic Ca(2+) concentration is responsible for activating mammalian eggs at fertilization, thus initiating embryo development. It has been proposed that the sperm causes these Ca(2+) oscillations after membrane fusion by delivering a soluble protein into the egg cytoplasm. We previously identified sperm-specific phospholipase C (PLC)-ζ as a protein that can trigger the same pattern of Ca(2+) oscillations in eggs seen at fertilization. PLCζ appears to be the elusive sperm factor mediating egg activation in mammals. It has potential therapeutic use in infertility treatments to improve the rate of egg activation and early embryo development after intra-cytoplasmic sperm injection. A stable form of recombinant human PLCζ could be a prototype for use in such in vitro fertilization (IVF) treatments. We do not yet understand exactly how PLCζ causes inositol 1,4,5-trisphosphate (InsP3) production in eggs. Sperm PLCζ is distinct among mammalian PI-specific PLCs in that it is far more potent in triggering Ca(2+) oscillations in eggs than other PLCs, but it lacks a PH domain that would otherwise be considered essential for binding to the phosphatidylinositol 4,5-bisphosphate (PIP2) substrate. PLCζ is also unusual in that it does not appear to interact with or hydrolyse plasma membrane PIP2. We consider how other regions of PLCζ may mediate its binding to PIP2 in eggs and how interaction of PLCζ with egg-specific factors could enable the hydrolysis of internal sources of PIP2. © 2016 Authors; published by Portland Press Limited.

  18. Phospholipase C from Pseudomonas aeruginosa and Bacillus cereus; characterization of catalytic activity.

    PubMed

    Elleboudy, Nooran Sherif; Aboulwafa, Mohammad Mabrouk; Hassouna, Nadia Abdel-Haleem

    2014-11-01

    To study characteristics of phospholipases C (PLCs), their importance for producing microorganisms as well as the potential of their use for industrial purposes. PLC from Bacillus cereus (B. cereus) D101 was selected as an example of Gram-positive PLCs and PLC from Pseudomonas aeruginosa (P. aeruginosa) D183 of Gram-negative ones. Enzymes were partially purified by ammonium sulfate precipitation followed by membrane dialysis. Partially purified preparations were used to study effect of different factors on activities as well as in substrate specificity tests which were conducted using a turbidimetric assay method. Maximum activity was at pH 7 and 8 and 40 °C for P. aeruginosa PLC, and pH 8-10 and 37 °C for B. cereus PLC. Both PLCs were inhibited by Pi at 5 mM or higher, whereas, PLC from B. cereus only was inhibited by EDTA. Activity of P. aeruginosa PLC was not affected by removing Zn(2+) ions from reaction mixture or their replacement with Ca(2+), Ba(2+), Mg(2+) or Mn(2+) ions. Vis-à-vis, activity of B. cereus PLC was found to be metal ion dependent. PLCs from both isolates were relatively thermostable and showed maximum affinity toward phosphatidylcholine. Sphingomyelin and phosphatidylethanolamine were not good substrates and phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and cardiolipin could be considered non-substrates. Human body physiological conditions could favor activity of P. aeruginosa and B. cereus PLCs. These enzymes may participate in phosphate scavenging and virulence of producing isolates but not in autolysis. PLCs from both isolates are potential candidates for industrial use. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  19. Phosphatidylcholine-specific phospholipase C and sphingomyelinase activities in bacteria of the Bacillus cereus group.

    PubMed

    Pomerantsev, A P; Kalnin, K V; Osorio, M; Leppla, S H

    2003-11-01

    Bacillus anthracis is nonhemolytic, even though it is closely related to the highly hemolytic Bacillus cereus. Hemolysis by B. cereus results largely from the action of phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelinase (SPH), encoded by the plc and sph genes, respectively. In B. cereus, these genes are organized in an operon regulated by the global regulator PlcR. B. anthracis contains a highly similar cereolysin operon, but it is transcriptionally silent because the B. anthracis PlcR is truncated at the C terminus. Here we report the cloning, expression, purification, and enzymatic characterization of PC-PLC and SPH from B. cereus and B. anthracis. We also investigated the effects of expressing PlcR on the expression of plc and sph. In B. cereus, PlcR was found to be a positive regulator of plc but a negative regulator of sph. Replacement of the B. cereus plcR gene by its truncated orthologue from B. anthracis eliminated the activities of both PC-PLC and SPH, whereas introduction into B. anthracis of the B. cereus plcR gene with its own promoter did not activate cereolysin expression. Hemolytic activity was detected in B. anthracis strains containing the B. cereus plcR gene on a multicopy plasmid under control of the strong B. anthracis protective antigen gene promoter or in a strain carrying a multicopy plasmid containing the entire B. cereus plc-sph operon. Slight hemolysis and PC-PLC activation were found when PlcR-producing B. anthracis strains were grown under anaerobic-plus-CO(2) or especially under aerobic-plus-CO(2) conditions. Unmodified parental B. anthracis strains did not demonstrate obvious hemolysis under the same conditions.

  20. Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes

    PubMed Central

    1987-01-01

    African trypanosomes contain a membrane-bound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic PercollR centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate. The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes. The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of Trypanosoma brucei, cultured procyclic Trypanosoma brucei and bloodstream Trypanosoma vivax had little or no mfVSG phospholipase activity. The activity found in bloodstream forms of Trypanosoma congolense was intermediate between Trypanosoma vivax and Trypanosoma brucei. PMID:3624307

  1. Cholesterol regulates HERG K+ channel activation by increasing phospholipase C β1 expression

    PubMed Central

    Chun, Yoon Sun; Oh, Hyun Geun; Park, Myoung Kyu; Cho, Hana; Chung, Sungkwon

    2013-01-01

    Human ether-a-go-go-related gene (HERG) K+ channel underlies the rapidly activating delayed rectifier K+ conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of β1 and β3 isoforms of PLC (PLCβ1, PLCβ3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCβ1 expression, we conclude that increased PLCβ1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels. PMID:23793622

  2. Phospholipase C mediated Suppression of Dark Noise Enables Single Photon Detection in Drosophila Photoreceptors

    PubMed Central

    Katz, Ben; Minke, Baruch

    2012-01-01

    Drosophila photoreceptor cells use the ubiquitous G-protein-mediated phospholipase C (PLC) cascade to achieve ultimate single photon sensitivity. This is manifested in the single photon responses (quantum bumps). In photoreceptor cells, dark activation of Gqα molecules occurs spontaneously and produces unitary dark events (dark bumps). A high rate of spontaneous Gqα activation and dark bump production potentially hampers single photon detection. We found that in wild type flies the in vivo rate of spontaneous Gqα activation is very high. Nevertheless, this high rate is not manifested in a substantially high rate of dark bumps. Therefore, it is unclear how phototransduction suppresses dark bump production, arising from spontaneous Gqα activation, while still maintaining high-fidelity representation of single photons. In this study we show that reduced PLC catalytic activity selectively suppressed production of dark bumps but not light-induced bumps. Manipulations of PLC activity using PLC mutant flies and Ca2+ modulations revealed that a critical level of PLC activity is required to induce bump production. The required minimal level of PLC activity, selectively suppressed random production of single Gqα-activated dark bumps despite a high rate of spontaneous Gqα activation. This minimal PLC activity level is reliably obtained by photon induced synchronized activation of several neighboring Gqα molecules activating several PLC molecules, but not by random activation of single Gqα molecules. We thus demonstrate how a G-protein-mediated transduction system, with PLC as its target, selectively suppresses its intrinsic noise while preserving reliable signaling. PMID:22357856

  3. Osmotic activation of phospholipase C triggers structural adaptation in osmosensitive rat supraoptic neurons.

    PubMed

    Shah, Love; Bansal, Vimal; Rye, Peter L; Mumtaz, Naima; Taherian, Amir; Fisher, Thomas E

    2014-10-01

    The magnocellular neurosecretory cells of the hypothalamus (MNCs) synthesize and secrete vasopressin or oxytocin. A stretch-inactivated cation current mediated by TRPV1 channels rapidly transduces increases in external osmolality into a depolarization of the MNCs leading to an increase in action potential firing and thus hormone release. Prolonged increases in external osmolality, however, trigger a reversible structural and functional adaptation that may enable the MNCs to sustain high levels of hormone release. One poorly understood aspect of this adaptation is somatic hypertrophy. We demonstrate that hypertrophy can be evoked in acutely isolated rat MNCs by exposure to hypertonic solutions lasting tens of minutes. Osmotically evoked hypertrophy requires activation of the stretch-inactivated cation channel, action potential firing, and the influx of Ca(2+). Hypertrophy is prevented by pretreatment with a cell-permeant inhibitor of exocytotic fusion and is associated with an increase in total membrane capacitance. Recovery is disrupted by an inhibitor of dynamin function, suggesting that it requires endocytosis. We also demonstrate that hypertonic solutions cause a decrease in phosphatidylinositol 4,5-bisphosphate in the plasma membranes of MNCs that is prevented by an inhibitor of phospholipase C (PLC). Inhibitors of PLC or protein kinase C (PKC) prevent osmotically evoked hypertrophy, and treatment with a PKC-activating phorbol ester can elicit hypertrophy in the absence of changes in osmolality. These studies suggest that increases in osmolality cause fusion of internal membranes with the plasma membrane of the MNCs and that this process is mediated by activity-dependent activation of PLC and PKC. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

  4. Juvenile hormone-activated phospholipase C pathway enhances transcriptional activation by the methoprene-tolerant protein

    PubMed Central

    Liu, Pengcheng; Peng, Hong-Juan; Zhu, Jinsong

    2015-01-01

    Juvenile hormone (JH) is a key regulator of a wide diversity of developmental and physiological events in insects. Although the intracellular JH receptor methoprene-tolerant protein (MET) functions in the nucleus as a transcriptional activator for specific JH-regulated genes, some JH responses are mediated by signaling pathways that are initiated by proteins associated with plasma membrane. It is unknown whether the JH-regulated gene expression depends on the membrane-mediated signal transduction. In Aedes aegypti mosquitoes, we found that JH activated the phospholipase C (PLC) pathway and quickly increased the levels of inositol 1,4,5-trisphosphate, diacylglycerol, and intracellular calcium, leading to activation and autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). When abdomens from newly emerged mosquitoes were cultured in vitro, the JH-activated gene expression was repressed substantially if specific inhibitors of PLC or CaMKII were added to the medium together with JH. In newly emerged female mosquitoes, RNAi-mediated depletion of PLC or CaMKII considerably reduced the expression of JH-responsive genes, including the Krüppel homolog 1 gene (AaKr-h1) and the early trypsin gene (AaET). JH-induced loading of MET to the promoters of AaKr-h1 and AaET was weakened drastically when either PLC or CaMKII was inactivated in the cultured tissues. Therefore, the results suggest that the membrane-initiated signaling pathway modifies the DNA-binding activity of MET via phosphorylation and thus facilitates the genomic responses to JH. In summary, this study reveals an interplay of genomic and nongenomic signaling mechanisms of JH. PMID:25825754

  5. Dopamine D1 Receptor Signaling: Does GαQ–Phospholipase C Actually Play a Role?

    PubMed Central

    Lee, Sang-Min; Yang, Yang

    2014-01-01

    Despite numerous studies showing therapeutic potential, no central dopamine D1 receptor ligand has ever been approved, because of potential limitations, such as hypotension, seizures, and tolerance. Functional selectivity has been widely recognized as providing a potential mechanism to develop novel therapeutics from existing targets, and a highly biased, functionally selective D1 ligand might overcome some of the past limitations. SKF-83959 [6-chloro-3-methyl-1-(m-tolyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepine-7,8-diol] is reported to be a highly biased D1 ligand, having full agonism at D1-mediated activation of phospholipase C (PLC) signaling (via GαQ) and antagonism at D1-mediated adenylate cyclase signaling (via GαOLF/S). For this reason, numerous studies have used this compound to elucidate the physiologic role of D1-PLC signaling, including a novel molecular mechanism (GαQ-PLC activation via D1-D2 heterodimers). There is, however, contradictory literature that suggests that SKF-83959 is actually a partial agonist at both D1-mediated adenylate cyclase and β-arrestin recruitment. Moreover, the D1-mediated PLC stimulation has also been questioned. This Minireview examines 30 years of relevant literature and proposes that the data strongly favor alternate hypotheses: first, that SKF-83959 is a typical D1 partial agonist; and second, that the reported activation of PLC by SKF-83959 and related benzazepines likely is due to off-target effects, not actions at D1 receptors. If these hypotheses are supported by future studies, it would suggest that caution should be used regarding the role of PLC and downstream pathways in D1 signaling. PMID:25052835

  6. [Phospholipid content of plasma membranes and phospholipase C activity in epithelial cells of the large intestine in colitis-associated carcinogenesis in rats].

    PubMed

    Drobins'ka, O V; Kravchenko, O O; Koval'ova, V A; Artemenko, O Iu; Ostapchenko, L I

    2009-01-01

    The decrease of major cytoplasmic membrane phospholipids (phosphatidylcholine and phosphatidylethanolamine) content was established in mucosal epithelial cell under colon inflammation pathology--ulcerative colitis. It was shown that aforementioned changes were associated with the increase of phospholipids' hydrolyzing enzyme--phospholipase C activity and intracellular Ca2+ concentration enlargement. Carcinogenesis stimulation under inflammation was accompanied by phospholipase C activity increase when quantity of investigated phospholipids (phosphatidylcholine, phosphatidylinozytol, phosphatidylserine) separately decreased and cytoplasmic Ca2+ value normalization was established.

  7. Activity of phospholipase C and release of prostaglandin F2 alpha by endometrial tissue from ovariectomized ewes receiving progesterone and estradiol.

    PubMed

    Raw, R E; Silvia, W J

    1991-03-01

    Progesterone and estradiol interact to regulate secretion of prostaglandin (PG) F2 alpha from the ovine endometrium in response to oxytocin. Two experiments were conducted to determine if these effects were due to changes in activity of phospholipase C or in the second messenger responsive pathways that regulate production of PGF2 alpha. In both experiments, ovariectomized ewes were assigned to one of four treatment groups (control, estradiol, progesterone, progesterone and estradiol). Steroids were administered, in vivo, to mimic the changes that occur during the estrous cycle. On Day 16 of steroid treatment, endometrial tissue was collected and incubated, in vitro, to measure activity of phospholipase C and release of PGF2 alpha. Treatment with progesterone, in vivo, enhanced basal and oxytocin-induced activity of phospholipase C and release of PGF2 alpha, in vitro. Estradiol suppressed oxytocin-induced activity of phospholipase C, both in the presence and absence of progesterone. In contrast to its effects on phospholipase C, estradiol inhibited basal and oxytocin-induced release of PGF2 alpha when administered alone, but not when administered with progesterone. Steroids had similar effects on the release of PGF2 alpha induced by phorbol 12-myristate 13-acetate and A23187. It was concluded that progesterone and estradiol regulate endometrial release of PGF2 alpha by affecting both the activity of phospholipase C and its associated second messenger responsive pathways that may regulate production of PGF2 alpha.

  8. Gene identification and evidence for expression of G protein alpha subunits, phospholipase C, and an inositol 1,4,5-trisphosphate receptor in Aplysia californica rhinophore.

    PubMed

    Cummins, Scott F; De Vries, Melissa R; Hill, Kristen S; Boehning, Darren; Nagle, Gregg T

    2007-07-01

    In the marine mollusk Aplysia californica, waterborne protein pheromones that are released during egg laying act in concert to stimulate mate attraction. However, molecular information concerning the cellular receptors and signaling mechanisms that may be involved in waterborne peptide and protein pheromonal communication is lacking. As a first step toward examining whether members of the G protein family and phosphoinositide signaling pathway are present in the primary peripheral chemosensory organs (i.e., rhinophores), we isolated five full-length cDNA clones from an A. californica central nervous system cDNA library. These clones encoded (1) the G protein alpha subunits of the Gq, Gi, and Go families, (2) a protein with homology to phospholipase C (PLC) isoforms, and (3) an inositol 1,4,5-trisphosphate receptor (IP3R). The expression of these genes was examined using laser capture microdissection/reverse transcription-polymerase chain reaction and in situ hybridization. All of them are expressed in the rhinophore sensory epithelium, suggesting that Galphaq, Galphai, Galphao, PLC-like protein, and IP3R may be involved in waterborne protein pheromone detection in Aplysia-possibly via a phosphoinositide signaling mechanism.

  9. Phospholipase C-independent effects of 3M3FBS in murine colon.

    PubMed

    Dwyer, Laura; Kim, Hyun Jin; Koh, Byoung Ho; Koh, Sang Don

    2010-02-25

    The muscarinic receptor subtype M(3) is coupled to Gq/11 proteins. Muscarinic receptor agonists such as carbachol stimulate these receptors that result in activation of phospholipase C (PLC) which hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and Ins(1,4,5)P(3). This pathway leads to excitation and smooth muscle contraction. In this study the PLC agonist, 2, 4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benezenesulfonamide (m-3M3FBS), was used to investigate whether direct PLC activation mimics carbachol-induced excitation. We examined the effects of m-3M3FBS and 2, 4, 6-trimethyl-N-(ortho-3-trifluoromethyl-phenyl)-benzenesulfonamide (o-3M3FBS), on murine colonic smooth muscle tissue and cells by performing conventional microelectrode recordings, isometric force measurements and patch clamp experiments. Application of m-3M3FBS decreased spontaneous contractility in murine colonic smooth muscle without affecting the resting membrane potential. Patch clamp studies revealed that delayed rectifier K(+) channels were reversibly inhibited by m-3M3FBS and o-3M3FBS. The PLC inhibitor, 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), did not prevent this inhibition by m-3M3FBS. Both m-3M3FBS and o-3M3FBS decreased two components of delayed rectifier K(+) currents in the presence of tetraethylammonium chloride or 4-aminopyridine. Ca(2+) currents were significantly suppressed by m-3M3FBS and o-3M3FBS with a simultaneous increase in intracellular Ca(2+). Pretreatment with U73122 did not prevent the decrease in Ca(2+) currents upon m-3M3FBS application. In conclusion, both m-3M3FBS and o-3M3FBS inhibit inward and outward currents via mechanisms independent of PLC acting in an antagonistic manner. In contrast, both compounds also caused an increase in [Ca(2+)](i) in an agonistic manner. Therefore caution must be employed when interpreting their effects at the tissue and cellular level.

  10. Phospholipase C-independent effects of 3M3FBS in murine colon

    PubMed Central

    Dwyer, Laura; Kim, Hyunjin; Koh, Byoung Ho; Koh, Sang Don

    2009-01-01

    The muscarinic receptor subtype M3 is coupled to Gq/11 proteins. Muscarinic receptor agonists such as carbachol stimulate these receptors that result in activation of phospholipase C (PLC) which hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and Ins(1,4,5)P3. This pathway leads to excitation and smooth muscle contraction. In this study the PLC agonist, 2, 4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benezenesulfonamide (m-3M3FBS), was used to investigate whether direct PLC activation mimics carbachol-induced excitation. We examined the effects of m-3M3FBS and 2, 4, 6-trimethyl-N-(ortho-3-trifluoromethyl-phenyl)-benzenesulfonamide (o-3M3FBS), on murine colonic smooth muscle tissue and cells by performing conventional microelectrode recordings, isometric force measurements and patch clamp experiments. Application of m-3M3FBS decreased spontaneous contractility in murine colonic smooth muscle without affecting the resting membrane potential. Patch clamp studies revealed that delayed rectifier K+ channels were reversibly inhibited by m-3M3FBS and o-3M3FBS. The PLC inhibitor, 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), did not prevent this inhibition by m-3M3FBS. Both m-3M3FBS and o-3M3FBS decreased two components of delayed rectifier K+ currents in the presence of tetraethylammonium chloride or 4-aminopyridine. Ca2+ currents were significantly suppressed by m-3M3FBS and o-3M3FBS with a simultaneous increase in intracellular Ca2+. Pretreatment with U73122 did not prevent the decrease in Ca2+ currents upon m-3M3FBS application. In conclusion, both m-3M3FBS and o-3M3FBS inhibit inward and outward currents via mechanisms independent of PLC acting in an antagonistic manner. In contrast, both compounds also caused an increase in [Ca2+]i in an agonistic manner. Therefore caution must be employed when interpreting their effects at the tissue and cellular level. PMID:19931239

  11. Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

    PubMed Central

    Falkenburger, Björn H.; Jensen, Jill B.

    2010-01-01

    G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of

  12. Oocyte activation and phospholipase C zeta (PLCζ): diagnostic and therapeutic implications for assisted reproductive technology

    PubMed Central

    2012-01-01

    Infertility affects one in seven couples globally and has recently been classified as a disease by the World Health Organisation (WHO). While in-vitro fertilisation (IVF) offers effective treatment for many infertile couples, cases exhibiting severe male infertility (19–57%) often remain difficult, if not impossible to treat. In such cases, intracytoplasmic sperm injection (ICSI), a technique in which a single sperm is microinjected into the oocyte, is implemented. However, 1–5% of ICSI cycles still fail to fertilise, affecting over 1000 couples per year in the UK alone. Pregnancy and delivery rates for IVF and ICSI rarely exceed 30% and 23% respectively. It is therefore imperative that Assisted Reproductive Technology (ART) protocols are constantly modified by associated research programmes, in order to provide patients with the best chances of conception. Prior to fertilisation, mature oocytes are arrested in the metaphase stage of the second meiotic division (MII), which must be alleviated to allow the cell cycle, and subsequent embryogenesis, to proceed. Alleviation occurs through a series of concurrent events, collectively termed ‘oocyte activation’. In mammals, oocytes are activated by a series of intracellular calcium (Ca2+) oscillations following gamete fusion. Recent evidence implicates a sperm-specific phospholipase C, PLCzeta (PLCζ), introduced into the oocyte following membrane fusion as the factor responsible. This review summarises our current understanding of oocyte activation failure in human males, and describes recent advances in our knowledge linking certain cases of male infertility with defects in PLCζ expression and activity. Systematic literature searches were performed using PubMed and the ISI-Web of Knowledge. Databases compiled by the United Nations and World Health Organisation databases (UNWHO), and the Human Fertilization and Embryology Authority (HFEA) were also scrutinised. It is clear that PLCζ plays a fundamental role in

  13. Mu-opioids activate phospholipase C in SH-SY5Y human neuroblastoma cells via calcium-channel opening.

    PubMed Central

    Smart, D; Smith, G; Lambert, D G

    1995-01-01

    We have recently reported that, in SH-SY5Y cells, mu-opioid receptor occupancy activates phospholipase C via a pertussis toxin-sensitive G-protein. In the present study we have further characterized the mechanisms involved in this process. Fentanyl (0.1 microM) caused a monophasic increase in inositol 1,4,5-trisphosphate mass formation, with a peak (20.5 +/- 3.6 pmol/mg of protein) at 15 s. Incubation in Ca(2+)-free buffer abolished this response, while Ca2+ replacement 1 min later restored the stimulation of inositol 1,4,5-trisphosphate formation (20.1 +/- 0.6 pmol/mg of protein). In addition, nifedipine (1 nM-0.1 mM), an L-type Ca(2+)-channel antagonist, caused a dose-dependent inhibition of inositol 1,4,5-trisphosphate formation, with an IC50 of 60.3 +/- 1.1 nM. Elevation of endogenous beta/gamma subunits by selective activation of delta-opioid and alpha 2 adrenoceptors failed to stimulate phospholipase C. Fentanyl also caused a dose-dependent (EC50 of 16.2 +/- 1.0 nM), additive enhancement of carbachol-induced inositol 1,4,5-trisphosphate formation. In summary, we have demonstrated that in SH-SY5Y cells activation of the mu-opioid receptor allows Ca2+ influx to activate phospholipase C. However, the possible role of this mechanism in the process of analgesia remains to be elucidated. PMID:7832776

  14. Inhibition of high-affinity gamma-aminobutyric acid uptake in primary astrocyte cultures by phorbol esters and phospholipase C.

    PubMed Central

    Gomeza, J; Casado, M; Gimenez, C; Aragon, C

    1991-01-01

    The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), on high-affinity Na(+)-dependent gamma-aminobutyric acid (GABA) uptake were investigated in primary cultures of neurons and glial cells from rat brain cortex. Incubation of glial cells with PMA led to concentration- and time-dependent decreases in the GABA transport in glial cells. This effect could be completely suppressed by addition of the PKC inhibitor H7. The PMA effects could be mimicked by oleoylacetylglycerol, the diacylglycerol kinase inhibitor R59022 and exogenous phospholipase C. Treatment with PMA did not affect GABA transport in neuronal cells. PMID:1902665

  15. An Autoinhibitory Helix in the C-Terminal Region of Phospholipase C-β Mediates Gαq Activation

    PubMed Central

    Lyon, Angeline M.; Tesmer, Valerie M.; Dhamsania, Vishan D.; Thal, David M.; Gutierrez, Joanne; Chowdhury, Shoaib; Suddala, Krishna C.; Northup, John K.; Tesmer, John J. G.

    2011-01-01

    Phospholipase C-β (PLCβ) is a key regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to Gq. We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCβ3 dramatically increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCβ. PMID:21822282

  16. Collagen type IV stimulates an increase in intracellular Ca2+ in pancreatic acinar cells via activation of phospholipase C.

    PubMed Central

    Somogyi, L; Lasić, Z; Vukicević, S; Banfić, H

    1994-01-01

    Intracellular Ca2+ responses to extracellular matrix molecules were studied in suspensions of pancreatic acinar cells loaded with Fura-2. Collagen type I, laminin, fibrinogen and fibronectin were unable to raise cytosolic free Ca2+ concentration ([Ca2+]i), whereas collagen type IV, at concentrations from 5 to 50 micrograms/ml, significantly increased it. The effect of collagen type IV was not due to possible contamination with type-I transforming growth factor beta or plasminogen, as neither of these agents was able to increase [Ca2+]i. Using highly specific mass assays, concentrations of inositol lipids, 1,2-diacylglycerol (DAG) and Ins(1,4,5) P3 were measured in pancreatic acinar cells stimulated with collagen type IV. A decrease in the concentrations of PtdIns(4,5) P2 and PtdIns4 P with a concomitant increase in the concentrations of DAG and InsP3 mass were observed, showing that collagen type IV increases [Ca2+]i by activation of phospholipase C. The observed [Ca2+]i signals had two components, the first resulting from Ca2+ release from the intracellular stores, and the second resulting from Ca2+ flux from the extracellular medium through the verapamil-insensitive channels. A tyrosine kinase inhibitor (tyrphostine) was able to block inositol lipid signalling caused by collagen type IV, which together with the insensitivity of this pathway to cholera toxin and pertussis toxin or to preactivation of protein kinase C, the longer duration of the increase in [Ca2+]i and a longer lag period needed for observation of increases in DAG and InsP3 concentration with collagen type IV than with carbachol (50 mM) suggest that activation of phospholipase C by collagen type IV is caused by tyrosine kinase activation. Inositol lipid signalling and increases in [Ca2+]i were also observed with Arg-Gly-Asp (RGD)-containing peptide but not with Arg-Asp-Gly (RDG)-containing peptide. Collagen type IV and RGD-containing peptide, but not carbachol, competed in increasing [Ca2+]i and

  17. NHERF2 specifically interacts with LPA2 receptor and defines the specificity and efficiency of receptor-mediated phospholipase C-beta3 activation.

    PubMed

    Oh, Yong-Seok; Jo, Nam Won; Choi, Jung Woong; Kim, Hyeon Soo; Seo, Sang-Won; Kang, Kyung-Ok; Hwang, Jong-Ik; Heo, Kyun; Kim, Sun-Hee; Kim, Yun-Hee; Kim, In-Hoo; Kim, Jae Ho; Banno, Yoshiko; Ryu, Sung Ho; Suh, Pann-Ghill

    2004-06-01

    Lysophosphatidic acid (LPA) activates a family of cognate G protein-coupled receptors and is involved in various pathophysiological processes. However, it is not clearly understood how these LPA receptors are specifically coupled to their downstream signaling molecules. This study found that LPA(2), but not the other LPA receptor isoforms, specifically interacts with Na(+)/H(+) exchanger regulatory factor2 (NHERF2). In addition, the interaction between them requires the C-terminal PDZ domain-binding motif of LPA(2) and the second PDZ domain of NHERF2. Moreover, the stable expression of NHERF2 potentiated LPA-induced phospholipase C-beta (PLC-beta) activation, which was markedly attenuated by either a mutation in the PDZ-binding motif of LPA(2) or by the gene silencing of NHERF2. Using its second PDZ domain, NHERF2 was found to indirectly link LPA(2) to PLC-beta3 to form a complex, and the other PLC-beta isozymes were not included in the protein complex. Consistently, LPA(2)-mediated PLC-beta activation was specifically inhibited by the gene silencing of PLC-beta3. In addition, NHERF2 increases LPA-induced ERK activation, which is followed by cyclooxygenase-2 induction via a PLC-dependent pathway. Overall, the results suggest that a ternary complex composed of LPA(2), NHERF2, and PLC-beta3 may play a key role in the LPA(2)-mediated PLC-beta signaling pathway.

  18. Vav1 transduces T cell receptor signals to the activation of phospholipase C-gamma1 via phosphoinositide 3-kinase-dependent and -independent pathways.

    PubMed

    Reynolds, Lucinda F; Smyth, Lesley A; Norton, Trisha; Freshney, Norman; Downward, Julian; Kioussis, Dimitris; Tybulewicz, Victor L J

    2002-05-06

    Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4(+)CD8(+) double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-gamma1 (PLCgamma1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCgamma1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCgamma1 and the adaptor molecule Src homology 2 domain-containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K.

  19. Vav1 Transduces T Cell Receptor Signals to the Activation of Phospholipase C-γ1 via Phosphoinositide 3-Kinase-dependent and -independent Pathways

    PubMed Central

    Reynolds, Lucinda F.; Smyth, Lesley A.; Norton, Trisha; Freshney, Norman; Downward, Julian; Kioussis, Dimitris; Tybulewicz, Victor L.J.

    2002-01-01

    Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4+CD8+ double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-γ1 (PLCγ1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCγ1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCγ1 and the adaptor molecule Src homology 2 domain–containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K. PMID:11994416

  20. Nuclear diacylglycerol lipase-α in rat brain cortical neurons: evidence of 2-arachidonoylglycerol production in concert with phospholipase C-β activity.

    PubMed

    García del Caño, Gontzal; Aretxabala, Xabier; González-Burguera, Imanol; Montaña, Mario; López de Jesús, Maider; Barrondo, Sergio; Barrio, Ramón J; Sampedro, Carmen; Goicolea, M Arantzazu; Sallés, Joan

    2015-03-01

    In this report, we describe the localization of diacylglycerol lipase-α (DAGLα) in nuclei from adult cortical neurons, as assessed by double-immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double-labeling assays using the anti-DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα-signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC-35- and NeuN-signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C-β (PLCβ) family, PLCβ1, PLCβ2, and PLCβ4 exhibited the same distribution with respect to chromatin, lamin B1, SC-35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of 2-arachidonoylglycerol (2-AG) by liquid chromatography and mass spectrometry (LC-MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for 2-AG production, and its PLCβ/DAGLα-dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for 2-AG locally produced within the neuronal nucleus. © 2014 International Society for Neurochemistry.

  1. Effects of bilayer composition and physical properties on the phospholipase C and sphingomyelinase activities of Clostridium perfringens α-toxin.

    PubMed

    Urbina, Patricia; Flores-Díaz, Marietta; Alape-Girón, Alberto; Alonso, Alicia; Goñi, Félix M

    2011-01-01

    α-Toxin, a major determinant of Clostridium perfringens toxicity, exhibits both phospholipase C and sphingomyelinase activities. Our studies with large unilamellar vesicles containing a variety of lipid mixtures reveal that both lipase activities are enhanced by cholesterol and by lipids with an intrinsic negative curvature, e.g. phosphatidylethanolamine. Conversely lysophospholipids, that possess a positive intrinsic curvature, inhibit the α-toxin lipase activities. Phospholipids with a net negative charge do not exert any major effect on the lipase activities, and the same lack of effect is seen with the lysosomal lipid bis (monoacylglycero) phosphate. Ganglioside GT1b has a clear inhibitory effect, while the monosialic ganglioside GM3 is virtually ineffectual even when incorporated at 6mol % in the vesicles. The length of the lag periods appears to be inversely related to the maximum (post-lag) enzyme activities. Moreover, and particularly in the presence of cholesterol, lag times increase with pH. Both lipase activities are sensitive to vesicle size, but in opposite ways: while phospholipase C is higher with larger vesicles, sphingomyelinase activity is lower. The combination of our results with previous structural studies suggests that α-toxin lipase activities have distinct, but partially overlapping and interacting active sites.

  2. Vasoactive intestinal polypeptide requires parallel changes in adenylate cyclase and phospholipase C to entrain circadian rhythms to a predictable phase

    PubMed Central

    An, Sungwon; Irwin, Robert P.; Allen, Charles N.; Tsai, Connie

    2011-01-01

    Circadian oscillations in the suprachiasmatic nucleus (SCN) depend on transcriptional repression by Period (PER)1 and PER2 proteins within single cells and on vasoactive intestinal polypeptide (VIP) signaling between cells. Because VIP is released by SCN neurons in a circadian pattern, and, after photic stimulation, it has been suggested to play a role in the synchronization to environmental light cycles. It is not known, however, if or how VIP entrains circadian gene expression or behavior. Here, we tested candidate signaling pathways required for VIP-mediated entrainment of SCN rhythms. We found that single applications of VIP reset PER2 rhythms in a time- and dose-dependent manner that differed from light. Unlike VIP-mediated signaling in other cell types, simultaneous antagonism of adenylate cyclase and phospholipase C activities was required to block the VIP-induced phase shifts of SCN rhythms. Consistent with this, VIP rapidly increased intracellular cAMP in most SCN neurons. Critically, daily VIP treatment entrained PER2 rhythms to a predicted phase angle within several days, depending on the concentration of VIP and the interval between VIP applications. We conclude that VIP entrains circadian timing among SCN neurons through rapid and parallel changes in adenylate cyclase and phospholipase C activities. PMID:21389307

  3. Aluminum ions alter the function of non-specific phospholipase C through the changes in plasma membrane physical properties.

    PubMed

    Pejchar, Přemysl; Martinec, Jan

    2015-01-01

    The first indication of the aluminum (Al) toxicity in plants growing in acidic soils is the cessation of root growth, but the detailed mechanism of Al effect is unknown. Here we examined the impact of Al stress on the activity of non-specific phospholipase C (NPC) in the connection with the processes related to the plasma membrane using fluorescently labeled phosphatidylcholine. We observed a rapid and significant decrease of labeled diacylglycerol (DAG), product of NPC activity, in Arabidopsis seedlings treated with AlCl₃. Interestingly, an application of the membrane fluidizer, benzyl alcohol, restored the level of DAG during Al treatment. Our observations suggest that the activity of NPC is affected by Al-induced changes in plasma membrane physical properties.

  4. A dynamic set point for thermal adaptation requires phospholipase C-mediated regulation of TRPM8 in vivo

    PubMed Central

    Brenner, Daniel S.; Golden, Judith P.; Vogt, Sherri K.; Dhaka, Ajay; Story, Gina M.; Gereau, Robert W.

    2014-01-01

    The ability to sense and respond to thermal stimuli at varied environmental temperatures is essential for survival in seasonal areas. In this study, we show that mice respond similarly to ramping changes in temperature from a wide range of baseline temperatures. Further investigation suggests that this ability to adapt to different ambient environments is based on rapid adjustments made to a dynamic temperature setpoint. The adjustment of this setpoint requires TRPM8 but not TRPA1 and is regulated by phospholipase C (PLC) activity. Overall, our findings suggest that temperature response thresholds in mice are dynamic, and that this ability to adapt to environmental temperature seems to mirror the in vitro findings that PLC-mediated hydrolysis of phosphoinositol 4,5-bisphosphate modulates TRPM8 activity and thereby regulates the response thresholds to cold stimuli. PMID:25109670

  5. Inositol 5'-phosphatase, SHIP1 interacts with phospholipase C-gamma1 and modulates EGF-induced PLC activity.

    PubMed

    Song, Minseok; Kim, Myung Jong; Ha, Sanghoon; Park, Jong Bae; Ryu, Sung Ho; Suh, Pann-Ghill

    2005-06-30

    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.

  6. Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1) in Cryptococcus neoformans reveals unique biochemical features.

    PubMed

    Henry, Jennifer; Guillotte, Aimee; Luberto, Chiara; Del Poeta, Maurizio

    2011-02-18

    In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.

  7. Bacillus anthracis phospholipases C facilitate macrophage-associated growth and contribute to virulence in a murine model of inhalation anthrax.

    PubMed

    Heffernan, Brian J; Thomason, Brendan; Herring-Palmer, Amy; Shaughnessy, Lee; McDonald, Rod; Fisher, Nathan; Huffnagle, Gary B; Hanna, Philip

    2006-07-01

    Several models of anthrax pathogenesis suggest that early in the infectious process Bacillus anthracis endospores germinate and outgrow into vegetative bacilli within phagocytes before being released into the blood. Here, we define the respective contributions of three phospholipases C (PLCs) to the pathogenesis of B. anthracis. Genetic deletions of the PLCs were made in the Sterne 7702 background, resulting in the respective loss of their activities. The PLCs were redundant both in tissue culture and in murine models of anthrax. Deletion of all three PLC genes was required for attenuation of virulence in mice after intratracheal inoculation. This attenuation may be attributed to the inability of the PLC-null strain to grow in association with the macrophage. Complementation of these defects in both models of anthrax was achieved by expression of the PLC genes in trans. The functional redundancy between PLCs in the virulence of B. anthracis implies that their activities are important for anthrax pathogenesis.

  8. Bacillus anthracis Phospholipases C Facilitate Macrophage-Associated Growth and Contribute to Virulence in a Murine Model of Inhalation Anthrax

    PubMed Central

    Heffernan, Brian J.; Thomason, Brendan; Herring-Palmer, Amy; Shaughnessy, Lee; McDonald, Rod; Fisher, Nathan; Huffnagle, Gary B.; Hanna, Philip

    2006-01-01

    Several models of anthrax pathogenesis suggest that early in the infectious process Bacillus anthracis endospores germinate and outgrow into vegetative bacilli within phagocytes before being released into the blood. Here, we define the respective contributions of three phospholipases C (PLCs) to the pathogenesis of B. anthracis. Genetic deletions of the PLCs were made in the Sterne 7702 background, resulting in the respective loss of their activities. The PLCs were redundant both in tissue culture and in murine models of anthrax. Deletion of all three PLC genes was required for attenuation of virulence in mice after intratracheal inoculation. This attenuation may be attributed to the inability of the PLC-null strain to grow in association with the macrophage. Complementation of these defects in both models of anthrax was achieved by expression of the PLC genes in trans. The functional redundancy between PLCs in the virulence of B. anthracis implies that their activities are important for anthrax pathogenesis. PMID:16790747

  9. Aluminum ions alter the function of non-specific phospholipase C through the changes in plasma membrane physical properties

    PubMed Central

    Pejchar, Přemysl; Martinec, Jan

    2015-01-01

    The first indication of the aluminum (Al) toxicity in plants growing in acidic soils is the cessation of root growth, but the detailed mechanism of Al effect is unknown. Here we examined the impact of Al stress on the activity of non-specific phospholipase C (NPC) in the connection with the processes related to the plasma membrane using fluorescently labeled phosphatidylcholine. We observed a rapid and significant decrease of labeled diacylglycerol (DAG), product of NPC activity, in Arabidopsis seedlings treated with AlCl3. Interestingly, an application of the membrane fluidizer, benzyl alcohol, restored the level of DAG during Al treatment. Our observations suggest that the activity of NPC is affected by Al-induced changes in plasma membrane physical properties. PMID:26024014

  10. Sensitivity to MK-801 in phospholipase C-β1 knockout mice reveals a specific NMDA receptor deficit.

    PubMed

    Gray, Laura; McOmish, Caitlin E; Scarr, Elizabeth; Dean, Brian; Hannan, Anthony J

    2009-08-01

    Phospholipase C-β1 (PLC-β1) is a critical component of multiple signalling pathways downstream of neurotransmitter receptors. Mice lacking this enzyme display a striking behavioural phenotype with relevance to human psychiatric disease. Glutamatergic dysfunction is strongly associated with several abnormal behavioural states and may underlie part of the phenotype of the phospholipase C-β1 knockout (KO) mouse. A heightened response to glutamatergic psychotomimetic drugs is a critical psychosis-related endophenotype, and in this study it was employed as a correlate of glutamatergic dysfunction. Control (n=8) and PLC-β1 KO mice (n=6) were treated with MK-801, a NMDA receptor (NMDAR) antagonist, following either standard housing or environmental enrichment, and the motor function and locomotor activity thus evoked was assessed. In addition, MK-801 binding to the NMDAR was evaluated through radioligand autoradiography in post-mortem tissue (on a drug-naive cohort). We have demonstrated a significantly increased sensitivity to the effects of the NMDA antagonist MK-801 in the PLC-β1 KO mouse. In addition, we found that this mouse line displays reduced hippocampal NMDAR expression, as measured by radioligand binding. We previously documented a reversal of specific phenotypes in this mouse line following housing in an enriched environment. Enrichment did not alter this heightened MK-801 response, nor NMDAR expression, indicating that this therapeutic intervention works on specific pathways only. These findings demonstrate the critical role of the glutamatergic system in the phenotype of the PLC-β1 KO mouse and highlight the role of these interconnected signalling pathways in schizophrenia-like behavioural disruption. These results also shed further light on the capacity of environmental factors to modulate subsets of these phenotypes.

  11. 2-aminohydroxamic acid derivatives as inhibitors of Bacillus cereus phosphatidylcholine preferred phospholipase C PC-PLC(Bc).

    PubMed

    González-Bulnes, Patricia; González-Roura, Albert; Canals, Daniel; Delgado, Antonio; Casas, Josefina; Llebaria, Amadeu

    2010-12-15

    Phosphatidylcholine preferring phospholipase C (PC-PLC) is an important enzyme that plays a key role in a variety of cellular events and lipid homoeostases. Bacillus cereus phospholipase C (PC-PLC(Bc)) has antigenic similarity with the elusive mammalian PC-PLC, which has not thus far been isolated and purified. Therefore the discovery of inhibitors of PC-PLC(Bc) is of current interest. Here, we describe the synthesis and biological evaluation of a new type of compounds inhibiting PC-PLC(Bc). These compounds have been designed by evolution of previously described 2-aminohydroxamic acid PC-PLC(Bc) inhibitors that block the enzyme by coordination of the zinc active site atoms present in PC-PLC(Bc) [Gonzalez-Roura, A.; Navarro, I.; Delgado, A.; Llebaria, A.; Casas, J. Angew. Chem. Int. Ed.2004, 43, 862]. The new compounds maintain the zinc coordinating groups and possess an extra trimethylammonium function, linked to the hydroxyamide nitrogen by an alkyl chain, which is expected to mimic the trimethylammonium group of the phosphatidylcholine PC-PLC(Bc) substrates. Some of the compounds described inhibit the enzyme with IC(50)'s in the low micromolar range. Unexpectedly, the most potent inhibitors found are those that possess a trimethylammonium group but have chemically blocked the zinc coordinating functionalities. The results obtained suggest that PC-PLC(Bc) inhibition is not due to the interaction of compounds with the phospholipase catalytic zinc atoms, but rather results from the inhibitor cationic group recognition by the PC-PLC(Bc) amino acids involved in choline lipid binding.

  12. Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: evaluation of the roles of phospholipases C and D.

    PubMed

    Booz, G W; Taher, M M; Baker, K M; Singer, H A

    1994-12-21

    Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In 32P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar1]angiotensin II was shown to rapidly induce formation of 32P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as 32P- or 3H-phosphatidylethanol was produced when cells labeled with [32P]H3PO4 or 1-O-[1,2- 3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar1]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca2+, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar1]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar1]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar1]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar1]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such as de novo synthesis, may contribute to [Sar1]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar1]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Hetero-oligomerization of C2 domains of phospholipase C-related but catalytically inactive protein and synaptotagmin-1.

    PubMed

    Wang, DaGuang; Takeuchi, Hiroshi; Gao, Jing; Zhang, Zhao; Hirata, Masato

    2015-01-01

    The C2 domain is a protein module often found in molecules that regulate exocytosis. C2 domains mediate interactions between the parental molecule and Ca(2+), phospholipids, and proteins. Although various molecules have been shown to interact with several C2 domains, no interactions between the C2 domains from different molecules have yet been reported. In the present study, we identified direct interactions between the C2 domain of PRIP (phospholipase C-related but catalytically inactive protein) and the C2 domains of other molecules. Among the C2 domains examined, those of synaptotagmin-1 (Syt1-C2A and Syt1-C2B) and phospholipase C δ-1 bound to the C2 domain of PRIP. We investigated the interactions between the C2 domain of PRIP (PRIP-C2) with Syt1-C2A and Syt1-C2B, and the mode of binding of each was Ca(2+)-dependent and -independent, respectively. We further demonstrated that the Ca(2+) dependence of the interaction between PRIP-C2 and Syt1-C2A was attributed to Ca(2+) binding with Syt1-C2A, but not PRIP-C2, using a series of mutants prepared from both C2 domains. We previously reported that the interaction between PRIP-C2 and the membrane fusion machinery suggested a critical role for PRIP in exocytosis; therefore, the results of the present study further support the importance of PRIP-C2 in the inhibitory function of PRIP in regulating exocytosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: A sup 31 P NMR study

    SciTech Connect

    Shashidhar, M.S.; Kuppe, A. ); Volwerk, J.J.; Griffith, O.H.

    1990-09-04

    The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by {sup 31}P NMR. {sup 31}P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are {minus}0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. The authors also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by {sup 31}P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B.cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. They propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.

  15. Neuropeptide Y reduces the expression of PLCB2, PLCD1 and selected PLC genes in cultured human endothelial cells.

    PubMed

    Lo Vasco, V R; Leopizzi, M; Puggioni, C; Della Rocca, C; Businaro, R

    2014-09-01

    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.

  16. Corticotropin-releasing factor induces phosphorylation of phospholipase C-gamma at tyrosine residues via its receptor 2beta in human epidermoid A-431 cells.

    PubMed

    Kiang, J G; Ding, X Z; Gist, I D; Jones, R R; Tsokos, G C

    1998-12-18

    This laboratory previously reported that corticotropin-releasing factor (CRF) increased intracellular free calcium concentrations, cellular cAMP, inositol 1,4,5-trisphosphate, protein kinase C activity, and protein phosphorylation in human A-431 cells. The increase was blocked by CRF receptor antagonist. In this study, we identified the type of CRF receptors present and investigated whether CRF induced tyrosine phosphorylation of phospholipase C-gamma via CRF receptors. Using novel primers in reverse transcriptase-polymerase chain reaction, we determined the CRF receptor type to be that of 2beta. The levels of the CRF receptor type 2beta were not altered in cells treated with activators of protein kinase C, Ca2+ ionophore, or cells overexpressing heat shock protein 70 kDa. Cells treated with CRF displayed increases in protein tyrosine phosphorylation approximately at 150 kDa as detected by immunoblotting using an antibody against phosphotyrosine. Immunoprecipitation with antibodies directed against phospholipase C-beta3, -gamma1, or -gamma2 isoforms (which have molecular weights around 150 kDa) followed by Western blotting using an anti-phosphotyrosine antibody showed that only phospholipase C-gamma1 and -gamma2 were phosphorylated. The increase in phospholipase C-gamma phosphorylation was concentration-dependent with an EC50 of 4.2+/-0.1 pM. The maximal phosphorylation by CRF at 1 nM occurred by 5 min. The CRF-induced phosphorylation was inhibited by the protein tyrosine kinase inhibitors genistein and herbimycin A, suggesting that CRF activates protein tyrosine kinases. Treatment of cells with CRF receptor antagonist, but not pertussis toxin, prior to treatment with CRF inhibited the CRF-induced phosphorylation, suggesting it is mediated by the CRF receptor type 2beta that is not coupled to pertussis toxin-sensitive G-proteins. Treatment with 1,2-bis(2iminophenoxy)ethane-N,N,N',N'-tetraacetic acid attenuated the phospholipase C-gamma phosphorylation. In summary

  17. Magnetic fields promote a pro-survival non-capacitative Ca2+ entry via phospholipase C signaling.

    PubMed

    Cerella, Claudia; Cordisco, Sonia; Albertini, Maria Cristina; Accorsi, Augusto; Diederich, Marc; Ghibelli, Lina

    2011-03-01

    The ability of magnetic fields (MFs) to promote/increase Ca(2+) influx into cells is widely recognized, but the underlying mechanisms remain obscure. Here we analyze how static MFs of 6 mT modulates thapsigargin-induced Ca(2+) movements in non-excitable U937 monocytes, and how this relates to the anti-apoptotic effect of MFs. Magnetic fields do not affect thapsigargin-induced Ca(2+) mobilization from endoplasmic reticulum, but significantly increase the resulting Ca(2+) influx; this increase requires intracellular signal transduction actors including G protein, phospholipase C, diacylglycerol lipase and nitric oxide synthase, and behaves as a non-capacitative Ca(2+) entry (NCCE), a type of influx with an inherent signaling function, rather than a capacitative Ca(2+) entry (CCE). All treatments abrogating the extra Ca(2+) influx also abrogate the anti-apoptotic effect of MFs, demonstrating that MF-induced NCCE elicits an anti-apoptotic survival pathway. Copyright © 2010 Elsevier Ltd. All rights reserved.

  18. Non-specific phospholipase C1 affects silicon distribution and mechanical strength in stem nodes of rice.

    PubMed

    Cao, Huasheng; Zhuo, Lin; Su, Yuan; Sun, Linxiao; Wang, Xuemin

    2016-05-01

    Silicon, the second abundant element in the crust, is beneficial for plant growth, mechanical strength, and stress responses. Here we show that manipulation of the non-specific phospholipase C1, NPC1, alters silicon content in nodes and husks of rice (Oryza sativa). Silicon content in NPC1-overexpressing (OE) plants was decreased in nodes but increased in husks compared to wild-type, whereas RNAi suppression of NPC1 resulted in the opposite changes to those of NPC1-OE plants. NPC1 from rice hydrolyzed phospholipids and galactolipids to generate diacylglycerol that can be phosphorylated to phosphatidic acid. Phosphatidic acid interacts with Lsi6, a silicon transporter that is expressed at the highest level in nodes. In addition, the node cells of NPC1-OE plants have lower contents of cellulose and hemicellulose, and thinner sclerenchyma and vascular bundle fibre cells than wild-type plants; whereas NPC1-RNAi plants displayed the opposite changes. These data indicate that NPC1 modulates silicon distribution and secondary cell wall deposition in nodes and grains, affecting mechanical strength and seed shattering. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  19. Rhodopsin 5- and Rhodopsin 6-mediated clock synchronization in Drosophila melanogaster is independent of retinal phospholipase C-β signaling.

    PubMed

    Szular, Joanna; Sehadova, Hana; Gentile, Carla; Szabo, Gisela; Chou, Wen-Hai; Britt, Steven G; Stanewsky, Ralf

    2012-02-01

    Circadian clocks of most organisms are synchronized with the 24-hour solar day by the changes of light and dark. In Drosophila, both the visual photoreceptors in the compound eyes as well as the blue-light photoreceptor Cryptochrome expressed within the brain clock neurons contribute to this clock synchronization. A specialized photoreceptive structure located between the retina and the optic lobes, the Hofbauer-Buchner (H-B) eyelet, projects to the clock neurons in the brain and also participates in light synchronization. The compound eye photoreceptors and the H-B eyelet contain Rhodopsin photopigments, which activate the canonical invertebrate phototransduction cascade after being excited by light. We show here that 2 of the photopigments present in these photoreceptors, Rhodopsin 5 (Rh5) and Rhodopsin 6 (Rh6), contribute to light synchronization in a mutant (norpA(P41) ) that disrupts canonical phototransduction due to the absence of Phospholipase C-β (PLC-β). We reveal that norpA(P41) is a true loss-of-function allele, resulting in a truncated PLC-β protein that lacks the catalytic domain. Light reception mediated by Rh5 and Rh6 must therefore utilize either a different (nonretinal) PLC-β enzyme or alternative signaling mechanisms, at least in terms of clock-relevant photoreception. This novel signaling mode may distinguish Rhodopsin-mediated irradiance detection from image-forming vision in Drosophila.

  20. Optimization of the degumming process for camellia oil by the use of phospholipase C in pilot-scale system.

    PubMed

    Jiang, Xiaofei; Chang, Ming; Jin, Qingzhe; Wang, Xingguo

    2015-06-01

    In present study, phospholipase C (PLC) was applied in camellia oil degumming and the response surface method (RSM) was used to determine the optimum degumming conditions (reaction time, reaction temperature and enzyme dosage) for this enzyme. The optimum conditions for the minimum residual phosphorus content (15.14 mg/kg) and maximum yield of camellia oil (98.2 %) were obtained at reaction temperature 53 ºC, reaction time 2.2 h, PLC dosage 400 mg/kg and pH 5.4. The application of phospholipase A (PLA) - assisted degumming process could further reduce the residual phosphorus content of camellia oil (6.84 mg/kg) to make the oil suitable for physical refining while maintaining the maximal oil yield (98.2 %). These results indicate that PLC degumming process in combination with PLA treatment can be a commercially viable alternative for traditional degumming process. Study on the quality changes of degummed oils showed that the oxidative stability of camellia oil was slightly deceased after the enzymatic treatment, thus more attention should be paid to the oxidative stability in the further application.

  1. Spiraeoside inhibits mast cells activation and IgE-mediated allergic responses by suppressing phospholipase C-γ-mediated signaling.

    PubMed

    Kim, Jung Kuk; Seo, Young-Kyo; Park, Sehoon; Park, Soo-Ah; Lim, Seyoung; Lee, Susie; Kwon, Ohman; Seo, Jeong Kon; Choi, Ung-Kyu; Ryu, Sung Ho; Suh, Pann-Ghill

    2015-06-01

    Mast cells are responsible for IgE-mediated allergic responses through the secretion of various inflammatory cytokines and mediators. Therefore, the pharmacological regulation of mast cell activation is an important goal in the development of novel anti-allergic drugs. In this study, we found that spiraeoside (SP) inhibits mast cell activation and allergic responses in vivo. SP dose-dependently inhibited the degranulation induced by IgE-antigen (Ag) stimulation in RBL-2H3 mast cells without cytotoxic effects. At the molecular level, SP reduced the Ag-induced phosphorylation and subsequent activation of phospholipase C-γ2 (PLC-γ2). Moreover, SP inhibited the phosphorylation of spleen tyrosine kinase (Syk), linker for activation of T cells (LAT), and downstream MAPKs, such as ERK1/2, p38, and JNK, eventually attenuating expression of TNF-α and IL-4. Finally, we found that SP significantly inhibited IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Taken together, our results strongly suggest that SP suppresses IgE-mediated mast cell activation and allergic responses by inhibiting Lyn-induced PLC-γ2/MAPK signaling in mast cells.

  2. Clostridium perfringens phospholipase C induced ROS production and cytotoxicity require PKC, MEK1 and NFκB activation.

    PubMed

    Monturiol-Gross, Laura; Flores-Díaz, Marietta; Pineda-Padilla, Maria Jose; Castro-Castro, Ana Cristina; Alape-Giron, Alberto

    2014-01-01

    Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis.

  3. Clostridium perfringens Phospholipase C Induced ROS Production and Cytotoxicity Require PKC, MEK1 and NFκB Activation

    PubMed Central

    Monturiol-Gross, Laura; Flores-Díaz, Marietta; Pineda-Padilla, Maria Jose; Castro-Castro, Ana Cristina; Alape-Giron, Alberto

    2014-01-01

    Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis. PMID:24466113

  4. The affinities of human platelet and Acanthamoeba profilin isoforms for polyphosphoinositides account for their relative abilities to inhibit phospholipase C.

    PubMed Central

    Machesky, L M; Goldschmidt-Clermont, P J; Pollard, T D

    1990-01-01

    In light of recent work implicating profilin from human platelets as a possible regulator of both cytoskeletal dynamics and inositol phospholipid-mediated signaling, we have further characterized the interaction of platelet profilin and the two isoforms of Acanthamoeba profilin with inositol phospholipids. Profilin from human platelets binds to phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) with relatively high affinity (Kd approximately 1 microM for PIP2 by equilibrium gel filtration), but interacts only weakly (if at all) with phosphatidylinositol (PI) or inositol trisphosphate IP3) in small-zone gel-filtration assays. The two isoforms of Acanthamoeba profilin both have a lower affinity for PIP2 than does human platelet profilin, but the more basic profilin isoform from Acanthamoeba (profilin-II) has a much higher (approximately 10-microM Kd) affinity than the acidic isoform (profilin-I, 100 to 500-microM Kd). None of the profilins bind to phosphatidylserine (PS) or phosphatidylcholine (PC) in small-zone gel-filtration experiments. The differences in affinity for PIP2 parallel the ability of these three profilins to inhibit PIP2 hydrolysis by soluble phospholipase C (PLC). The results show that the interaction of profilins with PIP2 is specific with respect to both the lipid and the proteins. In Acanthamoeba, the two isoforms of profilin may have specialized functions on the basis of their identical (approximately 10 microM) affinities for actin monomers and different affinities for PIP2. PMID:1966040

  5. Role of biotransformation in the activation of rat hepatic phospholipase C by carbon tetrachloride and related haloalkanes

    SciTech Connect

    Coleman, J.B.

    1987-01-01

    CCl/sub 4/ exerts its hepatotoxicity through a reactive metabolite. Phospholipid degradation has been proposed as a mechanism by which CCl/sub 4/-induced alterations at the endoplasmic reticulum result in damage to organelles distant from it. Activation of the hepatic phospholipid degradative enzyme phospholipase C (PLC) occurs rapidly after CCl/sub 4/ exposure, yet the role of CCl/sub 4/ metabolites in this activation has been uncertain. 1000 g rat hepatocellular fractions exposed to CCl/sub 4/ exhibited time- and concentration-dependent increases in the conversion of membrane bound /sup 14/C-phosphatic acid into /sup 14/C-neutral lipid when /sup 14/C-glycerol-3-phosphate was incubated with the fraction in the presence of Ca2+, CoA, ATP, and palmitate. CCl/sub 4/-induced PLC activation in the presence of NADPH (when CCl/sub 4/ metabolism occurred) was 2-3 fold greater than in its absence at CCl/sub 4/ concentrations below 1 mM. The metabolism-dependent activation occurred subsequent to the plateau of CCl/sub 4/ metabolism, and was inhibited by metyrapone,whereas the metabolism-independent component was not.

  6. Cloning and characterization of the human phosphoinositide-specific phospholipase C-beta 1 (PLC beta 1).

    PubMed

    Caricasole, A; Sala, C; Roncarati, R; Formenti, E; Terstappen, G C

    2000-12-15

    Phospholipase C-beta (PLC beta) catalyses the generation of inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (IP(2)), a key step in the intracellular transduction of a large number of extracellular signals, including neurotransmitters and hormones modulating diverse developmental and functional aspects of the mammalian central nervous system. Four mammalian isozymes are known (PLC beta 1-4), which differ in their function and expression patterns in vivo. We have characterized the human PLC beta 1 genomic locus (PLC beta 1), cloned two distinct PLC beta 1 cDNAs (PLC beta 1a and b) and analysed their respective expression patterns in a comprehensive panel of human tissues using quantitative TaqMan technology. The two cDNAs derive from transcripts generated through alternative splicing at their 3' end, and are predicted to encode for PLC beta 1 isoforms differing at their carboxy-terminus. The human PLC beta 1 isoforms are co-expressed in the same tissues with a distinctly CNS-specific profile of expression. Quantitative differences in PLC beta 1 isoform expression levels are observed in some tissues. Transient expression of epitope-tagged versions of the two isoforms followed by immunofluorescence revealed localization of the proteins to the cytoplasm and the inner side of the cell membrane. Finally, we characterized the structure of the PLC beta 1 locus and confirmed its mapping to human chromosome 20.

  7. Phospholipase C from two bacterial strains acts differently on pure phospholipids and membrane bound glycosylphosphatidylinositol (GPI) anchors.

    PubMed

    Rastogi, Arshi; Hutchinson, Tarun E; Pereira, Ben M J

    2005-04-01

    Phospholipase C (PLC) was purified to homogeneity from the culture filtrate of Bacillus cereus (65-fold, 540 U/mg protein) and B. thuringiensis (76-fold, 306 U/mg protein) by conventional techniques of enzyme purification. The purified enzymes have the molecular mass of 34 kDa and 38 kDa respectively, as determined by SDS-PAGE. Both the PLCs exhibited identical sensitivity to pH, temperature, cations, anions and inhibitors like glutathione and p-chloromercuribenzoate. PLC-Bc showed a preference for phosphatidylinositol, while PLC-Bt favoured phosphatidylcholine as the substrate. Although both the enzymes were able to hydrolyze pure phosphatidylinositol, distinct differences were observed in their activity on phosphatidylinositol-anchored membrane proteins. PLC-Bc cleaved and released alkaline phosphatase, a GPI-anchored marker enzyme from microsomal membranes to a greater extent, than PLC-Bt. Experiments with sperm membranes, followed by SDS-PAGE revealed that the pattern of proteins released from their GPI-anchors by PLC-Bc and PLC-Bt were dissimilar. Although some proteins were cleaved in common by both PLCs, some others including a prominent 57 kDa protein were resistant to PLC-Bt, but sensitive to cleavage by PLC-Bc. The type of modification in the GPI anchor, special environment on membranes, and relative charge of host plasma membrane to the charge of PLC may be the factors that are responsible for the differential action of two enzymes.

  8. Phospholipase C-gamma1 potentiates integrin-dependent cell spreading and migration through Pyk2/paxillin activation.

    PubMed

    Choi, Jang Hyun; Yang, Yong-Ryoul; Lee, Seul Ki; Kim, Il-Shin; Ha, Sang Hoon; Kim, Eung-Kyun; Bae, Yun Soo; Ryu, Sung Ho; Suh, Pann-Ghill

    2007-08-01

    Phospholipase C-gamma1 (PLC-gamma1), which generates two second messengers, namely, inositol-1, 4, 5-trisphosphate and diacylglycerol, is implicated in growth factor-mediated chemotaxis. However, the exact role of PLC-gamma1 in integrin-mediated cell adhesion and migration remains poorly understood. In this study, we demonstrate that PLC-gamma1 is required for actin cytoskeletal organization and cell motility through the regulation of Pyk2 and paxillin activation. After fibronectin stimulation, PLC-gamma1 directly interacted with the cytoplasmic tail of integrin beta1. In PLC-gamma1-silenced cells, integrin-induced Pyk2 and paxillin phosphorylation were significantly reduced and PLC-gamma1 potentiated the integrin-induced Pyk2/paxillin activation in its enzymatic activity-dependent manner. In addition, specific knock-down of PLC-gamma1 resulted in a failure to form focal adhesions dependent on fibronectin stimulation, which appeared to be caused by the suppression of Pyk2 and paxillin phosphorylation. Interestingly, PLC-gamma1 potentiated the activations of Rac, thus integrin-induced lamellipodia formation was up-regulated. Consequently, the strength of cell-substratum interaction and cell motility were profoundly up-regulated by PLC-gamma1. Taken together, these results suggest that PLC-gamma1 is a key player in integrin-mediated cell spreading and motility achieved by the activation of Pyk2/paxillin/Rac signaling.

  9. The direct interaction of phospholipase C-gamma 1 with phospholipase D2 is important for epidermal growth factor signaling.

    PubMed

    Jang, Il Ho; Lee, Sukmook; Park, Jong Bae; Kim, Jong Hyun; Lee, Chang Sup; Hur, Eun-Mi; Kim, Il Shin; Kim, Kyong-Tai; Yagisawa, Hitoshi; Suh, Pann-Ghill; Ryu, Sung Ho

    2003-05-16

    The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-gamma1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a phospholipase complex composed of PLC-gamma1 and phospholipase D2 (PLD2). PLC-gamma1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and co-immunoprecipitation analysis in COS-7 cells show that the Src homology (SH) 3 domain of PLC-gamma1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-gamma1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol 1,4,5-trisphosphate (IP(3)) formation and Ca(2+) increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-gamma1 and PLD2 and fails to potentiate EGF-induced IP(3) formation and Ca(2+) increase. However, neither PLD2 wild type nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation of PLC-gamma1. These findings suggest that, upon EGF stimulation, PLC-gamma1 directly interacts with PLD2 and this interaction is important for PLC-gamma1 activity.

  10. Coincident regulation of PKCdelta in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling.

    PubMed

    Hall, Kellie J; Jones, Matthew L; Poole, Alastair W

    2007-09-15

    PKC (protein kinase C)d plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCd. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCd with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCd is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCd. Both thrombin and PMA induce recruitment of PKCd to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCd, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCd is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity.

  11. Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice.

    PubMed

    Bakala N'Goma, Jean Claude; Le Moigne, Vincent; Soismier, Nathalie; Laencina, Laura; Le Chevalier, Fabien; Roux, Anne-Laure; Poncin, Isabelle; Serveau-Avesque, Carole; Rottman, Martin; Gaillard, Jean-Louis; Etienne, Gilles; Brosch, Roland; Herrmann, Jean-Louis; Canaan, Stéphane; Girard-Misguich, Fabienne

    2015-02-01

    Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.

  12. Activation of phospholipase C in SH-SY5Y neuroblastoma cells by potassium-induced calcium entry.

    PubMed Central

    Smart, D.; Wandless, A.; Lambert, D. G.

    1995-01-01

    1. We used SH-SY5Y human neuroblastoma cells to investigate whether depolarization with high K+ could stimulate inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) formation and, if so, the mechanism involved. 2. Ins(1,4,5)P3 was measured by a specific radioreceptor mass assay, whilst [Ca2+]i was measured fluorimetrically with the Ca2+ indicator dye, Fura-2. 3. Depolarization with K+ caused a time- and dose-dependent increase in [Ca2+]i (peak at 27 s, EC50 of 50.0 +/- 9.0 mM) and Ins(1,4,5)P3 formation (peak at 30 s, EC50 of 47.4 +/- 1.1 mM). 4. Both the K(+)-induced Ins(1,4,5)P3 formation and increase in [Ca2+]i were inhibited dose-dependently by the L-type voltage-sensitive Ca2+ channel closer, (R+)-BayK8644, with IC50 values of 53.4 nM and 87.9 nM respectively. 5. These data show a close temporal and dose-response relationship between Ca2+ entry via L-type voltage-sensitive Ca2+ channels and Ins(1,4,5)P3 formation following depolarization with K+, indicating that Ca2+ influx can activate phospholipase C in SH-SY5Y cells. PMID:8528562

  13. S-phase induction and transformation of quiescent NIH 3T3 cells by microinjection of phospholipase C

    SciTech Connect

    Smith, M.R.; Ryu, Sungho; Suh, Panghill; Rhee, Suegoo; Kung, Hsiangfu )

    1989-05-01

    Two inositol phospholipid-specific phospholipase C (PLC) isozymes (PLC-I and -II) have been purified from bovine brain. When PLC-I or PLC-II was microinjected into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by ({sup 3}H)thymidine incorporation into nuclear DNA. In addition, {approx} 8 hr after PLC injection, NIH 3T3 fibroblasts appeared spindle-shaped, refractile, and highly vacuolated, displaying a morphology similar to transformed cells. The morphologic transformation was apparent for 26-30 hr after which the injected cells reverted back to a normal phenotype. Microinjected PLC at a high concentration was cytotoxic, dissolving the cytoplasmic membrane and leaving behind cellular ghosts. PLC is a key regulatory enzyme involved in cellular membrane signal transduction. Introduction of exogenous PLC into NIH 3T3 cells by microinjection induced a growth and oncogenic potential, as demonstrated by the ability of microinjected PLC to override the cellular G{sub 0} block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.

  14. S-phase induction and transformation of quiescent NIH 3T3 cells by microinjection of phospholipase C.

    PubMed

    Smith, M R; Ryu, S H; Suh, P G; Rhee, S G; Kung, H F

    1989-05-01

    Two inositol phospholipid-specific phospholipase C (PLC) isozymes (PLC-I and -II) have been purified from bovine brain. When PLC-I or PLC-II was microinjected (100-700 micrograms/ml) into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by [3H]thymidine incorporation into nuclear DNA. In addition, approximately to 8 hr after PLC injection, NIH 3T3 fibroblasts appeared spindle-shaped, refractile, and highly vacuolated, displaying a morphology similar to transformed cells. The morphologic transformation was apparent for 26-30 hr after which the injected cells reverted back to a normal phenotype. Microinjected PLC at a high concentration (1 mg/ml) was cytotoxic, dissolving the cytoplasmic membrane and leaving behind cellular ghosts. PLC is a key regulatory enzyme involved in cellular membrane signal transduction. Introduction of exogenous PLC into NIH 3T3 cells by microinjection induced a growth and oncogenic potential, as demonstrated by the ability of microinjected PLC (approximately 10,000 molecules per cell) to override the cellular G0 block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.

  15. Expression Analysis of a Stress-Related Phosphoinositide-Specific Phospholipase C Gene in Wheat (Triticum aestivum L.)

    PubMed Central

    Wu, Lizhu; Hou, Mingyu; Dou, Shijuan; Pan, Yanyun

    2014-01-01

    Plant phosphoinositide-specific phospholipases C (PI-PLCs) function in several essential plant processes associated with either development or environmental stress. In this report, we examined the expression patterns of TaPLC1 under drought and high salinity stress at the transcriptional and post-transcriptional levels. TaPLC1 mRNA was expressed in all wheat organs examined. U73122 and edelfosine, the PLC inhibitor, impaired seedling growth and enhanced seedling sensitivity to drought and high salinity stress. Though TaPLC1 expression in wheat was lowest at the seedling stage, it was strongly induced under conditions of stress. When 6-day-old wheat seedlings were treated with 200 mM NaCl or 20% (w/v) PEG 6000 for 6 or 12 h, respectively, the TaPLC1 transcript level increased by 16-fold compared to the control. Western blotting showed that the TaPLC protein concentration was also maintained at a high level from 24 to 48 h during stress treatment. Together, our results indicate the possible biological functions of TaPLC1 in regulating seedling growth and the response to drought and salinity stress. PMID:25121594

  16. Two isoforms of eukaryotic phospholipase C in Paramecium affecting transport and release of GPI-anchored proteins in vivo.

    PubMed

    Klöppel, Christine; Müller, Alexandra; Marker, Simone; Simon, Martin

    2009-10-01

    Surface proteins anchored by a glycosylphosphatidylinositol (GPI) residue in the cell membrane are widely distributed among eukaryotic cells. The GPI anchor is cleavable by a phospholipase C (PLC) leading to the release of such surface proteins, and this process is postulated to be essential in several systems. For higher eukaryotes, the responsible enzymes have not been characterized in any detail as yet. Here we characterize six PLCs in the ciliated protozoan, Paramecium, which, in terms of catalytic domains and architecture, all show characteristics of PLCs involved in signal transduction in higher eukaryotes. We show that some of these endogenous PLCs can release GPI-anchored surface proteins in vitro: using RNA(i) to reduce PLC expression results in the same effects as the application of PLC inhibitors. With two enzymes, PLC2 and PLC6, RNA(i) phenotypes show strong defects in release of GPI-anchored surface proteins in vivo. Moreover, these RNA(i) lines also show abnormal surface protein distribution, suggesting that GPI cleavage may influence trafficking of anchored proteins. As we find GFP fusion proteins in the cytosol and in the surface protein extracts, these PLCs obviously show unconventional translocation mechanisms. This is the first molecular data on endogenous Paramecium PLCs with the described properties affecting GPI anchors in vitro and in vivo.

  17. End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

    PubMed Central

    Ibarguren, Maitane; Bomans, Paul H. H.; Frederik, Peter M.; Stonehouse, Martin; Vasil, Michael L.; Alonso, Alicia; Goñi, Félix M.

    2009-01-01

    A phospholipase C/ sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol, at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230–3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme. PMID:19891956

  18. Does advancing male age influence the expression levels and localisation patterns of phospholipase C zeta (PLCζ) in human sperm?

    PubMed Central

    Yeste, Marc; Jones, Celine; Amdani, Siti Nornadhirah; Yelumalai, Suseela; Mounce, Ginny; da Silva, Sarah J. Martins; Child, Tim; Coward, Kevin

    2016-01-01

    Socio-economic factors have led to an increasing trend for couples to delay parenthood. However, advancing age exerts detrimental effects upon gametes which can have serious consequences upon embryo viability. While such effects are well documented for the oocyte, relatively little is known with regard to the sperm. One fundamental role of sperm is to activate the oocyte at fertilisation, a process initiated by phospholipase C zeta (PLCζ), a sperm-specific protein. While PLCζ deficiency can lead to oocyte activation deficiency and infertility, it is currently unknown whether the expression or function of PLCζ is compromised by advancing male age. Here, we evaluate sperm motility and the proportion of sperm expressing PLCζ in 71 males (22–54 years; 44 fertile controls and 27 infertile patients), along with total levels and localisation patterns of PLCζ within the sperm head. Three different statistical approaches were deployed with male age considered both as a categorical and a continuous factor. While progressive motility was negatively correlated with male age, all three statistical models concurred that no PLCζ–related parameter was associated with male age, suggesting that advancing male age is unlikely to cause problems in terms of the sperm’s fundamental ability to activate an oocyte. PMID:27270687

  19. Dephosphorylation of the adaptor LAT and phospholipase C-γ by SHP-1 inhibits natural killer cell cytotoxicity.

    PubMed

    Matalon, Omri; Fried, Sophia; Ben-Shmuel, Aviad; Pauker, Maor H; Joseph, Noah; Keizer, Danielle; Piterburg, Marina; Barda-Saad, Mira

    2016-05-24

    Natural killer (NK) cells discriminate between healthy cells and virally infected or transformed self-cells by tuning activating and inhibitory signals received through cell surface receptors. Inhibitory receptors inhibit NK cell function by recruiting and activating the tyrosine phosphatase Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1) to the plasma membrane. However, to date, the guanine nucleotide exchange factor VAV1 is the only direct SHP-1 substrate identified in NK cells. We reveal that the adaptor protein linker for activation of T cells (LAT) as well as phospholipase C-γ1 (PLC-γ1) and PLC-γ2 are SHP-1 substrates. Dephosphorylation of Tyr(132) in LAT by SHP-1 in NK cells abrogated the recruitment of PLC-γ1 and PLC-γ2 to the immunological synapse between the NK cell and a cancer cell target, which reduced NK cell degranulation and target cell killing. Furthermore, the ubiquitylation of LAT by the E3 ubiquitin ligases c-Cbl and Cbl-b, which was induced by LAT phosphorylation, led to the degradation of LAT in response to the engagement of inhibitory receptors on NK cells, which abrogated NK cell cytotoxicity. Knockdown of the Cbl proteins blocked LAT ubiquitylation, which promoted NK cell function. Expression of a ubiquitylation-resistant mutant LAT blocked inhibitory receptor signaling, enabling cells to become activated. Together, these data identify previously uncharacterized SHP-1 substrates and inhibitory mechanisms that determine the response of NK cells.

  20. Tyrosine phosphorylation is involved in receptor coupling to phospholipase D but not phospholipase C in the human neutrophil.

    PubMed Central

    Uings, I J; Thompson, N T; Randall, R W; Spacey, G D; Bonser, R W; Hudson, A T; Garland, L G

    1992-01-01

    The tyrosine kinase inhibitors ST271, ST638 and erbstatin inhibited phospholipase D (PLD) activity in human neutrophils stimulated by fMet-Leu-Phe, platelet-activating factor and leukotriene B4. These compounds did not inhibit phorbol ester-stimulated PLD, indicating that they do not inhibit PLD per se, but probably act at a site between the receptor and the phospholipase. In contrast, the protein kinase C inhibitor Ro-31-8220 inhibited phorbol 12,13-dibutyrate- but not fMet-Leu-Phe-stimulated PLD activity, arguing against the involvement of protein kinase C in the receptor-mediated activation of PLD. ST271 did not inhibit Ins(1,4,5)P3 generation, but did inhibit protein tyrosine phosphorylation stimulated by fMet-Leu-Phe. The phosphotyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation and stimulated PLD. These results suggest that tyrosine kinase activity is involved in receptor coupling to PLD but not to PtdIns(4,5)P2-specific phospholipase C in the human neutrophil. Images Fig. 3. PMID:1371383

  1. End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa.

    PubMed

    Ibarguren, Maitane; Bomans, Paul H H; Frederik, Peter M; Stonehouse, Martin; Vasil, Adriana I; Vasil, Michael L; Alonso, Alicia; Goñi, Félix M

    2010-01-01

    A phospholipase C/sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties; the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230-3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme.

  2. Calcineurin phosphatase and phospholipase C are required for developmental and pathological functions in the citrus fungal pathogen Alternaria alternata.

    PubMed

    Tsai, Hsieh-Chin; Chung, Kuang-Ren

    2014-07-01

    Excessive Ca(2+) or compounds interfering with phosphoinositide cycling have been found to inhibit the growth of the tangerine pathotype of Alternaria alternata, suggesting a crucial role of Ca(2+) homeostasis in this pathotype. The roles of PLC1, a phospholipase C-coding gene and CAL1, a calcineurin phosphatase-coding gene were investigated. Targeted gene disruption showed that both PLC1 and CAL1 were required for vegetative growth, conidial formation and pathogenesis in citrus. Fungal strains lacking PLC1 or CAL1 exhibited extremely slow growth and induced small lesions on calamondin leaves. Δplc1 mutants produced fewer conidia, which germinated at slower rates than wild-type. Δcal1 mutants produced abnormal hyphae and failed to produce any mature conidia, but instead produced highly melanized bulbous hyphae with distinct septae. Fluorescence microscopy using Fluo-3 dye as a Ca(2+) indicator revealed that the Δplc1 mutant hyphae emitted stronger cytosolic fluorescence, and the Δcal1 mutant hyphae emitted less cytosolic fluorescence, than those of wild-type. Infection assessed on detached calamondin leaves revealed that application of CaCl2 or neomycin 24 h prior to inoculation provided protection against Alt. alternata. These data indicate that a dynamic equilibrium of cellular Ca(2+) is critical for developmental and pathological processes of Alt. alternata. © 2014 The Authors.

  3. Distinct Phospholipase C-β Isozymes Mediate Lysophosphatidic Acid Receptor 1 Effects on Intestinal Epithelial Homeostasis and Wound Closure

    PubMed Central

    Lee, Sei-Jung; Leoni, Giovanna; Neumann, Philipp-Alexander; Chun, Jerold; Nusrat, Asma

    2013-01-01

    Maintenance of the epithelial barrier in the intestinal tract is necessary to protect the host from the hostile luminal environment. Phospholipase C-β (PLC-β) has been implicated to control myriad signaling cascades. However, the biological effects of selective PLC-β isozymes are poorly understood. We describe novel findings that lysophosphatidic acid (LPA) regulates PLC-β1 and PLC-β2 via two distinct pathways to enhance intestinal epithelial cell (IEC) proliferation and migration that facilitate wound closure and recovery of the intestinal epithelial barrier. LPA acting on the LPA1 receptor promotes IEC migration by facilitating the interaction of Gαq with PLC-β2. LPA-induced cell proliferation is PLC-β1 dependent and involves translocation of Gαq to the nucleus, where it interacts with PLC-β1 to induce cell cycle progression. An in vivo study using LPA1-deficient mice (Lpar1−/−) shows a decreased number of proliferating IECs and migration along the crypt-luminal axis. Additionally, LPA enhances migration and proliferation of IECs in an LPA1-dependent manner, and Lpar1−/− mice display defective mucosal wound repair that requires cell proliferation and migration. These findings delineate novel LPA1-dependent lipid signaling that facilitates mucosal wound repair via spatial targeting of distinct PLC-βs within the cell. PMID:23478264

  4. Phospholipase C regulatory mutation of Pseudomonas aeruginosa that results in constitutive synthesis of several phosphate-repressible proteins.

    PubMed Central

    Gray, G L; Berka, R M; Vasil, M L

    1982-01-01

    We describe here a new mutant of Pseudomonas aeruginosa PAO, strain D10C (genotype plcB), which produces phospholipase C and alkaline phosphatase constitutively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracellular proteins produced by this mutant in high- and low-Pi media revealed that the mutation resulted in a marked deficiency of one major Pi-regulated protein of 41,000 molecular weight and constitutive synthesis of all other major extracellular Pi-regulated proteins. Furthermore, the plcB mutant was deficient in phosphate transport. A plcA mutation, which also led to a loss of the 41,000-molecular-weight protein, was similarly deficient in Pi transport. The genetic loci, plcA and plcB, located at 22 to 23 min on the PAO chromosome, were indistinguishable by conjugational and transductional mapping, and may therefore be in the same gene or in a cluster of genes which regulate the synthesis of Pi-repressible proteins. Images PMID:6804440

  5. The involvement of phospholipases C and D in the asymmetric division of subsidiary cell mother cells of Zea mays.

    PubMed

    Apostolakos, Panagiotis; Panteris, Emmanuel; Galatis, Basil

    2008-11-01

    In the present study, the involvement of phospholipase C and D (PLC and PLD) pathways in the asymmetric divisions that produce the stomatal complexes of Zea mays was investigated. In particular, the polar organization of microtubules (MTs) and actin filaments (AFs) and the process of asymmetric division were studied in subsidiary cell mother cells (SMCs) treated with PLC and PLD modulators. In SMCs treated with butanol-1 (but-1), which blocks phosphatidic acid (PA) production via PLDs, AF-patch formation laterally to the inducing guard cell mother cell (GMC) and the subsequent asymmetric division were inhibited. In these SMCs, cell division plane determination, as expressed by MT preprophase band (MT-PPB) formation, was not disturbed. Exogenously applied PA partially relieved the but-1 effects on SMCs. In contrast to SMCs, but-1 did not affect the symmetric GMC division. Inhibition of the PLC catalytic activity by neomycin or U73122 resulted in inhibition of asymmetric SMC division, while AF-patch and MT-PPB were organized as in control SMCs. These data show that the PLC and PLD signaling pathways are involved in the transduction and/or perception of the inductive stimulus that is emitted by the GMCs and induces the polar AF organization and asymmetric SMC division. In contrast, division plane determination in SMCs, as expressed by MT-PPB formation, does not depend on PLC and PLD signaling pathways.

  6. Tyrosine kinase activity is essential for the association of phospholipase C-gamma with the epidermal growth factor receptor.

    PubMed Central

    Margolis, B; Bellot, F; Honegger, A M; Ullrich, A; Schlessinger, J; Zilberstein, A

    1990-01-01

    Epidermal growth factor (EGF) treatment of NIH 3T3 cells transfected with wild-type EGF receptor induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). The EGF receptor and PLC-gamma were found to be physically associated such that antibodies directed against PLC-gamma or the EGF receptor coimmunoprecipitated both proteins. The association between PLC-gamma and wild-type EGF receptor was dependent on the concentration of EGF, but EGF did not enhance the association between PLC-gamma and a kinase-negative mutant of the EGF receptor. Oligomerization of the EGF receptor was not sufficient to induce association of the EGF receptor with PLC-gamma, since the kinase-negative mutant receptor underwent normal dimerization in response to EGF yet did not associate with PLC-gamma. The form of PLC-gamma associated with the EGF receptor appeared to be primarily the non-tyrosine-phosphorylated form. It is concluded that the kinase activity of the EGF receptor is essential for association of PLC-gamma with the EGF receptor, possibly by stimulating receptor autophosphorylation. Images PMID:2153914

  7. Phospholipase C not protein kinase C is required for the activation of TRPC5 channels by cholecystokinin.

    PubMed

    Grisanti, Laurel A; Kurada, Lalitha; Cilz, Nicholas I; Porter, James E; Lei, Saobo

    2012-08-15

    Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to G(q/11) proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca(2+). The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP(3) receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca(2+) stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.

  8. Enhanced phospholipase C-gamma1 activity produced by association of independently expressed X and Y domain polypeptides.

    PubMed Central

    Horstman, D A; DeStefano, K; Carpenter, G

    1996-01-01

    The X and Y domains of phospholipase C (PLC)-gamma1, which are conserved in all mammalian phosphoinositide-specific PLC isoforms and are proposed to interact to form the catalytic site, have been expressed as individual hexahistidine-tagged fusion proteins in the baculovirus system. Following coinfection of insect cells with recombinant viruses, association of X and Y polypeptides was demonstrated in coprecipitation assays. When enzyme activity was examined, neither domain possessed catalytic activity when expressed alone; however, coexpression of the X and Y polypeptides produced a functional enzyme. This reconstituted phospholipase activity remained completely dependent on the presence of free Ca2+. The specific activity of the X:Y complex was significantly greater (20- to 100-fold) than that of holoPLC-gamma1 and was only moderately influenced by varying the concentration of substrate. The enzyme activities of holoPLC-gamma1 and the X:Y complex exhibited distinct pH optima. For holoPLC-gamma1 maximal activity was detected at pH 5.0, while activity of the X:Y complex was maximal at pH 7.2. Images Fig. 1 PMID:8755506

  9. GABAB1 and GABAB2 receptor subunits co-expressed in cultured human RPE cells regulate intracellular Ca2+ via Gi/o-protein and phospholipase C pathways.

    PubMed

    Cheng, Z-Y; Wang, X-P; Schmid, K L; Han, X-G

    2014-11-07

    GABAB receptors associate with Gi/o-proteins that regulate voltage-gated Ca(2+) channels and thus the intracellular Ca(2+) concentration ([Ca(2+)]i), there is also reported cross-regulation of phospholipase C. These associations have been studied extensively in the brain and also shown to occur in non-neural cells (e.g. human airway smooth muscle). More recently GABAB receptors have been observed in chick retinal pigment epithelium (RPE). The aims were to investigate whether the GABAB receptor subunits, GABAB1 and GABAB2, are co-expressed in cultured human RPE cells, and then determine if the GABAB receptor similarly regulates the [Ca(2+)]i of RPE cells and if phospholipase C is involved. Human RPE cells were cultured from five donor eye cups. Evidence for GABAB1 and GABAB2 mRNAs and proteins in the RPE cell cultures was investigated using real time polymerase chain reaction, western blots and immunofluorescence. The effects of the GABAB receptor agonist baclofen, antagonist CGP46381, a Gi/o-protein inhibitor pertussis toxin, and the phospholipase C inhibitor U73122 on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo-3. Both GABAB1 and GABAB2 mRNA and protein were identified in cell cultures of human RPE; antibody staining was co-localized to the cell membrane and cytoplasm. One-hundred micromolars of baclofen caused a transient increase in the [Ca(2+)]i of RPE cells regardless of whether Ca(2+) was added to the buffer. Baclofen-induced increases in the [Ca(2+)]i were attenuated by pre-treatment with CGP46381, pertussis toxin, and U73122. GABAB1 and GABAB2 are co-expressed in cell cultures of human RPE. GABAB receptors in RPE regulate the [Ca(2+)]i via a Gi/o-protein and phospholipase C pathway. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. Subtype-specific roles of phospholipase C-β via differential interactions with PDZ domain proteins.

    PubMed

    Kim, Jung Kuk; Lim, Seyoung; Kim, Jinho; Kim, Sanguk; Kim, Jae Ho; Ryu, Sung Ho; Suh, Pann-Ghill

    2011-01-01

    Since we first identified the PLC-β isozyme, enormous studies have been conducted to investigate the functional roles of this protein (Min et al., 1993; Suh et al.,1988). It is now well-known that the four PLC-β subtypes are major effector molecules in GPCR-mediated signaling, especially for intracellular Ca2+ signaling. Nonetheless, it is still poorly understood why multiple PLC-β subtype exist. Most cells express multiple subtypes of PLC-β in different combinations, and each subtype is involved in somewhat different signaling pathways. Therefore, studying the differential roles of each PLC-β subtype is a very interesting issue. In this regard, we focus here on PDZ domain proteins which are novel PLC-β interacting proteins. As scaffolders, PDZ domain proteins recruit various target proteins ranging from membrane receptors to cytoskeletal proteins to assemble highly organized signaling complexes; this can give rise to efficiency and diversity in cellular signaling. Because PLC-β subtypes have different PDZ-binding motifs, it is possible that they are engaged with different PDZ domain proteins, and in turn participate in distinct physiological responses. To date, several PDZ domain proteins, such as the NHERF family, Shank2, and Par-3, have been reported to selectively interact with certain PLC-β subtypes and GPCRs. Systematic predictions of potential binding partners also suggests differential binding properties between PLC-β subtypes. Furthermore, we elucidated parallel signaling processes for multiple PLC-β subtypes, which still perform distinct functions resulting from differential interactions with PDZ domain proteins within a single cell. Therefore, these results highlight the novel function of PDZ domain proteins as intermediaries in subtype-specific role of PLC-β in GPCR-mediated signaling. Future studies will focus on the physiological meanings of this signaling complex formation by different PDZ domain proteins and PLC-β subtypes. It has been

  11. Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C.

    PubMed

    Su, Lingqia; Jiang, Qi; Yu, Lingang; Wu, Jing

    2017-02-08

    Our laboratory has reported a strategy for improving the extracellular production of recombinant proteins through co-expression with Thermobifida fusca cutinase, which increases membrane permeability via its phospholipid hydrolysis activity. However, the foam generated by the lysophospholipid product makes the fermentation process difficult to control in a fermentor. Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce sn1,2-diacylglycerides and organic phosphate, which do not induce foam formation. Therefore, co-expression with Bacillus cereus PLC was investigated as a method to improve the extracellular production of recombinant proteins. When B. cereus PLC was expressed in Escherichia coli without its signal peptide, 95.3% of the total PLC activity was detected in the culture supernatant. PLC expression enhanced membrane permeability without obvious cell lysis. Then, six test enzymes, three secretory and three cytosolic, were co-expressed with B. cereus PLC. The enhancement of extracellular production correlated strongly with the molecular mass of the test enzyme. Extracellular production of Streptomyces sp. FA1 xylanase (43 kDa), which had the lowest molecular mass among the secretory enzymes, was 4.0-fold that of its individual expression control. Extracellular production of glutamate decarboxylase (51 kDa), which had the lowest molecular mass among the cytosolic enzymes, reached 26.7 U/mL; 88.3% of the total activity produced. This strategy was effectively scaled up using a 3-L fermentor. No obvious foam was generated during this fermentation process. This is the first study to detail the enhanced extracellular production of recombinant proteins through co-expression with PLC. This new strategy, which is especially appropriate for lower molecular mass proteins, allows large-scale protein production in an easily controlled fermentation process.

  12. Phospholipase C epsilon 1 (PLCE1) Haplotypes are Associated with Increased Risk of Gastric Cancer in Kashmir Valley

    PubMed Central

    Malik, Manzoor A.; Srivastava, Priya; Zargar, Showkat A.; Mittal, Balraj

    2014-01-01

    Background/Aim: Phospholipase C epsilon 1 (PLCE1) plays a crucial role in carcinogenesis and progression of several types of cancers. A single nucleotide polymorphism (SNP, rs2274223) in PLCE1 has been identified as a novel susceptibility locus. The aim of the present study was to investigate the role of three potentially functional SNPs (rs2274223A > G, rs3765524C > T, and rs7922612C > T) of PLCE1 in gastric cancer patients from Kashmir Valley. Patients and Methods: The study was conducted in 108 GC cases and 195 healthy controls from Kashmir Valley. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism method. Data were statistically analyzed using χ2 test and logistic regression models. A P value of less than 0.05 was regarded as statistically significant. Results: The frequency of PLCE1 A2274223C3765524T7922612, G2274223C3765524T7922612, and G2274223T3765524C7922612 haplotypes were higher in patients compared with controls, conferred high risk for GC [odds ratio (OR) =6.29; P = 0.001; Pcorr = 0.003], (OR = 3.23; P = 0.011; Pcorr = 0.033), and (OR = 5.14; P = 0.011; Pcorr = 0.033), respectively. Smoking and salted tea are independent risk factors for GC, but we did not find any significant modulation of cancer risk by PLCE1 variants with smoking or excessive consumption of salted tea. Conclusion: These results suggest that variation in PLCE1 may be associated with GC risk in Kashmir Valley. PMID:25434319

  13. Phospholipase C-gamma 1 can induce DNA synthesis by a mechanism independent of its lipase activity.

    PubMed Central

    Smith, M R; Liu, Y L; Matthews, N T; Rhee, S G; Sung, W K; Kung, H F

    1994-01-01

    Inositol phospholipid-specific phospholipase C (PLC) is involved in several signaling pathways leading to cellular growth and differentiation. Our previous studies reported the induction of DNA synthesis in quiescent NIH 3T3 cells after microinjection of PLC and the inhibition of serum- or Ras-stimulated DNA synthesis by a mixture of monoclonal antibodies to PLC-gamma 1. In the course of our investigation of anti-PLC-gamma 1 monoclonal antibodies, we found that each antibody exerts different inhibitory effects on the phosphatidylinositol-hydrolyzing activity of PLC-gamma 1 and that the inhibition of enzymatic activity does not correlate with the inhibition of DNA synthesis observed in the microinjection assay. PLC-gamma 1 with defective enzymatic activity was synthesized by substituting phenylalanine for histidine within the PLC-gamma 1 catalytic domain at amino acids 335 and 380, and mutant enzymes were expressed using a vaccinia expression system. The mutant enzymes were purified and microinjected into quiescent NIH 3T3 cells to evaluate their mitogenic activity. A moderate induction of DNA synthesis occurred after injection of mutant PLC-gamma 1. This mitogenic activity was inhibited by an antibody (alpha E 8-4) that does not significantly inhibit PLC-gamma 1 enzyme activity, which indicates that something else has to be inhibited. Furthermore, the partial induction of DNA synthesis observed with mutant PLC-gamma 1 was increased to levels seen with wild-type PLC-gamma 1 by coinjection of mutant PLC-gamma 1 with two second messengers, diacylglycerol and inositol trisphosphate. These results suggest that the mitogenic activity of PLC-gamma 1 does not exclusively result from the enzymatic activity of the lipase and that another activity inherent to the PLC-gamma 1 molecule can also induce DNA synthesis in quiescent cells. Images PMID:8022819

  14. Regulation of lipid raft proteins by glimepiride- and insulin-induced glycosylphosphatidylinositol-specific phospholipase C in rat adipocytes.

    PubMed

    Müller, Günter; Schulz, Andrea; Wied, Susanne; Frick, Wendelin

    2005-03-01

    The insulin receptor-independent insulin-mimetic signalling provoked by the antidiabetic sulfonylurea drug, glimepiride, is accompanied by the redistribution and concomitant activation of lipid raft-associated signalling components, such as the acylated tyrosine kinase, pp59(Lyn), and some glycosylphosphatidylinositol-anchored proteins (GPI-proteins). We now found that impairment of glimepiride-induced lipolytic cleavage of GPI-proteins in rat adipocytes by the novel inhibitor of glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), GPI-2350, caused almost complete blockade of (i) dissociation from caveolin-1 of pp59(Lyn) and GPI-proteins, (ii) their redistribution from high cholesterol- (hcDIGs) to low cholesterol-containing (lcDIGs) lipid rafts, (iii) tyrosine phosphorylation of pp59(Lyn) and insulin receptor substrate-1 protein (IRS-1) and (iv) stimulation of glucose transport as well as (v) inhibition of isoproterenol-induced lipolysis in response to glimepiride. In contrast, blockade of the moderate insulin activation of the GPI-PLC and of lipid raft protein redistribution by GPI-2350 slightly reduced insulin signalling and metabolic action, only. Importantly, in response to both insulin and glimepiride, lipolytically cleaved hydrophilic GPI-proteins remain associated with hcDIGs rather than redistribute to lcDIGs as do their uncleaved amphiphilic versions. In conclusion, GPI-PLC controls the localization within lipid rafts and thereby the activity of certain GPI-anchored and acylated signalling proteins. Its stimulation is required and may even be sufficient for insulin-mimetic cross-talking to IRS-1 in response to glimepiride via redistributed and activated pp59(Lyn).

  15. Structural characterization of the split pleckstrin homology domain in phospholipase C-gamma1 and its interaction with TRPC3.

    PubMed

    Wen, Wenyu; Yan, Jing; Zhang, Mingjie

    2006-04-28

    Phospholipase C (PLC)-gamma is unique among the PLC enzymes because each PLC-gamma isozyme contains a split pleckstrin homology (PH) domain with an SH2SH2SH3 tandem repeat insertion (where SH indicates Src homology domain) in the middle of its sequence. Split PH domains exist in a number of other proteins that play crucial signaling roles. However, little is known about the structure and function of split PH domains. The C-terminal half of the PLC-gamma split PH domain has been implicated to interact directly with the TRPC3 calcium channel, thereby providing a direct coupling mechanism between PLC-gamma and agonist-induced calcium entry. However, this interaction has not been proved by direct biochemical or structural studies. Here we determined the three-dimensional structure of the split PH domain of PLC-gamma1, and we found that the split PH domain of the enzyme folds into a canonical PH domain fold with high thermostability. The SH2SH2SH3 insertion between the beta3 and beta4 strands does not change the structure of the split PH domain. In contrast to the majority of phospholipid-binding PH domains, the PLC-gamma1 split PH domain lacks the signature lipid-binding motif located between the beta1 and beta2 strands. Consistent with this structural feature, the split PH domain of PLC-gamma1 does not bind to phospholipids. Multiple biochemical and biophysical experiments have argued against a direct interaction between TRPC3 and the C-terminal half of the PLC-gamma1 split PH domain. Our data pointed to the existence of a yet to be elucidated interaction mechanism between TRPC3 and PLC-gamma1.

  16. Dual role of SLP-76 in mediating T cell receptor-induced activation of phospholipase C-gamma1.

    PubMed

    Beach, Dvora; Gonen, Ronnie; Bogin, Yaron; Reischl, Ilona G; Yablonski, Deborah

    2007-02-02

    Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.

  17. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

    PubMed

    Luangwedchakarn, Voravich; Day, Noorbibi K; Hitchcock, Remi; Brown, Pam G; Lerner, Danica L; Rucker, Rajivi P; Cianciolo, George J; Good, Robert A; Haraguchi, Soichi

    2003-05-01

    CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

  18. Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling.

    PubMed

    Kim, Jung Kuk; Choi, Jung Woong; Lim, Seyoung; Kwon, Ohman; Seo, Jeong Kon; Ryu, Sung Ho; Suh, Pann-Ghill

    2011-06-01

    Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca(2+) signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca(2+) source is enough for PLC-η1 activation. Furthermore, the IP(3) receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca(2+) mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca(2+) mobilization from the ER, and therefore amplifies GPCR-mediated signaling.

  19. Phospholipase C of Cryptococcus neoformans Regulates Homeostasis and Virulence by Providing Inositol Trisphosphate as a Substrate for Arg1 Kinase

    PubMed Central

    Lev, Sophie; Desmarini, Desmarini; Li, Cecilia; Chayakulkeeree, Methee; Traven, Ana; Sorrell, Tania C.

    2013-01-01

    Phospholipase C (PLC) of Cryptococcus neoformans (CnPlc1) is crucial for virulence of this fungal pathogen. To investigate the mechanism of CnPlc1-mediated signaling, we established that phosphatidylinositol 4,5-bisphosphate (PIP2) is a major CnPlc1 substrate, which is hydrolyzed to produce inositol trisphosphate (IP3). In Saccharomyces cerevisiae, Plc1-derived IP3 is a substrate for the inositol polyphosphate kinase Arg82, which converts IP3 to more complex inositol polyphosphates. In this study, we show that in C. neoformans, the enzyme encoded by ARG1 is the major IP3 kinase, and we further demonstrate that catalytic activity of Arg1 is essential for cellular homeostasis and virulence in the Galleria mellonella infection model. IP3 content was reduced in the CnΔplc1 mutant and markedly increased in the CnΔarg1 mutant, while PIP2 was increased in both mutants. The CnΔplc1 and CnΔarg1 mutants shared significant phenotypic similarity, including impaired thermotolerance, compromised cell walls, reduced capsule production and melanization, defective cell separation, and the inability to form mating filaments. In contrast to the S. cerevisiae ARG82 deletion mutant (ScΔarg82) strain, the CnΔarg1 mutant exhibited dramatically enlarged vacuoles indicative of excessive vacuolar fusion. In mammalian cells, PLC-derived IP3 causes Ca2+ release and calcineurin activation. Our data show that, unlike mammalian PLCs, CnPlc1 does not contribute significantly to calcineurin activation. Collectively, our findings provide the first evidence that the inositol polyphosphate anabolic pathway is essential for virulence of C. neoformans and further show that production of IP3 as a precursor for synthesis of more complex inositol polyphosphates is the key biochemical function of CnPlc1. PMID:23381992

  20. Calcium Signaling via Phospholipase C Is Essential for Proline Accumulation upon Ionic But Not Nonionic Hyperosmotic Stresses in Arabidopsis1

    PubMed Central

    Parre, Elodie; Ghars, Mohamed Ali; Leprince, Anne-Sophie; Thiery, Laurent; Lefebvre, Delphine; Bordenave, Marianne; Richard, Luc; Mazars, Christian; Abdelly, Chedly; Savouré, Arnould

    2007-01-01

    Proline (Pro) accumulation occurs in various plant organisms in response to environmental stresses. To identify the signaling components involved in the regulation of Pro metabolism upon water stress in Arabidopsis (Arabidopsis thaliana), a pharmacological approach was developed. The role of phosphoinositide-specific phospholipases C (PLCs) in Pro accumulation was assessed by the use of the aminosteroid U73122, a commonly employed specific inhibitor of receptor-mediated PLCs. We found that U73122 reduced pyrroline-5-carboxylate synthetase transcript and protein as well as Pro levels in salt-treated seedlings. Inhibition of PLC activity by U73122 was quantified by measuring the decrease of inositol 1,4,5-trisphosphate (InsP3) levels. Moreover, the utilization of diacylglycerol kinase and InsP3-gated calcium release receptor inhibitors suggested that InsP3 or its derivatives are essential for Pro accumulation upon salt stress, involving calcium as a second messenger in ionic stress signaling. This observation was further supported by a partial restoration of Pro accumulation in salt- and U73122-treated seedlings after addition of extracellular calcium, or when calcium homeostasis was perturbed by cyclopiazonic acid, a blocker of plant type IIA calcium pumps. Taken together, our data indicate that PLC-based signaling is a committed step in Pro biosynthesis upon salinity but not in the case of mannitol stress. Calcium acts as a molecular switch to trigger downstream signaling events. These results also demonstrated the specific involvement of lipid signaling pathway to discriminate between ionic and nonionic stresses. PMID:17369432

  1. Oxytocin- and aluminium fluoride-induced phospholipase C activity and prostaglandin F2 alpha secretion during the ovine luteolytic period.

    PubMed

    Graf, G A; Burns, P D; Silvia, W J

    1998-03-01

    A series of studies was conducted to characterize changes in components of the cell signalling cascade that mediates oxytocin-induced prostaglandin F2 alpha (PGF2 alpha) synthesis at the onset of luteolysis in sheep. In the first experiment, caruncular tissue was dissected from 20 ewes on days 12-15 of the oestrous cycle, and incubated for the measurement of phospholipase C (PLC) activity or secretion of PGF2 alpha. Activation of GTP-binding proteins with aluminium fluoride stimulated both inositol phosphate accumulation and PGF2 alpha secretion on all days examined. However, oxytocin did not stimulate PLC activity or PGF2 alpha accumulation until day 13. While the ability of oxytocin to stimulate PLC activity increased after day 13, oxytocin-induced PGF2 alpha secretion declined slightly from day 13 to 15, suggesting that cell signalling components downstream from PLC modulate the response to oxytocin after day 13. Oxytocin failed to stimulate PGF2 alpha synthesis on day 14 after oestrus. Secretion of endogenous luteal oxytocin may have rendered uterine tissues collected on day 14 refractory to oxytocin in vitro. Therefore, a second study was conducted in ovariectomized, steroid replaced ewes. Ovarian steroids were administered to mimic endogenous changes in progesterone and oestradiol. The temporal patterns of PGF2 alpha synthesis in response to oxytocin and pharmacological agents were similar to uterine tissues from cyclic ewes in the first experiment; however, the magnitude of the response was less. These data suggest that oxytocin receptors are absent or are not coupled to PLC until day 13 after oestrus.

  2. Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

    PubMed Central

    Wong, Raymond; Fabian, Lacramioara; Forer, Arthur; Brill, Julie A

    2007-01-01

    Background Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated. Results Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3. Conclusion We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring. PMID:17509155

  3. Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis.

    PubMed

    Wong, Raymond; Fabian, Lacramioara; Forer, Arthur; Brill, Julie A

    2007-05-17

    Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated. Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3. We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring.

  4. SKF-83959 is not a highly-biased functionally selective D1 dopamine receptor ligand with activity at phospholipase C.

    PubMed

    Lee, Sang-Min; Kant, Andrew; Blake, Daniel; Murthy, Vishakantha; Boyd, Kevin; Wyrick, Steven J; Mailman, Richard B

    2014-11-01

    SKF-83959 [6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine] is reported to be a functionally selective dopamine D1 receptor ligand with high bias for D1-mediated phospholipase C (PLC) versus D1-coupled adenylate cyclase signaling. This signaling bias is proposed to explain behavioral activity in both rat and primate Parkinson's disease models, and a D1-D2 heterodimer has been proposed as the underlying mechanism. We have conducted an in-depth pharmacological characterization of this compound in dopamine D1 and D2 receptors in both rat brain and heterologous systems expressing human D1 or D2 receptors. Contrary to common assumptions, SKF-83959 is similar to the classical, well-characterized partial agonist SKF38393 in all systems. It is a partial agonist (not an antagonist) at adenylate cyclase in vitro and ex vivo, and is a partial agonist in D1-mediated β-arrestin recruitment. Contrary to earlier reports, it does not have D1-mediated effects on PLC signaling in heterologous systems. Because drug metabolites can also contribute, its 3-N-demethylated analog also was synthesized and tested. As expected from the known structure-activity relationships of the benzazepines, this compound also had high affinity for the D1 receptor and somewhat higher intrinsic activity than the parent ligand, and also might contribute to in vivo effects of SKF-83959. Together, these data demonstrate that SKF-83959 is not a highly-biased functionally selective D1 ligand, and that its reported behavioral data can be explained solely by its partial D1 agonism in canonical signaling pathway(s). Mechanisms that have been proposed based on the purported signaling novelty of SKF-83959 at PLC should be reconsidered.

  5. Multimodal Recognition of Diverse Peptides by the C-Terminal SH2 Domain of Phospholipase C-γ1 Protein.

    PubMed

    McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

    2017-04-11

    SH2 domains recognize phosphotyrosine (pY)-containing peptide ligands and play key roles in the regulation of receptor tyrosine kinase pathways. Each SH2 domain has individualized specificity, encoded in the amino acids neighboring the pY, for defined targets that convey their distinct functions. The C-terminal SH2 domain (PLCC) of the phospholipase C-γ1 full-length protein (PLCγ1) typically binds peptides containing small and hydrophobic amino acids adjacent to the pY, including a peptide derived from platelet-derived growth factor receptor B (PDGFRB) and an intraprotein recognition site (Y783 of PLCγ1) involved in the regulation of the protein's lipase activity. Remarkably, PLCC also recognizes unexpected peptides containing amino acids with polar or bulky side chains that deviate from this pattern. This versatility in recognition specificity may allow PLCγ1 to participate in diverse, previously unrecognized, signaling pathways in response to binding chemically dissimilar partners. We have used structural approaches, including nuclear magnetic resonance and X-ray crystallography, to elucidate the mechanisms of noncognate peptide binding to PLCC by ligands derived from receptor tyrosine kinase ErbB2 and from the insulin receptor. The high-resolution peptide-bound structures reveal that PLCC has a relatively static backbone but contains a chemically rich protein surface comprised of a combination of hydrophobic pockets and amino acids with charged side chains. We demonstrate that this expansive and chemically diverse PLCC interface, in addition to peptide conformational plasticity, permits PLCC to recognize specific noncognate peptide ligands with multimodal specificity.

  6. Modulation of Bacillus thuringiensis Phosphatidylinositol-Specific Phospholipase C Activity by Mutations in the Putative Dimerization Interface

    SciTech Connect

    Shi, X.; Shao, C; Zhang, X; Zambonelli, C; Redfield, A; Head, J; Seaton, B; Roberts, M

    2009-01-01

    Cleavage of phosphatidylinositol (PI) to inositol 1,2-(cyclic)-phosphate (cIP) and cIP hydrolysis to inositol 1-phosphate by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C are activated by the enzyme binding to phosphatidylcholine (PC) surfaces. Part of this reflects improved binding of the protein to interfaces. However, crystallographic analysis of an interfacially impaired phosphatidylinositol-specific phospholipase (W47A/W242A) suggested protein dimerization might occur on the membrane. In the W47A/W242A dimer, four tyrosine residues from one monomer interact with the same tyrosine cluster of the other, forming a tight dimer interface close to the membrane binding regions. We have constructed mutant proteins in which two or more of these tyrosine residues have been replaced with serine. Phospholipid binding and enzymatic activity of these mutants have been examined to assess the importance of these residues to enzyme function. Replacing two tyrosines had small effects on enzyme activity. However, removal of three or four tyrosine residues weakened PC binding and reduced PI cleavage by the enzyme as well as PC activation of cIP hydrolysis. Crystal structures of Y247S/Y251S in the absence and presence of myo-inositol as well as Y246S/Y247S/Y248S/Y251S indicate that both mutant proteins crystallized as monomers, were very similar to one another, and had no change in the active site region. Kinetic assays, lipid binding, and structural results indicate that either (i) a specific PC binding site, critical for vesicle activities and cIP activation, has been impaired, or (ii) the reduced dimerization potential for Y246S/Y247S/Y248S and Y246S/Y247S/Y248S/Y251S is responsible for their reduced catalytic activity in all assay systems.

  7. Mycobacterium abscessus Phospholipase C Expression Is Induced during Coculture within Amoebae and Enhances M. abscessus Virulence in Mice

    PubMed Central

    Bakala N'Goma, Jean Claude; Le Moigne, Vincent; Soismier, Nathalie; Laencina, Laura; Le Chevalier, Fabien; Roux, Anne-Laure; Poncin, Isabelle; Serveau-Avesque, Carole; Rottman, Martin; Gaillard, Jean-Louis; Etienne, Gilles; Brosch, Roland; Canaan, Stéphane

    2014-01-01

    Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria. PMID:25486995

  8. Phospholipase C and protein kinase A mediate bradykinin sensitization of TRPA1: a molecular mechanism of inflammatory pain.

    PubMed

    Wang, Shenglan; Dai, Yi; Fukuoka, Tetsuo; Yamanaka, Hiroki; Kobayashi, Kimiko; Obata, Koichi; Cui, Xiuyu; Tominaga, Makoto; Noguchi, Koichi

    2008-05-01

    Bradykinin is an inflammatory mediator that plays a pivotal role in pain and hyperalgesia in inflamed tissues by exciting and/or sensitizing nociceptors. TRPA1 is an important component of the transduction machinery through which environmental irritants and endogenous proalgesic agents depolarize nociceptors to elicit inflammatory pain. Here, using electrophysiological, immunocytochemical and behavioural analyses, we showed a functional interaction of these two inflammation-related molecules in both heterologous expressing systems and primary sensory neurons. We found that bradykinin increased the TRPA1 currents evoked by allyl isothiocyanate (AITC) or cinnamaldehyde in HEK293 cells expressing TRPA1 and bradykinin receptor 2 (B2R). This potentiation was inhibited by phospholipase C (PLC) inhibitor or protein kinase A (PKA) inhibitor, and mimicked by PLC or PKA activator. The functional interaction between B2R and TRPA1, as well as the modulation mechanism, was also observed in rat dorsal root ganglia neurons. In an occlusion experiment, the PLC activator could enhance AITC-induced TRPA1 current further even in saturated PKA-mediated potentiation, indicating the additive potentiating effects of the PLC and PKA pathways. These data for the first time indicate that a cAMP-PKA signalling is involved in the downstream from B2R in dorsal root ganglia neurons in addition to PLC. Finally, subcutaneous pre-injection of a sub-inflammatory dose of bradykinin into rat hind paw enhanced AITC-induced pain behaviours, which was consistent with the observations in vitro. Collectively, these results represent a novel mechanism through which bradykinin released in response to tissue inflammation might trigger the sensation of pain by TRPA1 activation.

  9. Differences in rapid desensitization of 5-hydroxytryptamine2A and 5-hydroxytryptamine2C receptor-mediated phospholipase C activation.

    PubMed

    Berg, K A; Stout, B D; Maayani, S; Clarke, W P

    2001-11-01

    The serotonin (5-HT)2A and 5-HT2C receptors share a high degree of sequence homology and have very similar pharmacological profiles. Although it is generally believed that the cellular signal transduction mechanisms activated by these receptors are indistinguishable, recent data suggest significant differences in their signaling cascades. In this study we explored differences in the characteristics and mechanisms of rapid desensitization between the 5-HT2A and 5-HT2C receptor systems. For both receptor systems, pretreatment with 5-HT reduced the ability of a maximal concentration of 5-HT to stimulate phospholipase C-mediated inositol phosphate accumulation by about 65%, although the 5-HT2C receptor system was more sensitive to the desensitizing stimulus. Differences in the concentration dependence of the rate constant for desensitization (k(des)) suggested different mechanisms of desensitization for the 5-HT2A and 5-HT2C receptor systems. At very high receptor occupancy (>99%), the responsiveness of the 5-HT2A, but not the 5-HT2C, receptor system returned to control levels despite the continued presence of the agonist. This resensitization was dependent upon the activity of protein kinase C (PKC). Agonist-induced desensitization of the 5-HT2A, but not the 5-HT2C, receptor system was reduced by the PKC inhibitors staurosporine and bisindolylmaleimide, and by down-regulation of PKC. In addition, inhibitors of calmodulin (W-7) or of calmodulin-dependent protein kinase II, reduced 5-HT2A, but not 5-HT2C, desensitization. Desensitization of the 5-HT2C, but not the 5-HT2A, receptor system was dependent on G protein receptor kinase activity. These data further emphasize the major differences in the signaling systems coupled to 5-HT2A/2C receptors.

  10. Phospholipase C of Cryptococcus neoformans regulates homeostasis and virulence by providing inositol trisphosphate as a substrate for Arg1 kinase.

    PubMed

    Lev, Sophie; Desmarini, Desmarini; Li, Cecilia; Chayakulkeeree, Methee; Traven, Ana; Sorrell, Tania C; Djordjevic, Julianne T

    2013-04-01

    Phospholipase C (PLC) of Cryptococcus neoformans (CnPlc1) is crucial for virulence of this fungal pathogen. To investigate the mechanism of CnPlc1-mediated signaling, we established that phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a major CnPlc1 substrate, which is hydrolyzed to produce inositol trisphosphate (IP(3)). In Saccharomyces cerevisiae, Plc1-derived IP(3) is a substrate for the inositol polyphosphate kinase Arg82, which converts IP(3) to more complex inositol polyphosphates. In this study, we show that in C. neoformans, the enzyme encoded by ARG1 is the major IP(3) kinase, and we further demonstrate that catalytic activity of Arg1 is essential for cellular homeostasis and virulence in the Galleria mellonella infection model. IP(3) content was reduced in the CnΔplc1 mutant and markedly increased in the CnΔarg1 mutant, while PIP(2) was increased in both mutants. The CnΔplc1 and CnΔarg1 mutants shared significant phenotypic similarity, including impaired thermotolerance, compromised cell walls, reduced capsule production and melanization, defective cell separation, and the inability to form mating filaments. In contrast to the S. cerevisiae ARG82 deletion mutant (ScΔarg82) strain, the CnΔarg1 mutant exhibited dramatically enlarged vacuoles indicative of excessive vacuolar fusion. In mammalian cells, PLC-derived IP(3) causes Ca(2+) release and calcineurin activation. Our data show that, unlike mammalian PLCs, CnPlc1 does not contribute significantly to calcineurin activation. Collectively, our findings provide the first evidence that the inositol polyphosphate anabolic pathway is essential for virulence of C. neoformans and further show that production of IP(3) as a precursor for synthesis of more complex inositol polyphosphates is the key biochemical function of CnPlc1.

  11. Serotonin-2A homodimers are needed for signalling via both phospholipase A2 and phospholipase C in transfected CHO cells.

    PubMed

    Iglesias, Alba; Cimadevila, Marta; Cadavid, María Isabel; Loza, María Isabel; Brea, José

    2017-04-05

    Different ligands differentially activate phospholipase A2 (PLA2) and phospholipase C (PLC) signalling pathways that are coupled to the serotonin 2A (5-HT2A) receptor, a class-A G-protein coupled receptor (GPCR). The serotonin 5-HT2A receptor has been shown to be expressed as a homodimer displaying some ligands negative cooperativity between protomers in the PLA2 signalling pathway. We hypothesized that the homodimeric complex is the minimum functional unit required for activation of the PLA2 and PLC pathways by the serotonin 5-HT2A receptor. To investigate this hypothesis, we partially blocked the serotonin 5-HT2A receptors with ritanserin and measured PLA2 and PLC activity simultaneously. We subsequently added the competitive antagonist spiperone to release the inactivator through a crosstalk mechanism and thus allow the dimer to return to a reactive state. Partial inactivation of the homodimer by ritanserin binding decreased the activity of the receptor by 59±13% and 70±4% in the PLA2 and PLC pathways respectively (P<0.001), with no difference in the potency of the serotonin (5-HT) was observed. The subsequent binding of spiperone released ritanserin due to the crosstalk between protomers and recovery of the receptor activity to 74±7% and 72±4%. Negative cooperativity between protomers in the dimer was maintained during arachidonic acid (AA) release after blocking ritanserin, as indicated by the biphasic inhibition curves for clozapine over 1μM serotonin (5-HT) in these conditions. These findings provide evidence that serotonin 5-HT2A receptors must be expressed as homodimers in order to activate both the PLA2 and PLC signalling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Methylmercury-induced toxicity is mediated by enhanced intracellular calcium through activation of phosphatidylcholine-specific phospholipase C

    SciTech Connect

    Kang, Mi Sun; Jeong, Ju Yeon; Seo, Ji Heui; Jeon, Hyung Jun; Jung, Kwang Mook; Chin, Mi-Reyoung; Moon, Chang-Kiu; Bonventre, Joseph V.; Jung, Sung Yun; Kim, Dae Kyong . E-mail: proteinlab@hanmail.net

    2006-10-15

    Methylmercury (MeHg) is a ubiquitous environmental toxicant to which humans can be exposed by ingestion of contaminated food. MeHg has been suggested to exert its toxicity through its high reactivity to thiols, generation of arachidonic acid and reactive oxygen species (ROS), and elevation of free intracellular Ca{sup 2+} levels ([Ca{sup 2+}]{sub i}). However, the precise mechanism has not been fully defined. Here we show that phosphatidylcholine-specific phospholipase C (PC-PLC) is a critical pathway for MeHg-induced toxicity in MDCK cells. D609, an inhibitor of PC-PLC, significantly reversed the toxicity in a time- and dose-dependent manner with concomitant inhibition of the diacylglycerol (DAG) generation and the phosphatidylcholine (PC)-breakdown. MeHg activated the group IV cytosolic phospholipase A{sub 2} (cPLA{sub 2}) and acidic form of sphingomyelinase (A-SMase) downstream of PC-PLC, but these enzymes as well as protein kinase C (PKC) were not linked to the toxicity by MeHg. Furthermore, MeHg produced ROS, which did not affect the toxicity. Addition of EGTA to culture media resulted in partial decrease of [Ca{sup 2+}]{sub i} and partially blocked the toxicity. In contrast, when the cells were treated with MeHg in the presence of Ca{sup 2+} in the culture media, D609 completely prevented cell death with parallel decrease in [Ca{sup 2+}]{sub i}. Our results demonstrated that MeHg-induced toxicity was linked to elevation of [Ca{sup 2+}]{sub i} through activation of PC-PLC, but not attributable to the signaling pathways such as cPLA{sub 2}, A-SMase, and PKC, or to the generation of ROS.

  13. Mechanosensitive activation of K+ channel via phospholipase C-induced depletion of phosphatidylinositol 4,5-bisphosphate in B lymphocytes.

    PubMed

    Nam, Joo Hyun; Lee, Hoo-Se; Nguyen, Yen Hoang; Kang, Tong Mook; Lee, Sung Won; Kim, Hye-Young; Kim, Sang Jeong; Earm, Yung E; Kim, Sung Joon

    2007-08-01

    In various types of cells mechanical stimulation of the plasma membrane activates phospholipase C (PLC). However, the regulation of ion channels via mechanosensitive degradation of phosphatidylinositol 4,5-bisphosphate (PIP(2)) is not known yet. The mouse B cells express large conductance background K(+) channels (LK(bg)) that are inhibited by PIP(2). In inside-out patch clamp studies, the application of MgATP (1 mm) also inhibited LK(bg) due to the generation of PIP(2) by phosphoinositide (PI)-kinases. In the presence of MgATP, membrane stretch induced by negative pipette pressure activated LK(bg), which was antagonized by PIP(2) (> 1 microm) or higher concentration of MgATP (5 mm). The inhibition by PIP(2) was partially reversible. However, the application of methyl-beta-cyclodextrin, a cholesterol scavenger disrupting lipid rafts, induced the full recovery of LK(bg) activity and facilitated the activation by stretch. In cell-attached patches, LK(bg) were activated by hypotonic swelling of B cells as well as by negative pressure. The mechano-activation of LK(bg) was blocked by U73122, a PLC inhibitor. Neither actin depolymerization nor the inhibition of lipid phosphatase blocked the mechanical effects. Direct stimulation of PLC by m-3M3FBS or by cross-linking IgM-type B cell receptors activated LK(bg). Western blot analysis and confocal microscopy showed that the hypotonic swelling of WEHI-231 induces tyrosine phosphorylation of PLCgamma2 and PIP(2) hydrolysis of plasma membrane. The time dependence of PIP(2) hydrolysis and LK(bg) activation were similar. The presence of LK(bg) and their stretch sensitivity were also proven in fresh isolated mice splenic B cells. From the above results, we propose a novel mechanism of stretch-dependent ion channel activation, namely, that the degradation of PIP(2) caused by stretch-activated PLC releases LK(bg) from the tonic inhibition by PIP(2).

  14. Inhibition of phosphatidylcholine-specific phospholipase C results in loss of mesenchymal traits in metastatic breast cancer cells

    PubMed Central

    2012-01-01

    Introduction Acquisition of mesenchymal characteristics confers to breast cancer (BC) cells the capability of invading tissues different from primary tumor site, allowing cell migration and metastasis. Regulators of the mesenchymal-epithelial transition (MET) may represent targets for anticancer agents. Accruing evidence supports functional implications of choline phospholipid metabolism in oncogene-activated cell signaling and differentiation. We investigated the effects of D609, a xanthate inhibiting phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS), as a candidate regulator of cell differentiation and MET in the highly metastatic BC cell line MDA-MB-231. Methods PC-PLC expression and activity were investigated using confocal laser scanning microscopy (CLSM), immunoblotting and enzymatic assay on human MDA-MB-231 compared with MCF-7 and SKBr3 BC cells and a nontumoral immortalized counterpart (MCF-10A). The effects of D609 on PC-PLC and SMS activity, loss of mesenchymal markers and changes in migration and invasion potential were monitored in MDA-MB-231 cells by enzymatic assays, CLSM, immunoblotting and transwell chamber invasion combined with scanning electron microscopy examinations. Cell proliferation, formation and composition of lipid bodies and cell morphology were investigated in D609-treated BC cells by cell count, CLSM, flow-cytometry of BODIPY-stained cells, nuclear magnetic resonance and thin-layer chromatography. Results PC-PLC (but not phospholipase D) showed 2- to 6-fold activation in BC compared with nontumoral cells, the highest activity (up to 0.4 pmol/μg protein/min) being detected in the poorly-differentiated MDA-MB-231 cells. Exposure of the latter cells to D609 (50 μg/mL, 24-72 h) resulted into 60-80% PC-PLC inhibition, while SMS was transiently inhibited by a maximum of 21%. These features were associated with progressive decreases of mesenchymal traits such as vimentin and N-cadherin expression

  15. Vomeronasal sensory neurons from Sternotherus odoratus (stinkpot/musk turtle) respond to chemosignals via the phospholipase C system

    PubMed Central

    Brann, Jessica H.; Fadool, Debra A.

    2009-01-01

    Summary The mammalian signal transduction apparatus utilized by vomeronasal sensory neurons (VSNs) in the vomeronasal organ (VNO) has been richly explored, while that of reptiles, and in particular, the stinkpot or musk turtle Sternotherus odoratus, is less understood. Given that the turtle’s well-known reproductive and mating behaviors are governed by chemical communication, 247 patch-clamp recordings were made from male and female S. odoratus VSNs to study the chemosignal-activated properties as well as the second-messenger system underlying the receptor potential. Of the total neurons tested, 88 (35%) were responsive to at least one of five complex natural chemicals, some of which demonstrated a degree of sexual dimorphism in response selectivity. Most notably, male VSNs responded to male urine with solely outward currents. Ruthenium Red, an IP3 receptor (IP3R) antagonist, failed to block chemosignal-activated currents, while the phospholipase C (PLC) inhibitor, U73122, abolished the chemosignal-activated current within 2 min, implicating the PLC system in the generation of a receptor potential in the VNO of musk turtles. Dialysis of several second messengers or their analogues failed to elicit currents in the whole-cell patch-clamp configuration, negating a direct gating of the transduction channel by cyclic adenosine monophosphate (cAMP), inositol 1,4,5-trisphosphate (IP3), arachidonic acid (AA), or diacylglycerol (DAG). Reversal potential analysis of chemosignal-evoked currents demonstrated that inward currents reversed at −5.7±7.8 mV (mean ± s.e.m.; N=10), while outward currents reversed at −28.2±2.4 mV (N=30). Measurements of conductance changes associated with outward currents indicated that the outward current represents a reduction of a steady state inward current by the closure of an ion channel when the VSN is exposed to a chemical stimulus such as male urine. Chemosignal-activated currents were significantly reduced when a peptide mimicking a

  16. Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

    SciTech Connect

    Chung, Eric; Jakinovich, Paul; Bae, Aekyung; Rebecchi, Mario

    2012-10-01

    Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1} knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a

  17. Pyrimidinoceptor-mediated activation of phospholipase C and phospholipase A2 in RAW 264.7 macrophages.

    PubMed Central

    Lin, W. W.; Lee, Y. T.

    1996-01-01

    1. As well as the presence of P2Z purinoceptors previously found in macrophages, we identified pyrimidinoceptors in RAW 264.7 cells, which activate phospholipase C (PLC) and phospholipase A2 (PLA2). 2. The relative potency of agonists to stimulate inositol phosphate (IP) formation and arachidonic acid (AA) release was UTP = UDP > > ATP, ATP gamma S, 2MeSATP. For both signalling pathways, the EC50 values for UTP and UDP (3 microM) were significantly lower than that for ATP and all other analogues tested (> 100 microM). 3. UTP and UDP displayed no additivity in terms of IP formation and AA release at maximally effective concentrations. 4. UTP-, but not ATP-, evoked AA release was 60% inhibited by pertussis toxin (PTX), while stimulation of IP formation by both agonists was unaffected. Short-term treatment with phorbol 12-myristate 13-acetate (PMA) led to a dose-dependent inhibition of IP responses to UTP and UDP, but failed to affect the AA responses. Removal of extracellular Ca2+ inhibited the PI response to UTP, but abolished its AA response. 5. ATP-induction of these two transmembrane signal pathways was decreased in high Mg(2+)-containing medium but potentiated by the removal of extracellular Mg2+. 6. Suramin and reactive blue displayed equal potency to inhibit the IP responses of UTP and ATP. 7. Both UTP and UDP (0.1-100 microM) induced a sustained increase in [Ca2+]i which lasted for more than 10 min. 8. Taken together, these results indicate that in mouse RAW 264.7 macrophages, pyrimidinoceptors with specificity for UTP and UDP mediate the activation of PLC and cytosolic (c) PLA2. The activation of PLC is via a PTX-insensitive G protein, whereas that of cPLA2 is via a PTX-sensitive G protein-dependent pathway. The sustained Ca2+ influx caused by UTP contributes to the activation of cPLA2. RAW 264.7 cells also possess P2z purinoceptors which mediate ATP(4-)-induced PLC and PLA2 activation. Images Figure 3 PMID:8886407

  18. pH-regulated activation and release of a bacteria-associated phospholipase C during intracellular infection by Listeria monocytogenes

    PubMed Central

    Marquis, Hélène; Hager, Elizabeth J.

    2006-01-01

    Summary Listeria monocytogenes grows in the cytosol of mammalian cells and spreads from cell to cell without exiting the intracellular milieu. During cell–cell spread, bacteria become transiently entrapped in double-membrane vacuoles. Escape from these vacuoles is mediated in part by a bacterial phospholipase C (PC-PLC), whose activation requires cleavage of an N-terminal peptide. PC-PLC activation occurs in the acidified vacuolar environment. In this study, the pH-dependent mechanism of PC-PLC activation was investigated by manipulating the intracellular pH of the host. PC-PLC secreted into infected cells was immunoprecipitated, and both forms of the protein were identified by SDS–PAGE fluorography. PC-PLC activation occurred at pH 7.0 and lower, but not at pH 7.3. Total amounts of PC-PLC secreted into infected cells increased several-fold over controls within 5 min of a decrease in intracellular pH, and the active form of PC-PLC was the most abundant species detected. Bacterial release of active PC-PLC was dependent on Mpl, a bacterial metalloprotease that processes the proform (proPC-PLC), and did not require de novo protein synthesis. The amount of proPC-PLC released in response to a decrease in pH was the same in wild-type and Mpl-minus-infected cells. Immunofluorescence detection of PC-PLC in infected cells was performed. When fixed and permeabilized infected cells were treated with a bacterial cell wall hydrolase, over 97% of wild-type and Mpl-minus bacteria stained positively for PC-PLC, in contrast to less than 5% in untreated cells. These results indicate that intracellular bacteria carry pools of proPC-PLC. Upon cell–cell spread, a decrease in vacuolar pH triggers Mpl activation of proPC-PLC, resulting in bacterial release of active PC-PLC. PMID:10652090

  19. Phospholipase C gamma mediates endogenous brain-derived neurotrophic factor-regulated calcitonin gene-related peptide expression in colitis-induced visceral pain

    PubMed Central

    Hashmi, Fiza; Liu, Miao; Shen, Shanwei

    2016-01-01

    Background Visceral hypersensitivity is a complex pathophysiological paradigm with unclear mechanisms. Primary afferent neuronal plasticity marked by alterations in neuroactive compounds such as calcitonin gene-related peptide is suggested to underlie the heightened sensory responses. Signal transduction that leads to calcitonin gene-related peptide expression thereby sensory neuroplasticity during colitis remains to be elucidated. Results In a rat model with colitis induced by 2,4,6-trinitrobenzene sulfonic acid, we found that endogenously elevated brain-derived neurotrophic factor elicited an up-regulation of calcitonin gene-related peptide in the lumbar L1 dorsal root ganglia. At seven days of colitis, neutralization of brain-derived neurotrophic factor with a specific brain-derived neurotrophic factor antibody reversed calcitonin gene-related peptide up-regulation in the dorsal root ganglia. Colitis-induced calcitonin gene-related peptide transcription was also inhibited by brain-derived neurotrophic factor antibody treatment. Signal transduction studies with dorsal root ganglia explants showed that brain-derived neurotrophic factor-induced calcitonin gene-related peptide expression was mediated by the phospholipase C gamma, but not the phosphatidylinositol 3-kinase/Akt or the mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway. Application of PLC inhibitor U73122 in vivo confirmed that colitis-induced and brain-derived neurotrophic factor-mediated calcitonin gene-related peptide up-regulation in the dorsal root ganglia was regulated by the phospholipase C gamma pathway. In contrast, suppression of the phosphatidylinositol 3-kinase activity in vivo had no effect on colitis-induced calcitonin gene-related peptide expression. During colitis, calcitonin gene-related peptide also co-expressed with phospholipase C gamma but not with p-Akt. Calcitonin gene-related peptide up-regulation during colitis correlated to the activation

  20. Protein kinase C-independent expression of stromelysin by platelet-derived growth factor, ras oncogene, and phosphatidylcholine-hydrolyzing phospholipase C.

    PubMed

    Diaz-Meco, M T; Quiñones, S; Municio, M M; Sanz, L; Bernal, D; Cabrero, E; Saus, J; Moscat, J

    1991-11-25

    Changes in the expression of several genes play critical roles in cell growth and tumor transformation. A number of proteases are increased in some tumors, and the level of these enzymes correlates with the metastatic potential of several cancer cell lines. Stromelysin, with the widest substrate specificity, can degrade the extracellular matrix conferring metastatic potential to tumor cells. The mechanisms whereby growth factors and oncogenes control the expression of stromelysin are beginning to be characterized. In the study shown here we also identify a region in the stromelysin promoter which is involved in the induction of stromelysin in response to platelet-derived growth factor, phosphatidylcholine-hydrolyzing phospholipase C, and ras oncogene. Our results are consistent with the notion that platelet-derived growth factor/phosphatidylcholine-hydrolyzing phospholipase C induces stromelysin gene expression through a phorbol myristate acetate/protein kinase C-independent mechanism by acting through elements in the stromelysin promoter distinct from the 12-O-tetradecanoylphorbol-13-acetate-responsive element.

  1. Kinetic Model for Surface-Active Enzymes Based on the Langmuir Adsorption Isotherm: Phospholipase C (Bacillus cereus) Activity toward Dimyristoyl Phosphatidylcholine/Detergent Micelles

    NASA Astrophysics Data System (ADS)

    Burns, Ramon A.; El-Sayed, Maha Y.; Roberts, Mary F.

    1982-08-01

    A simple kinetic model for the enzymatic activity of surface-active proteins against mixed micelles has been developed. This model uses the Langmuir adsorption isotherm, the classic equation for the binding of gas molecules to metal surfaces, to characterize enzyme adsorption to micelles. The number of available enzyme binding sites is equated with the number of substrate and inhibitor molecules attached to micelles; enzyme molecules are attracted to the micelle due to the affinity of the enzyme active site for the molecules in the micelle. Phospholipase C (Bacillus cereus) kinetics in a wide variety of dimyristoyl phosphatidylcholine/detergent micelles are readily explained by this model and the assumption of competitive binding of the detergent at the enzyme active site. Binding of phospholipase C to pure detergent micelles is demonstrated by gel filtration chromatography. The experimentally determined enzyme-detergent micelle binding constants are used directly in the rate equation. The Langmuir adsorption model predicts a variety of the characteristics observed for phospholipase kinetics, such as differential inhibition by various charged, uncharged, and zwitterionic detergents and surface-dilution inhibition. The essential idea of this model, that proteins can be attracted and bound to bilayers or micelles by possessing a binding site for the molecules composing the surface, may have wider application in the study of water-soluble (extrinsic) protein-membrane interactions.

  2. Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

    PubMed Central

    Burgess, W H; Dionne, C A; Kaplow, J; Mudd, R; Friesel, R; Zilberstein, A; Schlessinger, J; Jaye, M

    1990-01-01

    Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma. Images PMID:2167438

  3. The effects of acute exposure to ethanol on neurotensin and guanine nucleotide-stimulation of phospholipase C activity in intact NIE-115 neuroblastoma cells

    SciTech Connect

    Smith, T.L. )

    1990-01-01

    Both ethanol and neurotensin produce sedation and hypothermia. When administered in combination the behavioral effects of these two substances are potentiated. In order to better understand the biochemical nature of this interaction, the direct effects of ethanol on neurotensin receptors and an associated signal transduction process were determined in NIE-115 neuroblastoma cells. Ethanol in physiologically relevant concentrations significantly reduced neurotensin stimulated ({sup 3}H)inositol phosphate production while having no effect on the specific binding of ({sup 3}H)neurotensin. In addition, ethanol up to 200 mM had no effect on GTPYS mediated ({sup 3}H)inositol phosphate production. The results indicate that acute exposure ethanol partially disrupts the normal coupling of activated neurotensin receptors to the guanine nucleotide binding protein associated with phospholipase C.

  4. Phospholipase C{gamma}1 stimulates transcriptional activation of the matrix metalloproteinase-3 gene via the protein kinase C/Raf/ERK cascade

    SciTech Connect

    Shin, Soon Young; Choi, Ha Young; Ahn, Bong-Hyun; Son, Sang Wook; Lee, Young Han . E-mail: younghan@hanyang.ac.kr

    2007-02-16

    The phospholipid hydrolase phospholipase C{gamma}1 (PLC{gamma}1) plays a major role in regulation of cell proliferation, development, and cell motility. Overexpression of PLC{gamma}1 is associated with tumor development, and it is overexpressed in some tumors. Matrix metalloproteinase-3 (MMP-3) is a protein involved in tumor invasion and metastasis. Here, we demonstrate that overexpression of PLC{gamma}1 stimulates MMP-3 expression at the transcriptional level via the PKC-mediated Raf/MEK1/ERK signaling cascade. We propose that modulation of PLC{gamma}1 activity might be of value in controlling the activity of MMPs, which are important regulators of invasion and metastasis in malignant tumors.

  5. Activation of the neuroprotective ERK signaling pathway by fructose-1,6-bisphosphate during hypoxia involves intracellular Ca2+ and phospholipase C.

    PubMed

    Fahlman, C S; Bickler, P E; Sullivan, Breandan; Gregory, G A

    2002-12-20

    The mechanism of the neuroprotective action of the glycolytic pathway intermediate fructose-1,6-bisphosphate (FBP) may involve activation of a phospholipase-C (PLC) dependent MAP kinase signaling pathway. In this study, we determined whether FBP's capacity to decrease delayed cell death in hippocampal slice cultures is dependent on PLC signaling or activation of the intracellular Ca(2+)-MEK/ERK neuroprotective signaling cascade. FBP (3.5 mM) reduced delayed death from oxygen/glucose deprivation in CA1, CA3 and dentate neurons in slice cultures. The phospholipase-C inhibitor U73122 and the MEK1/2 inhibitor U0126 prevented this protection. In hippocampal and cortical neurons, FBP increased phospho-ERK1/2 (p42/44) immunostaining during hypoxic, but not normoxic conditions. Increased phospho-ERK immunostaining was dependent on PLC and also on MEK 1/2, an upstream regulator of ERK. Further, we found that FBP enhancement of phospho-ERK immunostaining depended on [Ca(2+)](i): PLC inhibition and the IP(3) receptor blocker xestospongin C prevented FBP from increasing [Ca(2+)](i) and increasing phospho-ERK levels. However, while FBP-induced increases in [Ca(2+)](i) were blocked by xestospongin and a PLC inhibitor, [Ca(2+)](i) increases induced by the neuroprotective growth factor BDNF were not prevented. We conclude that during hypoxia FBP initiates a series of neuroprotective signals which include PLC activation, small increases in [Ca(2+)](i), and increased activity of the MEK/ERK signaling pathway.

  6. The Legionella pneumophila tatB Gene Facilitates Secretion of Phospholipase C, Growth under Iron-Limiting Conditions, and Intracellular Infection

    PubMed Central

    Rossier, Ombeline; Cianciotto, Nicholas P.

    2005-01-01

    Our previous mutational analysis of Legionella pneumophila demonstrated a role for type II protein (Lsp) secretion and iron acquisition in intracellular infection and virulence. In gram-negative bacteria, the twin-arginine translocation (Tat) pathway is involved in secretion of proteins, including components of respiratory complexes, across the inner membrane to the periplasm. To assess the significance of Tat for L. pneumophila, tatB mutants were characterized. The mutants exhibited normal growth in standard media but grew slowly under low-iron conditions. They were also impaired in the Nadi assay, indicating that the function of cytochrome c oxidase is influenced by tatB. Consistent with this observation, a subunit of the cytochrome c reductase was shown to be a Tat substrate. Supernatants of the tatB mutants showed a 30% reduction in phospholipase C activity while maintaining normal levels of other Lsp secreted activities. When tested for infection of U937 macrophages, the tatB mutants showed a 10-fold reduction in growth. Double mutants lacking tatB and Lsp secretion were even more defective, suggesting tatB has an intracellular role that is independent of Lsp. tatB mutants were also impaired 20-fold in Hartmannella vermiformis amoebae cultured in the presence of an iron chelator. All mutant phenotypes were complemented by reintroduction of an intact tatB. Thus, L. pneumophila tatB plays a role in the formation of a respiratory complex, growth under low-iron conditions, the secretion of a phospholipase C activity, and intracellular infection. PMID:15784543

  7. Leakage-free membrane fusion induced by the hydrolytic activity of PlcHR(2), a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa.

    PubMed

    Montes, L-Ruth; Ibarguren, Maitane; Goñi, Félix M; Stonehouse, Martin; Vasil, Michael L; Alonso, Alicia

    2007-10-01

    PlcHR(2) is the paradigm member of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial species. This paper describes the phospholipase C and sphingomyelinase activities of PlcHR(2) when the substrate is in the form of large unilamellar vesicles, and the subsequent effects of lipid hydrolysis on vesicle and bilayer stability, including vesicle fusion. PlcHR(2) cleaves phosphatidylcholine and sphingomyelin at equal rates, but is inactive on phospholipids that lack choline head groups. Calcium in the millimolar range does not modify in any significant way the hydrolytic activity of PlcHR(2) on choline-containing phospholipids. The catalytic activity of the enzyme induces vesicle fusion, as demonstrated by the concomitant observation of intervesicular total lipid mixing, inner monolayer-lipid mixing, and aqueous contents mixing. No release of vesicular contents is detected under these conditions. The presence of phosphatidylserine in the vesicle composition does not modify significantly PlcHR(2)-induced liposome aggregation, as long as Ca(2+) is present, but completely abolishes fusion, even in the presence of the cation. Each of the various enzyme-induced phenomena have their characteristic latency periods, that increase in the order lipid hydrolysis

  8. Hydrolysis of erythrocyte membrane phospholipids by a preparation of phospholipase C from Clostridium Welchii. Deactivation of (Ca-2+, Mg-2+)-ATPase and its reactivation by added lipids.

    PubMed

    Coleman, R; Bramley, T A

    1975-04-08

    1. Haemoglobin-free erythrocyte ghosts were prepared in 40 imosM bicarbonate buffer, pH 7.4, containing 1 mM EDTA (40 imosM/l mM EDTA). The ghost preparation was highly permeable on preparation but partially resealed on incubation in media containing Ca-2+. 2. A partially purified preparation of phospholipase C from Clostridum welchii caused an increase in observed Mg-2+-ATPase activity, reflecting a change in the permeability of the ghost to substrate. The phospholipase did not decrease Mg-2+-ATPase even at the highest levels tested. Mg-2+-ATPase activity could therefore be used as a permeability indicatior in these experiments. 3. Both (Ca-2+, Mg-2+)-ATPase activities of the ghosts were progressively lost as a result of the phospholipid hydrolysis induced by phospholipase C. 4. When a haemolysin in the commercial preparation was destroyed by heat-treatment, deactivation of the (Ca-2+, Mg-2+)-ATPase and (Na+, K+, Mg-2+)-ATPases were still observed but permeability changes were greatly reduced. 5. The products of phospholipase action were not inhibitory to the Ca-2+, Mg-2+)-ATPase. 6. Lysolecithin brought about a reactivation of the (Ca-2+, Mg-2+)-ATPase which was superimposed upon permeability changes in the preparation. 7. Reactivation of the (Ca-2+, Mg-2+)-ATPase was brought about by a nonlytic, mixed lipid preparation without significant effect upon permeability. 8. Human erythrocyte (Ca-2+, Mg-2+)-ATPase therefore appears to be an enzyme which responds to perturbation of the lipid environment in the membrane and is a "lipid-dependant" enzyme.

  9. The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection.

    PubMed

    Rossier, Ombeline; Cianciotto, Nicholas P

    2005-04-01

    Our previous mutational analysis of Legionella pneumophila demonstrated a role for type II protein (Lsp) secretion and iron acquisition in intracellular infection and virulence. In gram-negative bacteria, the twin-arginine translocation (Tat) pathway is involved in secretion of proteins, including components of respiratory complexes, across the inner membrane to the periplasm. To assess the significance of Tat for L. pneumophila, tatB mutants were characterized. The mutants exhibited normal growth in standard media but grew slowly under low-iron conditions. They were also impaired in the Nadi assay, indicating that the function of cytochrome c oxidase is influenced by tatB. Consistent with this observation, a subunit of the cytochrome c reductase was shown to be a Tat substrate. Supernatants of the tatB mutants showed a 30% reduction in phospholipase C activity while maintaining normal levels of other Lsp secreted activities. When tested for infection of U937 macrophages, the tatB mutants showed a 10-fold reduction in growth. Double mutants lacking tatB and Lsp secretion were even more defective, suggesting tatB has an intracellular role that is independent of Lsp. tatB mutants were also impaired 20-fold in Hartmannella vermiformis amoebae cultured in the presence of an iron chelator. All mutant phenotypes were complemented by reintroduction of an intact tatB. Thus, L. pneumophila tatB plays a role in the formation of a respiratory complex, growth under low-iron conditions, the secretion of a phospholipase C activity, and intracellular infection.

  10. Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5 prime -O-(3-thiotriphosphate)

    SciTech Connect

    Baldassare, J.J.; Henderson, P.A.; Fisher, G.J. )

    1989-01-10

    The effects of thrombin and GTP{gamma}S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous ({sup 3}H)inositol-labeled membranes or with lipid vesicles containing either ({sup 3}H)phosphatidylinositol or ({sup 3}H)phosphatidylinositol 4,5-bisphosphate. GTP{gamma}S (1 {mu}M) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP{sub 3}), inositol bisphosphate (IP{sub 2}), or inositol phosphate (IP) from ({sup 3}H)inositol-labeled membranes. IP{sub 2} and IP{sub 3}, but not IP, from ({sup 3}H)inositol-labeled membranes were, however, stimulated 3-fold by GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). A higher concentration of GTP{gamma}S (100 {mu}M) alone also stimulated IP{sub 2} and IP{sub 3}, but not IP, release. In the presence of 1 mM calcium, release of IP{sub 2} and IP{sub 3} was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) by platelet membrane associated PLC was also markedly enhanced by GTP{gamma}S (100 {mu}M) or GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP{sub 2} was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP{gamma}S (100 {mu}M) or calcium (1 mM) dependent PIP{sub 2} breakdown, while TPA inhibited GTP{gamma}S-dependent but not calcium-dependent phospholipase C activity.

  11. Human- and mouse-inducible nitric oxide synthase promoters require activation of phosphatidylcholine-specific phospholipase C and NF-kappa B.

    PubMed Central

    Spitsin, S. V.; Farber, J. L.; Bertovich, M.; Moehren, G.; Koprowski, H.; Michaels, F. H.

    1997-01-01

    BACKGROUND: The production of nitric oxide by type II inducible nitric oxide synthase (type II NOS) gene is controlled at least in part by transcriptional activation. Although the murine and human type II NOS genes share significant sequence homology, they differ in the induction stimuli required for activation. MATERIALS AND METHODS: The A549 human and murine RAW 264.7 cell lines were cultured in the presence of inducers of the type II NOS gene and exposed to specific inhibitors of phosphatidyl choline-specific phospholipase C, NF-kappa B, and endocytosis, as well as to reagents that deplete stores of ATP or prevent the acidification of endosomes. The effect of these reagents on the induction of the type II NOS gene transcription, translation, and NO expression was studied using electromobility shift assays, Western blotting, and the detection of NO as nitrates, as appropriate. Additionally, the ability of the native human type II NOS NF-kappa B recognition sequence to bind NF-kappa B was compared with a concensus sequence and with a mutated oligomer. RESULTS: Type II NOS production by both human and mouse cells could be prevented by the addition of the specific inhibitor of phosphatidylcholine-specific phospholipase C, D609, and of agents that interfere with the activation of NF-kappa B. Both mouse and human cells also required acidic endosome formation and the production of 1,2-diacylglycerol for type II NOS expression. Additionally, the native human type II NOS NF-kappa B recognition sequence bound NF-kappa B with significantly less affinity than did the recognition sequence derived from the human immunoglobulin light-chain gene promoter. CONCLUSIONS: These experiments show that whereas mouse cells can be activated by lipopolysaccharide to produce nitric oxide, and human cells require activation by a mixture of cytokines to produce nitric oxide, the intracellular activation pathway following receptor binding of these heterologous stimuli is shared. Additionally

  12. Phospholipase C-related Catalytically Inactive Protein Is a New Modulator of Thermogenesis Promoted by β-Adrenergic Receptors in Brown Adipocytes*

    PubMed Central

    Oue, Kana; Zhang, Jun; Harada-Hada, Kae; Asano, Satoshi; Yamawaki, Yosuke; Hayashiuchi, Masaki; Furusho, Hisako; Takata, Takashi; Irifune, Masahiro; Hirata, Masato; Kanematsu, Takashi

    2016-01-01

    Phospholipase C-related catalytically inactive protein (PRIP) was first identified as an inositol 1,4,5-trisphosphate-binding protein, and was later found to be involved in a variety of cellular events, particularly those related to protein phosphatases. We previously reported that Prip knock-out (KO) mice exhibit a lean phenotype with a small amount of white adipose tissue. In the present study, we examined whether PRIP is involved in energy metabolism, which could explain the lean phenotype, using high-fat diet (HFD)-fed mice. Prip-KO mice showed resistance to HFD-induced obesity, resulting in protection from glucose metabolism dysfunction and insulin resistance. Energy expenditure and body temperature at night were significantly higher in Prip-KO mice than in wild-type mice. Gene and protein expression of uncoupling protein 1 (UCP1), a thermogenic protein, was up-regulated in Prip-KO brown adipocytes in thermoneutral or cold environments. These phenotypes were caused by the promotion of lipolysis in Prip-KO brown adipocytes, which is triggered by up-regulation of phosphorylation of the lipolysis-related proteins hormone-sensitive lipase and perilipin, followed by activation of UCP1 and/or up-regulation of thermogenesis-related genes (e.g. peroxisome proliferator-activated receptor-γ coactivator-1α). The results indicate that PRIP negatively regulates UCP1-mediated thermogenesis in brown adipocytes. PMID:26706316

  13. cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C{beta}4 (PLCB4)

    SciTech Connect

    Alvarez, R.A.; Ghalayini, A.J.; Anderson, R.E.

    1995-09-01

    Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLC{beta}4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC{beta}4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC{beta}4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by florescence in situ hybridization. 4 refs., 5 figs.

  14. Substance P Activates Ca2+-Permeable Nonselective Cation Channels through a Phosphatidylcholine-Specific Phospholipase C Signaling Pathway in nNOS-Expressing GABAergic Neurons in Visual Cortex.

    PubMed

    Endo, Toshiaki; Yanagawa, Yuchio; Komatsu, Yukio

    2016-02-01

    To understand the functions of the neocortex, it is essential to characterize the properties of neurons constituting cortical circuits. Here, we focused on a distinct group of GABAergic neurons that are defined by a specific colocalization of intense labeling for both neuronal nitric oxide synthase (nNOS) and substance P (SP) receptor [neurokinin 1 (NK1) receptors]. We investigated the mechanisms of the SP actions on these neurons in visual cortical slices obtained from young glutamate decarboxylase 67-green fluorescent protein knock-in mice. Bath application of SP induced a nonselective cation current leading to depolarization that was inhibited by the NK1 antagonists in nNOS-immunopositive neurons. Ruthenium red and La(3+), transient receptor potential (TRP) channel blockers, suppressed the SP-induced current. The SP-induced current was mediated by G proteins and suppressed by D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), but not by inhibitors of phosphatidylinositol-specific PLC, adenylate cyclase or Src tyrosine kinases. Ca(2+) imaging experiments under voltage clamp showed that SP induced a rise in intracellular Ca(2+) that was abolished by removal of extracellular Ca(2+) but not by depletion of intracellular Ca(2+) stores. These results suggest that SP regulates nNOS neurons by activating TRP-like Ca(2+)-permeable nonselective cation channels through a PC-PLC-dependent signaling pathway.

  15. Efficient pig ICSI using Percoll-selected spermatozoa; evidence for the essential role of phospholipase C-ζ in ICSI success.

    PubMed

    Nakai, Michiko; Suzuki, Shun-Ichi; Ito, Junya; Fuchimoto, Dai-Ichiro; Sembon, Shoichiro; Noguchi, Junko; Onishi, Akira; Kashiwazaki, Naomi; Kikuchi, Kazuhiro

    2016-12-20

    In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used. A proportion of the oocytes injected with control sperm were subjected to electrical stimulation at 1 h after ICSI (Cont + ES group). It was found that the Percoll group showed a large amount of PLCζ in comparison with the Control group. Furthermore, application of Percoll-selected sperm for ICSI increased the efficiency of fertilization and embryo development. Thus, these results indicate the Percoll-selected sperm have sufficient PLCζ and high oocyte-activating ability after ICSI in pigs.

  16. Protein kinase C involvement in homologous desensitization of delta-opioid receptor coupled to Gi1-phospholipase C activation in Xenopus oocytes.

    PubMed

    Ueda, H; Miyamae, T; Hayashi, C; Watanabe, S; Fukushima, N; Sasaki, Y; Iwamura, T; Misu, Y

    1995-11-01

    We have developed the coexpression system of both delta-opioid receptor (DOR1) and M2-muscarinic receptor (M2) which mediate agonist-evoked currents due to common post-receptor mechanisms including Gi1 and phospholipase C (PLC) activation in Xenopus oocytes reconstituted with Gi1 alpha. The DOR1-currents by 100 nM D-Ser2-leu-enkephalin-Thr6 (DSLET) were selectively desensitized by 10 nM phorbol 12-myristate 13-acetate (PMA). The PMA-desensitization of DSLET-currents was abolished in the presence of calphostin C, a protein kinase C inhibitor, or reversed by an intracellular injection of calcineurin, a protein phosphatase 2B. When a higher concentration (3 microM) of DSLET was used, DSLET-currents were rapidly desensitized by repeated challenges of DSLET itself. However, repeated challenges of 10 microM ACh caused no influence on such DSLET- or M2-currents. The desensitization of DSLET-currents was selectively reversed by protein kinase C inhibitors. Similar results were also obtained with various delta-opioid agonists. These results suggest that protein kinase C is involved in the homologous desensitization of delta-opioid receptors.

  17. Molecular Characterization and Comparison of Phospholipase C zeta (PLCZ1) Gene Between Swamp (Bubalus carabanensis) and Riverine (Bubalus bubalis) Buffaloes: Its Implications and Future Perspectives.

    PubMed

    Atabay, Eufrocina P; Tadeo, Roseline D; Atabay, Edwin C; Venturina, Emma V; Fissore, Rafael A; Mingala, Claro N

    2017-08-11

    Phospholipase C zeta, a novel sperm-specific protein which is widely known to induce oocyte activation following fertilization, had already been characterized in various mammalian species, but not in water buffaloes thus far. The present study was conducted to initially characterize and compare the sequences of PLCZ1 gene of swamp and riverine buffaloes. Semen samples were collected; total RNA was extracted and reverse-transcribed. PLCZ1 cDNA was then amplified, and submitted for sequencing. Buffalo PLCZ1 gene yielded a sequence of 1905 base pair nucleotides translated into 634 bp amino acids. In general, the buffalo PLCZ1 gene was found to have high sequence identity with cattle and other domestic species. Similarly, significant residues and motifs in PLCZ1 gene sequence are found conserved in water buffaloes. However, there are variations in sequences identified between types of water buffaloes that may play a role in species-specific differences in terms of gene and protein expression, physiological mechanisms, and biological functions. The molecular information on buffalo PLCZ1 gene is highly valuable in subsequent works such as correlation studies on the identified gene variations with semen quality and fertility, and the development of biomarkers for bull fertility.

  18. Rhodopsin 5– and Rhodopsin 6–Mediated Clock Synchronization in Drosophila melanogaster Is Independent of Retinal Phospholipase C-β Signaling

    PubMed Central

    Szular, Joanna; Sehadova, Hana; Gentile, Carla; Szabo, Gisela; Chou, Wen-Hai; Britt, Steven G.; Stanewsky, Ralf

    2015-01-01

    Circadian clocks of most organisms are synchronized with the 24-hour solar day by the changes of light and dark. In Drosophila, both the visual photoreceptors in the compound eyes as well as the blue-light photoreceptor Cryptochrome expressed within the brain clock neurons contribute to this clock synchronization. A specialized photoreceptive structure located between the retina and the optic lobes, the Hofbauer-Buchner (H-B) eyelet, projects to the clock neurons in the brain and also participates in light synchronization. The compound eye photoreceptors and the H-B eyelet contain Rhodopsin photopigments, which activate the canonical invertebrate phototransduction cascade after being excited by light. We show here that 2 of the photopigments present in these photoreceptors, Rhodopsin 5 (Rh5) and Rhodopsin 6 (Rh6), contribute to light synchronization in a mutant (norpAP41) that disrupts canonical phototransduction due to the absence of Phospholipase C-β (PLC-β). We reveal that norpAP41 is a true loss-of-function allele, resulting in a truncated PLC-β protein that lacks the catalytic domain. Light reception mediated by Rh5 and Rh6 must therefore utilize either a different (nonretinal) PLC-β enzyme or alternative signaling mechanisms, at least in terms of clock-relevant photoreception. This novel signaling mode may distinguish Rhodopsin-mediated irradiance detection from image-forming vision in Drosophila. PMID:22306971

  19. Involvement of protein kinase C, phospholipase C, and protein tyrosine kinase pathways in oxygen radical generation by asbestos-stimulated alveolar macrophage.

    PubMed

    Lim, Y; Kim, S H; Kim, K A; Oh, M W; Lee, K H

    1997-09-01

    Although asbestos stimulates oxygen radical generation in alveolar macrophages, the exact mechanism is still not clear. The purpose of this study was to compare the ability of three asbestos fibers (amosite, chrysotile, and crocidolite) to generate oxygen radicals in macrophages and examine the mechanism of this action. All asbestos fibers were able to induce chemiluminescence but chrysotile induced maximal chemiluminescence at higher concentrations than amosite and crocidolite. Protein kinase C (PKC) inhibitors (sphingosine and staurosporine) suppressed the ability of asbestos to induce oxygen radical generation. Phospholipase C (PLC) inhibitors (U73122 and neomycin) and protein tyrosine kinase (PTK) inhibitors (erbstatin and genistein) decreased oxygen radical generation of asbestos-stimulated alveolar macrophages. Oxygen radical generation was not suppressed by an adenylate cyclase activator (forskolin), a protein kinase A inhibitor (H-8), and a protein serine-threonine phosphatase inhibitor (okadaic acid). PLC and PTK inhibitors suppressed the increment of phosphoinositide turnover by amosite. These results suggest that asbestos fibers induce the generation of oxygen radicals through PTK, PLC, and PKC pathways in a dose-response pattern.

  20. Phospholipase C-gamma1 is a guanine nucleotide exchange factor for dynamin-1 and enhances dynamin-1-dependent epidermal growth factor receptor endocytosis.

    PubMed

    Choi, Jang Hyun; Park, Jong Bae; Bae, Sun Sik; Yun, Sanguk; Kim, Hyeon Soo; Hong, Won-Pyo; Kim, Il-Shin; Kim, Jae Ho; Han, Mi Young; Ryu, Sung Ho; Patterson, Randen L; Snyder, Solomon H; Suh, Pann-Ghill

    2004-08-01

    Phospholipase C-gamma1 (PLC-gamma1), which interacts with a variety of signaling molecules through its two Src homology (SH) 2 domains and a single SH3 domain has been implicated in the regulation of many cellular functions. We demonstrate that PLC-gamma1 acts as a guanine nucleotide exchange factor (GEF) of dynamin-1, a 100 kDa GTPase protein, which is involved in clathrin-mediated endocytosis of epidermal growth factor (EGF) receptor. Overexpression of PLC-gamma1 increases endocytosis of the EGF receptor by increasing guanine nucleotide exchange activity of dynamin-1. The GEF activity of PLC-gamma1 is mediated by the direct interaction of its SH3 domain with dynamin-1. EGF-dependent activation of ERK and serum response element (SRE) are both up-regulated in PC12 cells stably overexpressing PLC-gamma1, but knockdown of PLC-gamma1 by siRNA significantly reduces ERK activation. These results establish a new role for PLC-gamma1 in the regulation of endocytosis and suggest that endocytosis of activated EGF receptors may mediate PLC-gamma1-dependent proliferation.

  1. Phospholipase C-epsilon augments epidermal growth factor-dependent cell growth by inhibiting epidermal growth factor receptor down-regulation.

    PubMed

    Yun, Sanguk; Hong, Won-Pyo; Choi, Jang Hyun; Yi, Kye Sook; Chae, Suhn-Kee; Ryu, Sung Ho; Suh, Pann-Ghill

    2008-01-04

    The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-epsilon. The overexpression of PLC-epsilon led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-epsilon RA2 domain independently of Ras. The interaction of PLC-epsilon with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-epsilon led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-epsilon in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-epsilon could promote EGF-dependent cell growth by suppressing receptor down-regulation.

  2. Subtype-specific role of phospholipase C-beta in bradykinin and LPA signaling through differential binding of different PDZ scaffold proteins.

    PubMed

    Choi, Jung Woong; Lim, Seyoung; Oh, Yong-Seok; Kim, Eung-Kyun; Kim, Sun-Hee; Kim, Yun-Hee; Heo, Kyun; Kim, Jaeyoon; Kim, Jung Kuk; Yang, Yong Ryul; Ryu, Sung Ho; Suh, Pann-Ghill

    2010-07-01

    Among phospholipase C (PLC) isozymes (beta, gamma, delta, epsilon, zeta and eta), PLC-beta plays a key role in G-protein coupled receptor (GPCR)-mediated signaling. PLC-beta subtypes are often overlapped in their distribution, but have unique knock-out phenotypes in organism, suggesting that each subtype may have the different role even within the same type of cells. In this study, we examined the possibility of the differential coupling of each PLC-beta subtype to GPCRs, and explored the molecular mechanism underlying the specificity. Firstly, we found that PLC-beta1 and PLC-beta 3 are activated by bradykinin (BK) or lysophosphatidic acid (LPA), respectively. BK-triggered phosphoinositides hydrolysis and subsequent Ca(2+) mobilization were abolished specifically by PLC-beta1 silencing, whereas LPA-triggered events were by PLC-beta 3 silencing. Secondly, we showed the evidence that PDZ scaffold proteins is a key mediator for the selective coupling between PLC-beta subtype and GPCR. We found PAR-3 mediates physical interaction between PLC-beta1 and BK receptor, while NHERF2 does between PLC-beta 3 and LPA(2) receptor. Consistently, the silencing of PAR-3 or NHERF2 blunted PLC signaling induced by BK or LPA respectively. Taken together, these data suggest that each subtype of PLC-beta is selectively coupled to GPCR via PDZ scaffold proteins in given cell types and plays differential role in the signaling of various GPCRs.

  3. Determination of the kinetic parameters for phospholipase C (Bacillus cereus) on different phospholipid substrates using a chromogenic assay based on the quantitation of inorganic phosphate.

    PubMed

    Hergenrother, P J; Martin, S F

    1997-08-15

    The kinetic parameters of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) have been evaluated for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine substrates with a new assay based on the quantitation of inorganic phosphate (Pi). Treatment of the phosphomonoester product of the PLCBc-catalyzed hydrolysis of these phospholipids with alkaline phosphatase releases Pi. This Pi forms a complex with ammonium molybdate that is then reduced by ascorbic acid to provide a blue molybdenum chromogen with an absorbance maximum at 700 nm. This highly sensitive assay may be used to determine accurately less than 5 nmol of Pi in solution. Performing the assay in 96-well plates provides a rapid and convenient method to evaluate a variety of phospholipids as substrates for PLCBc. The assay has been utilized to ascertain the kinetic constants for the PLCBc-catalyzed hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphocholine, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dihexanoyl-sn-glycero-3-phospho-L-serine. It is found that these compounds are substrates for the enzyme with their VmaxS being in the order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine.

  4. Mutation of the phospholipase C-γ1–binding site of LAT affects both positive and negative thymocyte selection

    PubMed Central

    Sommers, Connie L.; Lee, Jan; Steiner, Kevin L.; Gurson, Jordan M.; DePersis, Corinne L.; El-Khoury, Dalal; Fuller, Claudette L.; Shores, Elizabeth W.; Love, Paul E.; Samelson, Lawrence E.

    2005-01-01

    Linker for activation of T cells (LAT) is a scaffolding adaptor protein that is critical for T cell development and function. A mutation of LAT (Y136F) that disrupts phospholipase C-γ1 activation and subsequent calcium influx causes a partial block in T cell development and leads to a severe lymphoproliferative disease in homozygous knock-in mice. One possible contribution to the fatal disease of LAT Y136F knock-in mice could be from autoreactive T cells generated in these mice because of altered thymocyte selection. To examine the impact of the LAT Y136F mutation on thymocyte positive and negative selection, we bred this mutation onto the HY T cell receptor (TCR) transgenic, recombination activating gene-2 knockout background. Female mice with this genotype showed a severe defect in positive selection, whereas male mice exhibited a phenotype resembling positive selection (i.e., development and survival of CD8hi HY TCR-specific T cells) instead of negative selection. These results support the hypothesis that in non-TCR transgenic, LAT Y136F knock-in mice, altered thymocyte selection leads to the survival and proliferation of autoreactive T cells that would otherwise be negatively selected in the thymus. PMID:15795236

  5. Roles of brain phosphatidylinositol-specific phospholipase C and diacylglycerol lipase in centrally administered histamine-induced adrenomedullary outflow in rats.

    PubMed

    Shimizu, Takahiro; Yamaguchi, Naoko; Okada, Shoshiro; Lu, Lianyi; Sasaki, Tsuyoshi; Yokotani, Kunihiko

    2007-10-01

    Recently, we reported that intracerebroventricularly (i.c.v.) administered histamine evokes the secretion of noradrenaline and adrenaline from adrenal medulla by brain cyclooxygenase-1- and thromboxane A2-mediated mechanisms in rats. These results suggest the involvement of brain arachidonic acid cascade in the histamine-induced activation of the central adrenomedullary outflow. Arachidonic acid is released mainly by phospholipase A2 (PLA2)-dependent pathway or phospholipase C (PLC)/diacylglycerol lipase-dependent pathway. In the present study, histamine (27 nmol/animal, i.c.v.) -induced elevation of plasma noradrenaline and adrenaline was dose-dependently reduced by U-73122 (PLC inhibitor) (10 and 100 nmol/animal, i.c.v.), ET-18-OCH3 (phosphatidylinositol-specific PLC inhibitor) (10 and 30 nmol/animal, i.c.v.) and RHC-80267 (diacylglycerol lipase inhibitor) (1.3 and 2.6 micromol/animal, i.c.v.). However, mepacrine (PLA2 inhibitor) (1.1 and 2.2 micromol/animal, i.c.v.) and D609 (phosphatidylcholine-specific PLC inhibitor) (30, 100 and 300 nmol/animal, i.c.v.) had no effect. These results suggest the involvement of brain phosphatidylinositol-specific PLC and diacylglycerol lipase in the centrally administered histamine-induced activation of the adrenomedullary outflow in rats.

  6. Phospholipase C Signaling via the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Is Essential for Normal Bone Responses to PTH

    PubMed Central

    Guo, Jun; Liu, Minlin; Yang, Dehong; Bouxsein, Mary L.; Thomas, Clare C.; Schipani, Ernestina; Bringhurst, F. Richard; Kronenberg, Henry M.

    2010-01-01

    We have previously shown that differentiation of hypertrophic chondrocytes is delayed in mice expressing a mutated PTH/PTHrP receptor (PTHR) (called DSEL here) that stimulates adenylyl cyclase normally but fails to activate phospholipase C (PLC). To better understand the role of PLC signaling via the PTHR in skeletal and mineral homeostasis, we examined these mice fed a normal or calcium-deficient diet. On a standard diet, DSEL mice displayed a modest decrease in bone mass. Remarkably, when fed a low-calcium diet or infused with PTH, DSEL mice exhibited strikingly curtailed peritrabecular stromal cell responses and attenuated new bone formation when compared with Wt mice. Attenuated in vitro colony formation was also observed in bone marrow cells derived from DSEL mice fed a low-calcium diet. Furthermore, PTH stimulated proliferation and increased mRNAs encoding cyclin D1 in primary osteoblasts derived from Wt but not from DSEL mice. Our data indicate that PLC signaling through the PTHR is required for skeletal homeostasis. PMID:20501677

  7. Phospholipase C signaling via the parathyroid hormone (PTH)/PTH-related peptide receptor is essential for normal bone responses to PTH.

    PubMed

    Guo, Jun; Liu, Minlin; Yang, Dehong; Bouxsein, Mary L; Thomas, Clare C; Schipani, Ernestina; Bringhurst, F Richard; Kronenberg, Henry M

    2010-08-01

    We have previously shown that differentiation of hypertrophic chondrocytes is delayed in mice expressing a mutated PTH/PTHrP receptor (PTHR) (called DSEL here) that stimulates adenylyl cyclase normally but fails to activate phospholipase C (PLC). To better understand the role of PLC signaling via the PTHR in skeletal and mineral homeostasis, we examined these mice fed a normal or calcium-deficient diet. On a standard diet, DSEL mice displayed a modest decrease in bone mass. Remarkably, when fed a low-calcium diet or infused with PTH, DSEL mice exhibited strikingly curtailed peritrabecular stromal cell responses and attenuated new bone formation when compared with Wt mice. Attenuated in vitro colony formation was also observed in bone marrow cells derived from DSEL mice fed a low-calcium diet. Furthermore, PTH stimulated proliferation and increased mRNAs encoding cyclin D1 in primary osteoblasts derived from Wt but not from DSEL mice. Our data indicate that PLC signaling through the PTHR is required for skeletal homeostasis.

  8. Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence.

    PubMed

    Drayer, A L; Meima, M E; Derks, M W; Tuik, R; van Haastert, P J

    1995-10-15

    Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.

  9. Diacylglycerol kinase δ phosphorylates phosphatidylcholine-specific phospholipase C-dependent, palmitic acid-containing diacylglycerol species in response to high glucose levels.

    PubMed

    Sakai, Hiromichi; Kado, Sayaka; Taketomi, Akinobu; Sakane, Fumio

    2014-09-19

    Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. WDR26 functions as a scaffolding protein to promote Gβγ-mediated phospholipase C β2 (PLCβ2) activation in leukocytes.

    PubMed

    Sun, Zhizeng; Smrcka, Alan V; Chen, Songhai

    2013-06-07

    We have recently identified WDR26 as a novel WD40 repeat protein that binds Gβγ and promotes Gβγ signaling during leukocyte migration. Here, we have determined the mechanism by which WDR26 enhances Gβγ-mediated phospholipase C β2 (PLCβ2) activation in leukocytes. We show that WDR26 not only directly bound Gβγ but also PLCβ2. The binding sites of WDR26 and PLCβ2 on Gβ1γ2 were overlapping but not identical. WDR26 used the same domains for binding Gβγ and PLCβ but still formed a signaling complex with Gβγ and PLCβ2 probably due to the fact that WDR26 formed a higher order oligomer through its Lis homology and C-terminal to LisH (LisH-CTLH) and WD40 domains. Additional studies indicated that the formation of higher order oligomers was required for WDR26 to promote PLCβ2 interaction with and activation by Gβγ. Moreover, WDR26 was required for PLCβ2 translocation from the cytosol to the membrane in polarized leukocytes, and the translocation of PLCβ2 was sufficient to cause partial activation of PLCβ2. Collectively, our data indicate that WDR26 functions as a scaffolding protein to promote PLCβ2 membrane translocation and interaction with Gβγ, thereby enhancing PLCβ2 activation in leukocytes. These findings have identified a novel mechanism of regulating Gβγ signaling through a scaffolding protein.

  11. PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain*

    PubMed Central

    Zhang, Zhao; Takeuchi, Hiroshi; Gao, Jing; Wang, DaGuang; James, Declan J.; Martin, Thomas F. J.; Hirata, Masato

    2013-01-01

    Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K+-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation. PMID:23341457

  12. The Metabotropic Glutamate Receptor mGlu7 Activates Phospholipase C, Translocates Munc-13-1 Protein, and Potentiates Glutamate Release at Cerebrocortical Nerve Terminals*

    PubMed Central

    Martín, Ricardo; Durroux, Thierry; Ciruela, Francisco; Torres, Magdalena; Pin, Jean-Philippe; Sánchez-Prieto, José

    2010-01-01

    At synaptic boutons, metabotropic glutamate receptor 7 (mGlu7 receptor) serves as an autoreceptor, inhibiting glutamate release. In this response, mGlu7 receptor triggers pertussis toxin-sensitive G protein activation, reducing presynaptic Ca2+ influx and the subsequent depolarization evoked release. Here we report that receptor coupling to signaling pathways that potentiate release can be seen following prolonged exposure of nerve terminals to the agonist l-(+)-phosphonobutyrate, l-AP4. This novel mGlu7 receptor response involves an increase in the release induced by the Ca2+ ionophore ionomycin, suggesting a mechanism that is independent of Ca2+ channel activity, but dependent on the downstream exocytotic release machinery. The mGlu7 receptor-mediated potentiation resists exposure to pertussis toxin, but is dependent on phospholipase C, and increased phosphatidylinositol (4,5)-bisphosphate hydrolysis. Furthermore, the potentiation of release does not depend on protein kinase C, although it is blocked by the diacylglycerol-binding site antagonist calphostin C. We also found that activation of mGlu7 receptors translocate the active zone protein essential for synaptic vesicle priming, munc13-1, from soluble to particulate fractions. We propose that the mGlu7 receptor can facilitate or inhibit glutamate release through multiple pathways, thereby exerting homeostatic control of presynaptic function. PMID:20375012

  13. Diacylglycerol Kinase δ Phosphorylates Phosphatidylcholine-specific Phospholipase C-dependent, Palmitic Acid-containing Diacylglycerol Species in Response to High Glucose Levels*

    PubMed Central

    Sakai, Hiromichi; Kado, Sayaka; Taketomi, Akinobu; Sakane, Fumio

    2014-01-01

    Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels. PMID:25112873

  14. Efficient pig ICSI using Percoll-selected spermatozoa; evidence for the essential role of phospholipase C-ζ in ICSI success

    PubMed Central

    NAKAI, Michiko; SUZUKI, Shun-ichi; ITO, Junya; FUCHIMOTO, Dai-ichiro; SEMBON, Shoichiro; NOGUCHI, Junko; ONISHI, Akira; KASHIWAZAKI, Naomi; KIKUCHI, Kazuhiro

    2016-01-01

    In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used. A proportion of the oocytes injected with control sperm were subjected to electrical stimulation at 1 h after ICSI (Cont + ES group). It was found that the Percoll group showed a large amount of PLCζ in comparison with the Control group. Furthermore, application of Percoll-selected sperm for ICSI increased the efficiency of fertilization and embryo development. Thus, these results indicate the Percoll-selected sperm have sufficient PLCζ and high oocyte-activating ability after ICSI in pigs. PMID:27725346

  15. Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation

    PubMed Central

    Liu, Ta-Ming; Woyach, Jennifer A.; Zhong, Yiming; Lozanski, Arletta; Lozanski, Gerard; Dong, Shuai; Strattan, Ethan; Lehman, Amy; Zhang, Xiaoli; Jones, Jeffrey A.; Flynn, Joseph; Andritsos, Leslie A.; Maddocks, Kami; Jaglowski, Samantha M.; Blum, Kristie A.; Byrd, John C.; Dubovsky, Jason A.

    2015-01-01

    Ibrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinib’s capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2R665W confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance. PMID:25972157

  16. Phospholipase C gamma 1 (PLCG1) R707Q mutation is counterselected under targeted therapy in a patient with hepatic angiosarcoma

    PubMed Central

    Prenen, Hans; Smeets, Dominiek; Mazzone, Massimiliano; Lambrechts, Diether; Sagaert, Xavier; Sciot, Raf; Debiec-Rychter, Maria

    2015-01-01

    Hepatic angiosarcoma is a rare and aggressive vascular neoplasm. Pathogenic driver mutations are largely unknown. We present the case of a patient with recurrent hepatic angiosarcoma, who initially showed good response to sunitinib, followed by progression. Using comprehensive molecular techniques, we explored the potential mechanisms of resistance. By low-read-depth whole-genome sequencing, the comparison of copy number aberrations (CNAs) of the primary tumor to the skin metastatic lesion that developed after progression on sunitinib, revealed high-level amplification of the 4q11-q13.1 region (containing KIT, PDGFRA and VEGFR2 genes) that was sustained in both lesions. Whole exome sequencing on the germline, primary and metastatic tumor DNAs, resulted in 27 confirmed mutations, 19 of which (including TP53 mutation) presented in both primary and metastatic lesions. One mutation, ZNF331 frameshift deletion, was detected only in the primary tumor. In contrast, seven other mutations, including phospholipase C-gamma1 (PLCG1) R707Q mutation, were found only in the metastatic tumor, indicating selection of cells with the resistant genotype under sunitinib pressure. Our study supports the notion that PLCG1-R707Q mutation may confer VEGFR2-independent signaling and may thus cause resistance against VEGF(R)-directed therapies. This case illustrates also the advantages of using next-generation technologies in identifying individualized targeted therapy. PMID:26474454

  17. ARF1(2-17) does not specifically interact with ARF1-dependent pathways. Inhibition by peptide of phospholipases C beta, D and exocytosis in HL60 cells.

    PubMed

    Fensome, A; Cunningham, E; Troung, O; Cockcroft, S

    1994-07-25

    The small GTP-binding protein ARF has been shown recently to regulate phospholipase D (PLD). In order to investigate the role of ARF proteins in regulated exocytosis, we have used the N-terminal peptide ARF1(2-17) of the ARF1 protein. ARF1 reconstituted PLD activity in cytosol-depleted HL60 cells was inhibited by ARF1(2-17). In the presence of endogenous cytosol, ARF1(2-17) also inhibited GTP-gamma-S-stimulated PLD activity and exocytosis. Mastoparan Politses jadwagae and mastoparan Vespula lewisii which exhibit similar structural properties to ARF1(2-17) also inhibited GTP-gamma-S-stimulated PLD and exocytosis. GTP-gamma-S-stimulated phospholipase C-beta (PLC-beta) was also inhibited by ARF(2-17) and mastoparan. In cytosol-depleted HL60 cells, the ARF(2-17) inhibited the reconstitution of GTP-gamma-S-stimulated PLC-beta activity with exogenously-added PLC-beta 1 and phosphatidylinositol transfer protein. We conclude that the widely-used ARF1(2-17) peptide inhibits both ARF-independent (i.e. PLC-beta) and ARF-dependent pathways (i.e. PLD) and therefore cannot be regarded as a specific inhibitor of ARF function.

  18. Cholinergic Autoantibodies from Primary Sjögren's Syndrome Inhibit Mucin Production via Phospholipase C and Cyclooxygenase-2 In the Rat Submandibular Gland

    PubMed Central

    Passafaro, Daniela; Sterin-Borda, Leonor; Reina, Silvia; Borda, Enri

    2011-01-01

    Background: Patients with primary Sjögren's syndrome (pSS) produce functional IgG against cholinoreceptor of exocrine glands modifying their activity. The aim of the present work was to demonstrate pSS IgG antibodies (pSS IgG) interacting with M3 muscarinic acetylcholine receptors (mAChR) of rats submandibular glands that alter mucin release and production via phospholipase C (PLC) and cyclooxigenase-2 (COX-2) pathways. Methods: Mucin release and production of prostaglandin E2 (PGE2), and total inositol phosphates (InsP) were measured in rat submandibular gland in the presence of pSS IgG auto antibodies. Results: The auto antibodies interacting with M3 mAChR decreased mucin release and production through stimulation of PLC and COX-2. This stimulation leads to an incremental increase in InsP production and in PGE2 generation, inducing signalling through the prostaglandin membrane receptors subtype 2 (EP2). Moreover, the decrease in mucin production had negative correlation with PGE2 generation and InsP accumulation. Conclusion: IgG in patients with pSS could play an important role in the pathoetiology of dry mouth, decreasing the salivary mucin through the production of proinflammatory substances and leading to the reduction in the protection of the oral tissues. PMID:22013477

  19. Carboxyl-terminal basic amino acids in the X domain are essential for the nuclear import of phospholipase C delta1.

    PubMed

    Okada, Masashi; Fujii, Makoto; Yamaga, Masaki; Sugimoto, Hiroaki; Sadano, Hiroyuki; Osumi, Takashi; Kamata, Hideaki; Hirata, Hajime; Yagisawa, Hitoshi

    2002-09-01

    Although phospholipase C (PLC)delta1 containing a functional nuclear export signal (NES) is normally localized at the plasma membrane and in the cytoplasm, it shuttles between the nucleus and the cytoplasm. Since nucleocytoplasmic shuttling of a molecule is generally regulated by a balance between its NES and the nuclear localization signal (NLS), we examined whether PLCdelta1 contains an NLS sequence. A region corresponding to the C terminus of the X domain and the XY-linker, which contains clusters of basic amino acid residues, was essential for the nuclear import of PLCdelta1 in Madin-Darby canine kidney cells. A series of point mutations on lysine residues in this region revealed that K432 and K434 in combination were important for the nuclear import. A short synthetic peptide corresponding to residues 429-442, however, was not able to function as an NLS sequence when they were injected into the cytoplasm in a carrier-conjugated form. Neither a longer peptide equivalent to PLCdelta1 412-498 fused to a protein tag consisting of glutathione S-transferase and green fluorescent protein was imported to the nucleus after microinjection into the cytoplasm. The nuclear import of PLCdelta1 requires the C-terminus of the X domain, particularly the amino acid residues K432 and K434, and the XY-linker. The region alone, however, cannot serve as a functional NLS. The machinery for nuclear transport may require additional structural component(s) of the enzyme.

  20. Requirement of Phospholipase C and Protein Kinase C in Cholecystokinin-mediated Facilitation of NMDA Channel Function and Anxiety-like Behavior

    PubMed Central

    Xiao, Zhaoyang; Jaiswal, Manoj; Deng, Pan-Yue; Matsui, Toshimitsu; Shin, Hee-Sup; Porter, James E.; Lei, Saobo

    2011-01-01

    Whereas cholecystokinin (CCK) has long been known to exert anxiogenic effects in both animal anxiety models and humans, the underlying cellular and molecular mechanisms are ill-defined. CCK interacts with CCK-1 and CCK-2 receptors resulting in up-regulation of phospholipase C (PLC) and protein kinase C (PKC). However, the roles of PLC and PKC in CCK-mediated anxiogenic effects have not been determined. We have shown previously that CCK facilitates glutamate release in the hippocampus especially at the synapses formed by the perforant path and dentate gyrus granule cells via activations of PLC and PKC. Here we further demonstrated that CCK enhanced NMDA receptor function in dentate gyrus granule cells via activation of PLC and PKC pathway. At the single-channel level, CCK increased NMDA single-channel open probability and mean open time, reduced the mean close time and had no effects on the conductance of NMDA channels. Because elevation of glutamatergic functions results in anxiety, we explored the roles of PLC and PKC in CCK-induced anxiogenic actions using the Vogel Conflict Test (VCT). Our results from both pharmacological approach and knockout mice demonstrated that microinjection of CCK into the dentate gyrus concentration-dependently increased anxiety-like behavior via activation of PLC and PKC. Our results provide a novel unidentified signaling mechanism whereby CCK increases anxiety. PMID:22072552

  1. Novel interrelationship between salicylic acid, abscisic acid, and PIP2-specific phospholipase C in heat acclimation-induced thermotolerance in pea leaves.

    PubMed

    Liu, Hong-Tao; Liu, Yan-Yan; Pan, Qiu-Hong; Yang, Hao-Ru; Zhan, Ji-Cheng; Huang, Wei-Dong

    2006-01-01

    Increasing evidence suggests that heat acclimation and exogenous salicylic acid (SA) and abscisic acid (ABA) may lead to the enhancement of thermotolerance in plants. In this study, the roles that free SA, conjugated SA, ABA, and phosphatidylinositol-4,5-bisphosphate (PIP(2))-specific phospholipase C (PLC) play in thermotolerance development induced by heat acclimation (38 degrees C) were investigated. To evaluate their potential functions, three inhibitors of synthesis or activity were infiltrated into pea leaves prior to heat acclimation treatment. The results showed that the burst of free SA in response to heat acclimation could be attributed to the conversion of SA 2-O-D-glucose, the main conjugated form of SA, to free SA. Inhibition of ABA biosynthesis also resulted in a defect in the free SA peak during heat acclimation. In acquired thermotolerance assessment, the greatest weakness of antioxidant enzyme activity and the most severe heat injury (malondialdehyde content and degree of wilting) were found in pea leaves pre-treated with neomycin, a well-known inhibitor of PIP(2)-PLC activity. PsPLC gene expression was activated by exogenous ABA, SA treatments, and heat acclimation after pre-treatments with a SA biosynthesis inhibitor. From these results, PIP(2)-PLC appears to play a key role in free SA- and ABA-associated reinforcement of thermotolerance resulting from heat acclimation.

  2. Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

    PubMed Central

    Ricci, Alessandro; Pacella, Aurora; Cigliana, Giovanni; Bozzuto, Giuseppina; Podo, Franca; Carpinelli, Giulia

    2017-01-01

    Background The chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM), the most aggressive glioma. Phosphatidylcholine-specific phospholipase C (PC-PLC), a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways. In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells. Methods Confocal microscopy, immunoprecipitation, western blot analyses, and the evaluation of migration and invasion potential were performed on U87MG cells after PC-PLC inhibition with the xanthate D609. The intracellular metabolome was investigated by magnetic resonance spectroscopy; lactate levels and lactate dehydrogenase (LDH) activity were analyzed by colorimetric assay. Results Our studies demonstrated that CXCR4 and PC-PLC co-localize and are associated on U87MG cell membrane. D609 reduced CXCR4 expression, cell proliferation and invasion, interfering with AKT and EGFR activation and expression. Metabolic analyses showed a decrease in intracellular lactate concentration together with a decrement in LDH activity. Conclusions Our data suggest that inhibition of PC-PLC could represent a new molecular approach in glioma biology not only for its ability in modulating cell metabolism, glioma growth and motility, but also for its inhibitory effect on crucial molecules involved in cancer progression. PMID:28423060

  3. Expression of val-12 mutant ras p21 in an IL-3-dependent murine myeloid cell line is associated with loss of serum-dependence and increases in membrane PIP2-specific phospholipase C activity.

    PubMed

    Rizzo, M T; Boswell, H S; English, D; Gabig, T G

    1991-01-01

    We previously showed that the proliferative response of a serum- and interleukin-3 (IL-3)-dependent murine myeloid cell line, NFS/N1-H7, was partially inhibited by pertussis toxin as a result of toxin-induced increased adenylate cyclase activity. In the present studies, we examined the role of the phosphoinositide cycle in the proliferative response of these cells and demonstrated that there was no change in PIP (phosphatidylinositol bisphosphate)-specific phospholipase C activity in response to IL-3 alone. However, serum caused a pertussis toxin-insensitive increase in PIP2-specific phospholipase C activity as reflected by decreased cellular levels of 32P-labelled PIP2. Proliferation of a subline selected from val-12-mutant H-ras-transfected NFS-H7 cells, clone E5, was insensitive to pertussis toxin, occurred in the absence of serum but remained serum-stimulatable and absolutely dependent on IL-3. This val-12 mutant ras-expressing cell line showed an increase in 32P-labelled PIP (phosphatidylinositol phosphate) in response to serum whereas the parent cell line did not. Membrane fractions from 32P-labelled ras-transfected cells displayed higher GTP gamma S-, GTP-, or F(-)-stimulated PIP2-specific phospholipase C activity compared to membranes from the parent cell line. Thus serum-dependence and adenylate cyclase-mediated pertussis toxin-sensitivity of the parent cell line was bypassed by val-12 mutant ras p21, possibly as a result of increased PIP2-specific phospholipase C activity.

  4. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens

    SciTech Connect

    Tomavo, S.; Schwarz, R.T.; Dubremetz, J.F. )

    1989-10-01

    The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with {sup 3}H-fatty acids, ({sup 3}H)ethanolamine, and ({sup 3}H)carbohydrates. Treatment of {sup 3}H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment.

  5. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens.

    PubMed Central

    Tomavo, S; Schwarz, R T; Dubremetz, J F

    1989-01-01

    The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with 3H-fatty acids, [3H]ethanolamine, and [3H]carbohydrates. Treatment of 3H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment. Images PMID:2531282

  6. Negative regulation of parathyroid hormone (PTH)-activated phospholipase C by PTH/PTH-related peptide receptor phosphorylation and protein kinase A.

    PubMed

    Tawfeek, Hesham A W; Abou-Samra, Abdul B

    2008-08-01

    PTH binding to the PTH/PTHrP receptor activates adenylate cyclase/protein kinase A (PKA) and phospholipase C (PLC) pathways and increases receptor phosphorylation. The mechanisms regulating PTH activation of PLC signaling are poorly understood. In the current study, we explored the role of PTH/PTHrP receptor phosphorylation and PKA in PTH activation of PLC. When treated with PTH, LLCPK-1 cells stably expressing a green fluorescent protein (GFP)-tagged wild-type (WT) PTH/PTHrP receptor show a small dose-dependent increase in PLC signaling as measured by inositol trisphosphate accumulation assay. In contrast, PTH treatment of LLCPK-1 cells stably expressing a GFP-tagged receptor mutated in its carboxyl-terminal tail so that it cannot be phosphorylated (PD-GFP) results in significantly higher PLC activation (P<0.001). The effects of PTH on PLC activation are dose dependent and reach maximum at the 100 nm PTH dose. When WT receptor-expressing cells are pretreated with H89, a specific inhibitor of PKA, PTH activation of PLC signaling is enhanced in a dose-dependent manner. H89 pretreatment in PD-GFP cells causes a further increase in PLC activation in response to PTH treatment. Interestingly, PTH and forskolin (adenylate cyclase/PKA pathway activator) treatment causes an increase in PLCbeta3 phosphorylation at the Ser1105 inhibitory site and that increase is blocked by the PKA inhibitor, H89. Expression of a mutant PLCbeta3 in which Ser1105 was mutated to alanine (PLCbeta3-SA), in WT or PD cells increases PTH stimulation of inositol 1,4,5-trisphosphate formation. Altogether, these data suggest that PTH signaling to PLC is negatively regulated by PTH/PTHrP receptor phosphorylation and PKA. Furthermore, phosphorylation at Ser1105 is demonstrated as a regulatory mechanism of PLCbeta3 by PKA.

  7. Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium

    SciTech Connect

    Henriksen, E.J.; Rodnick, K.J.; Holloszy, J.O. )

    1989-12-25

    It has been hypothesized on the basis of studies on BC3H-1 myocytes that diacylglycerol generation with activation of protein kinase C (PKC) is involved in the stimulation of glucose transport in muscle by insulin. In the present study, we used the rat epitrochlearis muscle to evaluate the possibility that PKC activity mediates the stimulation of glucose transport by insulin in mammalian skeletal muscle. Phospholipase C from Clostridium perfringens (PLC-Cp), which generates diacylglycerol from membrane phospholipids, and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) induced increases in glucose transport activity (assessed using 3-O-methylglucose transport) that were approximately 80 and approximately 20% as great, respectively, as that induced by a maximal insulin stimulus. PLC-Cp and PMA both caused a approximately 2-fold increase in membrane-associated PKC activity. In contrast, insulin did not affect PKC activity. These findings argue against a role of diacylglycerol-mediated PKC activation in the stimulation of skeletal muscle glucose transport by insulin. They also show that the BC3H-1 myocyte is not a good model for studying regulation of glucose transport in skeletal muscle. Neither the submaximal nor maximal effects of PLC-Cp and insulin on glucose transport were additive, suggesting that PLC-Cp interferes with insulin action. The maximal effects of PLC-Cp and hypoxia or muscle contractions were also not additive. However, the submaximal effects of hypoxia and PLC-Cp were completely additive. These findings raise the possibility that PLC-Cp stimulates glucose transport by the exercise/hypoxia-activated, not the insulin-activated, pathway in skeletal muscle.

  8. Critical roles of Gi/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels.

    PubMed

    Thakur, Dhananjay P; Tian, Jin-bin; Jeon, Jaepyo; Xiong, Jian; Huang, Yu; Flockerzi, Veit; Zhu, Michael X

    2016-01-26

    Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCβ pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and Gi/o-coupled µ opioid receptor, µ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca(2+) and PIP2. Reducing PIP2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak Gi/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of Gi/o-mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident Gi/o, Ca(2+), and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems.

  9. Phospholipase C-Related Catalytically Inactive Protein (PRIP) Regulates Lipolysis in Adipose Tissue by Modulating the Phosphorylation of Hormone-Sensitive Lipase

    PubMed Central

    Okumura, Toshiya; Harada, Kae; Oue, Kana; Zhang, Jun; Asano, Satoshi; Hayashiuchi, Masaki; Mizokami, Akiko; Tanaka, Hiroto; Irifune, Masahiro; Kamata, Nobuyuki; Hirata, Masato; Kanematsu, Takashi

    2014-01-01

    Phosphorylation of hormone-sensitive lipase (HSL) and perilipin by protein kinase A (PKA) promotes the hydrolysis of lipids in adipocytes. Although activation of lipolysis by PKA has been well studied, inactivation via protein phosphatases is poorly understood. Here, we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a binding partner for protein phosphatase 1 and protein phosphatase 2A (PP2A), is involved in lipolysis by regulating phosphatase activity. PRIP knockout (PRIP-KO) mice displayed reduced body-fat mass as compared with wild-type mice fed with standard chow ad libitum. Most other organs appeared normal, suggesting that mutant mice had aberrant fat metabolism in adipocytes. HSL in PRIP-KO adipose tissue was highly phosphorylated compared to that in wild-type mice. Starvation of wild-type mice or stimulation of adipose tissue explants with the catabolic hormone, adrenaline, translocated both PRIP and PP2A from the cytosol to lipid droplets, but the translocation of PP2A was significantly reduced in PRIP-KO adipocytes. Consistently, the phosphatase activity associated with lipid droplet fraction in PRIP-KO adipocytes was significantly reduced and was independent of adrenaline stimulation. Lipolysis activity, as assessed by measurement of non-esterified fatty acids and glycerol, was higher in PRIP-KO adipocytes. When wild-type adipocytes were treated with a phosphatase inhibitor, they showed a high lipolysis activity at the similar level to PRIP-KO adipocytes. Collectively, these results suggest that PRIP promotes the translocation of phosphatases to lipid droplets to trigger the dephosphorylation of HSL and perilipin A, thus reducing PKA-mediated lipolysis. PMID:24945349

  10. Binding of phosphoinositide-specific phospholipase C-zeta (PLC-zeta) to phospholipid membranes: potential role of an unstructured cluster of basic residues.

    PubMed

    Nomikos, Michail; Mulgrew-Nesbitt, Anna; Pallavi, Payal; Mihalyne, Gyongyi; Zaitseva, Irina; Swann, Karl; Lai, F Anthony; Murray, Diana; McLaughlin, Stuart

    2007-06-01

    Phospholipase C-zeta (PLC-zeta) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-delta1, but lacks the PH domain that anchors PLC-delta1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-zeta interacts with membranes. The linker region between the X and Y catalytic domains of PLC-zeta, however, contains a cluster of basic residues not present in PLC-delta1. Application of electrostatic theory to a homology model of PLC-zeta suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-zeta to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2:1 PC/PS vesicles increases K about 10-fold, to 5x10(3) M-1, a biologically relevant value. Expressed fragments corresponding to the PLC-zeta X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge=+7) within the linker region, PLC-zeta-(374-385), binds to PC/PS vesicles with higher affinity than PLC-zeta, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-zeta may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.

  11. Signal-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate without activation of phospholipase C: implications on gating of Drosophila TRPL (transient receptor potential-like) channel.

    PubMed

    Lev, Shaya; Katz, Ben; Tzarfaty, Vered; Minke, Baruch

    2012-01-06

    In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P(2) in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P(2) was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P(2) is not an inhibitor of TRPL channel activation. PI(4,5)P(2) hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P(2) levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P(2) is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.

  12. Platelet activation by bacterial phospholipase C involves phosphoinositide turnover and phosphorylation of 47,000 dalton but not 20,000 dalton protein

    SciTech Connect

    Huzoor-Akbar; Anwer, K.

    1986-05-01

    This study was conducted to examine the role of phosphoinositides (PIns) and phosphorylation of 47,000 dalton (P47) and 20,000 dalton (P20) proteins in platelet activation by bacterial phospholipase C (PLC). PLC induced serotonin secretion (SS) and platelet aggregation (PA) in a concentration dependent manner. PLC (0.02 U/ml) caused phosphorylation of P47 in a time dependent manner (27% at 0.5 min to 378% at 7 min). PLC did not induce more than 15% phosphorylation of P20 by 7 min. Aspirin (500 ..mu..M) blocked phosphorylation of P20 but did not inhibit SS, PA or phosphorylation of P47. PLC (0.04 U/ml) decreased radioactivity (cpm) in /sup 32/P labeled phosphatidylinositol (PI), PI-4,5-bis-PO4 (PIP2) and PI-4-PO4 (PIP) by 20%, 12% and 7.5% respectively at 15 sec. The level of PI but not that of PIP2 returned to base line in 3 min. PIP level increased above control values within one min. PLC increased phosphatidic acid level (75% at 0.5 min. to 1545% at 3 min). In other experiments PLC produced diacylglycerol (DAG) in a time and concentration dependent manner. However, no DAG was detectable in the first 60 sec. These data suggest that: (a) PIns turnover and phosphorylation of P47 but not that of P20 is involved in platelet activation by PLC; and (b) DAG production from outer membrane phospholipids is not a prerequisite for platelet activation by PLC.

  13. Cloning of a novel phospholipase C-delta isoform from pacific purple sea urchin (Strongylocentrotus purpuratus) gametes and its expression during early embryonic development.

    PubMed

    Coward, Kevin; Owen, Helen; Poustka, Albert J; Hibbitt, Olivia; Tunwell, Richard; Kubota, Hiroki; Swann, Karl; Parrington, John

    2004-01-23

    Calcium (Ca(2+)) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, including fertilization and development of the embryo. One of the key mechanisms involved in triggering intracellular calcium release is the generation of the second messenger inositol-1,4,5-phosphate (IP(3)) by the phospholipase C (PLC) class of enzymes. Although five distinct forms of PLC have been identified in mammals (beta, gamma, delta, epsilon, and zeta), only one, PLCgamma, has thus far been detected in echinoderms. In the present study, we describe the isolation of a cDNA encoding a novel PLC isoform of the delta (delta) subclass, PLC-deltasu, from the egg of the Pacific purple sea urchin Strongylocentrotus purpuratus. We also demonstrate the presence of this PLC within the sperm and in the early embryo. The PLC-deltasu cDNA (2.44kb) encodes a 742 amino acid polypeptide with an open reading frame of 84.6kDa and a pI of 6.04. All of the characteristic domains found in mammalian PLCdelta isoforms (PH domain, EF hands, an X-Y catalytic region, and a C2 domain) are present in PLC-deltasu. A homology search revealed that PLC-deltasu shares most sequence identity with bovine PLCdelta2 (39%). We present evidence that PLC-deltasu is expressed in unfertilized eggs, fertilized eggs, and in the early embryo. In addition to Northern and polymerase chain reaction (PCR) analyses, in situ hybridization experiments further demonstrated that the embryonic regions within which the PLC-deltasu transcript can be detected during early embryonic development are associated with the highest levels of proliferative activity, suggesting a possible involvement with metabolism or cell cycle regulation.

  14. The roles of phospholipase C activation and alternative ADAR1 and ADAR2 pre-mRNA splicing in modulating serotonin 2C-receptor editing in vivo

    PubMed Central

    Schmauss, Claudia; Zimnisky, Ross; Mehta, Mukti; Shapiro, Lauren P.

    2010-01-01

    The serotonin 2C receptor (5-HT2CR), a Gq-protein-coupled neurotransmitter receptor, exists in multiple isoforms that result from RNA editing of five exonic adenosines that are converted to inosines. In the adult brain, editing of 5-HT2C pre-mRNA exhibits remarkable plasticity in response to environmental and neurochemical stimuli. Here, we investigated two potential mechanisms underlying these plastic changes in adult 5-HT2CR editing phenotypes in vivo: activation of phospholipase C (PLC) and alternative splicing of pre-mRNA encoding the editing enzymes ADAR1 and ADAR2. Studies on two inbred strains of mice (C57Bl/6 and Balb/c) revealed that sustained stimulation of PLC—a downstream effector of activated Gαq protein—increased editing of forebrain neocortical 5-HT2C pre-mRNA at two sites known to be targeted by ADAR2. Moreover, changes in relative expression of the alternatively spliced “a” and “b” mRNA isoforms of ADAR1 and ADAR2 also correlate with changes in 5-HT2CR editing. The site-specific changes in 5-HT2CR editing detected in mice with different “a” over “b” ADAR mRNA isoform ratios only partially overlap with those evoked by sustained PLC activation and are best explained by the increased editing efficiency of ADAR1. Thus, activation of PLC and alternative splicing of ADAR pre-mRNA have both overlapping and specific roles in modulating 5-HT2CR editing phenotypes. PMID:20651031

  15. Kinetics of bacterial phospholipase C activity at micellar interfaces: effect of substrate aggregate microstructure and a model for the kinetic parameters.

    PubMed

    Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

    2008-12-25

    Activity at micellar interfaces of bacterial phospholipase C from Bacillus cereus on phospholipids solubilized in micelles was investigated with the goal of elucidating the role of the interface microstructure and developing further an existing kinetic model. Enzyme kinetics and physicochemical characterization of model substrate aggregates were combined, thus enabling the interpretation of kinetics in the context of the interface. Substrates were diacylphosphatidylcholine of different acyl chain lengths in the form of mixed micelles with dodecyldimethylammoniopropanesulfonate. An early kinetic model, reformulated to reflect the interfacial nature of the kinetics, was applied to the kinetic data. A better method of data treatment is proposed, use of which makes the presence of microstructure effects quite transparent. Models for enzyme-micelle binding and enzyme-lipid binding are developed, and expressions incorporating the microstructural properties are derived for the enzyme-micelle dissociation constant K(s) and the interface Michaelis-Menten constant, K(M). Use of these expressions in the interface kinetic model brings excellent agreement between the kinetic data and the model. Numerical values for the thermodynamic and kinetic parameters are determined. Enzyme-lipid binding is found to be an activated process with an acyl chain length dependent free energy of activation that decreases with micelle lipid molar fraction with a coefficient of about -15RT and correlates with the tightness of molecular packing in the substrate aggregate. Thus, the physical insight obtained includes a model for the kinetic parameters that shows that these parameters depend on the substrate concentration and acyl chain length of the lipid. Enzyme-micelle binding is indicated to be hydrophobic and solvent mediated with a dissociation constant of 1.2 mM.

  16. Phospholipase C delta-4 overexpression upregulates ErbB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells.

    PubMed

    Leung, David W; Tompkins, Chris; Brewer, Jim; Ball, Alexey; Coon, Mike; Morris, Valerie; Waggoner, David; Singer, Jack W

    2004-05-13

    The expression of the rodent phosphoinositide-specific phospholipase C delta-4 (PLCdelta4) has been found to be elevated upon mitogenic stimulation and expression analysis have linked the upregulation of PLCdelta4 expression with rapid proliferation in certain rat transformed cell lines. The human homologue of PLCdelta4 has not been extensively characterized. Accordingly, we investigate the effects of overexpression of human PLCdelta4 on cell signaling and proliferation in this study. The cDNA for human PLCdelta4 has been isolated and expressed ectopically in breast cancer MCF-7 cells. Overexpression of PLCdelta4 selectively activates protein kinase C-phi and upregulates the expression of epidermal growth factor receptors EGFR/erbB1 and HER2/erbB2, leading to constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in MCF-7 cells. MCF-7 cells stably expressing PLCdelta4 demonstrates several phenotypes of transformation, such as rapid proliferation in low serum, formation of colonies in soft agar, and capacity to form densely packed spheroids in low-attachment plates. The growth signaling responses induced by PLCdelta4 are not reversible by siRNA. Overexpression or dysregulated expression of PLCdelta4 may initiate oncogenesis in certain tissues through upregulation of ErbB expression and activation of ERK pathway. Since the growth responses induced by PLCdelta4 are not reversible, PLCdelta4 itself is not a suitable drug target, but enzymes in pathways activated by PLCdelta4 are potential therapeutic targets for oncogenic intervention.

  17. A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways

    PubMed Central

    1994-01-01

    The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein- GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI- PLC, cell-associated gp63 could not be detected in immunoblots. Pulse- chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed. PMID:8132715

  18. Innate defense regulator IDR-1018 activates human mast cells through G protein-, phospholipase C-, MAPK- and NF-ĸB-sensitive pathways.

    PubMed

    Yanashima, Kensuke; Chieosilapatham, Panjit; Yoshimoto, Eri; Okumura, Ko; Ogawa, Hideoki; Niyonsaba, François

    2017-08-01

    Host defense (antimicrobial) peptides not only display antimicrobial activities against numerous pathogens but also exert a broader spectrum of immune-modulating functions. Innate defense regulators (IDRs) are a class of host defense peptides synthetically developed from natural or endogenous cationic host defense peptides. Of the IDRs developed to date, IDR-1018 is more efficient not only in killing bacteria but also in regulating the various functions of macrophages and neutrophils and accelerating the wound healing process. Because mast cells intimately participate in wound healing and a number of host defense peptides involved in wound healing are also known to activate mast cells, this study aimed to investigate the effects of IDR-1018 on mast cell activation. Here, we showed that IDR-1018 induced the degranulation of LAD2 human mast cells and caused their production of leukotrienes, prostaglandins and various cytokines and chemokines, including granulocyte-macrophage colony-stimulating factor, interleukin-8, monocyte chemoattractant protein-1 and -3, macrophage-inflammatory protein-1α and -1β, and tumor necrosis factor-α. Furthermore, IDR-1018 increased intracellular calcium mobilization and induced mast cell chemotaxis. The mast cell activation was markedly suppressed by pertussis toxin, U-73122, U0126, SB203580, JNK inhibitor II, and NF-κB activation inhibitor II, suggesting the involvement of G-protein, phospholipase C, ERK, p38, JNK and NF-κB pathways, respectively, in IDR-1018-induced mast cell activation. Notably, we confirmed that IDR-1018 caused the phosphorylation of MAPKs and IκB. Altogether, the current study suggests a novel immunomodulatory role of IDR-1018 through its ability to recruit and activate human mast cells at the sites of inflammation and wounds. We report that IDR-1018 stimulates various functions of human mast cells. IDR-1018-induced mast cell activation is mediated through G protein, PLC, MAPK and NF-κB pathways. IDR-1018

  19. Interaction of phospholipase C with liposome: A conformation transition of the enzyme is critical and specific to liposome composition for burst hydrolysis and fusion in concert.

    PubMed

    Patra, Samir Kumar; Sengupta, Dipta; Deb, Moonmoon; Kar, Swayamsiddha; Kausar, Chahat

    2017-02-15

    Phospholipase C (PLC)(1) is known to help the pathogen B. cereus entry to the host cell and human PLC is over expressed in multiple cancers. Knowledge of dynamic activity of the enzyme PLC while in action on membrane lipids is essential and helpful to drug design and delivery. In view of this, interactions of PLC with liposome of various lipid compositions have been visualized by testing enzyme activity and microenvironments around the intrinsic fluorophores of the enzyme. Overall change of the protein's conformation has been monitored by fluorescence spectroscopy and circular dichroism (CD). Liposome aggregation and fusion were predicted by increase in turbidity and vesicle size. PLC in solution has high fluorescence and exhibit appreciable shift in its emission maxima, upon gradual change in excitation wavelength towards the red edge of the absorption band. REES fluorescence studies indicated that certain Trp fluorophores of inactive PLC are in motionally restricted compact/rigid environments in solution conformation. PLC fluorescence decreased in association with liposome and Trps loosed rigidity where liposome aggregation and fusion occurred. We argue that the structural flexibility is the cause of decrease of fluorescence, mostly to gain optimum conformation for maximum activity of the enzyme PLC. Further studies deciphered that the enzyme PLC undergoes change of conformation when mixed to LUVs prepared with specific lipids. CD data at the far-UV and near-UV regions of PLC in solution are in excellent agreement with the previous reports. CD analyses of PLC with LUVs, showed significant reduction of α-helices, increase of β-sheets; and confirmed dramatic change of orientations of Trps. In case of liposome composed of lipid raft like composition, the enzyme binds very fast, hydrolyze PC with higher rate, exhibit highest structural flexibility and promote vesicle fusion. These data strongly suggest marked differences in conformation transition induced PLC

  20. T cell receptor-induced activation of phospholipase C-gamma1 depends on a sequence-independent function of the P-I region of SLP-76.

    PubMed

    Gonen, Ronnie; Beach, Dvora; Ainey, Carmit; Yablonski, Deborah

    2005-03-04

    SLP-76 forms part of a hematopoietic-specific adaptor protein complex, and is absolutely required for T cell development and activation. T cell receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) depends on three features of SLP-76: the N-terminal tyrosine phosphorylation sites, the Gads-binding site, and an intervening sequence, denoted the P-I region, which binds to the SH3 domain of PLC-gamma1 (SH3(PLC)) via a low affinity interaction. Despite extensive research, the mechanism whereby SLP-76 regulates PLC-gamma1 remains uncertain. In this study, we uncover and explore an apparent paradox: whereas the P-I region as a whole is essential for TCR-induced activation of PLC-gamma1 and nuclear factor of activated T cells (NFAT), no particular part of this region is absolutely required. To better understand the contribution of the P-I region to PLC-gamma1 activation, we mapped the PLC-gamma1-binding site within the region, and created a SLP-76 mutant that fails to bind SH3(PLC), but is fully functional, mediating TCR-induced phosphorylation of PLC-gamma1 at tyrosine 783, calcium flux, and nuclear factor of activated T cells activation. Unexpectedly, full functionality of this mutant was maintained even under less than optimal stimulation conditions, such as a low concentration of the anti-TCR antibody. Another SLP-76 mutant, in which the P-I region was scrambled to abolish any sequence-dependent protein-binding motifs, also retained significant functionality. Our results demonstrate that SLP-76 need not interact with SH3(PLC) to activate PLC-gamma1, and further suggest that the P-I region of SLP-76 serves a structural role that is sequence-independent and is not directly related to protein-protein interactions.

  1. Phospholipase C-delta1 modulates sustained contraction of rat mesenteric small arteries in response to noradrenaline, but not endothelin-1.

    PubMed

    Clarke, Christopher J; Forman, Simon; Pritchett, James; Ohanian, Vasken; Ohanian, Jacqueline

    2008-08-01

    Vasoconstrictors activate phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP(2)), leading to calcium mobilization, protein kinase C activation, and contraction. Our aim was to investigate whether PLC-delta(1), a PLC isoform implicated in alpha(1)-adrenoreceptor signaling and the pathogenesis of hypertension, is involved in noradrenaline (NA) or endothelin (ET-1)-induced PIP(2) hydrolysis and contraction. Rat mesenteric small arteries were studied. Contractility was measured by pressure myography, phospholipids or inositol phosphates were measured by radiolabeling with (33)Pi or myo-[(3)H]inositol, and caveolae/rafts were prepared by discontinuous sucrose density centrifugation. PLC-delta(1) was localized by immunoblot analysis and neutralized by delivery of PLC-delta(1) antibody. The PLC inhibitor U73122, but not the negative control U-73342, markedly inhibited NA and ET-1 contraction but had no effect on potassium or phorbol ester contraction, implicating PLC activity in receptor-mediated smooth muscle contraction. PLC-delta(1) was present in caveolae/rafts, and NA, but not ET-1, stimulated a rapid twofold increase in PLC-delta(1) levels in these domains. PLC-delta(1) is calcium dependent, and removal of extracellular calcium prevented its association with caveolae/rafts in response to NA, concomitantly reducing NA-induced [(33)P]PIP(2) hydrolysis and [(3)H]inositol phosphate formation but with no effect on ET-1-induced [(33)P]PIP(2) hydrolysis. Neutralization of PLC-delta(1) by PLC-delta(1) antibody prevented its caveolae/raft association and attenuated the sustained contractile response to NA compared with control antibodies. In contrast, ET-1-induced contraction was not affected by PLC-delta(1) antibody. These results indicate the novel and selective role of caveolae/raft localized PLC-delta(1) in NA-induced PIP(2) hydrolysis and sustained contraction in intact vascular tissue.

  2. Genomic organization and complete cDNA sequence of the human phosphoinositide-specific phospholipase C {beta}3 gene (PLCB3)

    SciTech Connect

    Lagercrantz, J.; Carson, E.; Phelan, C.

    1995-04-10

    We have characterized the complete cDNA sequence, genomic structure, and expression of the human phosphoinositide-specific phospholipase C {beta}3 (PLC {beta}3) gene (gene symbol PLCB3). PLC {beta}3 plays an important role in initiating receptor-mediated signal transduction. Activation of PLC takes place in many cells as a response to stimulation by hormones, growth factors, neurotransmitters, and other ligands. The partial cDNA sequence of PLC {beta}3, previously published, was extended with 876 bp in the 5{prime} direction, giving a transcript of 4400 bp and a total open reading frame of 1234 amino acids. This was in accordance with expression analysis by Northern blotting that revealed a single 4.4-kb transcript in all tissues tested. Genomic data were obtained by sequencing plasmid subclones of a cosmid that contained the whole gene. The size of the complete transcription unit was estimated to be on the order of 15 kb. The gene contains 31 exons, with all splice donor and acceptor sites conforming to the GT/AG rule. No exon exceeds 571 bp in length, and the shortest exon spans only 36 bp. More than half of the introns are smaller than 200 bp, with the smallest being only 79 bp long. The transcription initiation site was determined to be within an 8-bp cluster 328-321 bp upstream of the translation initiation site. The 5{prime} flanking region is highly GC rich, with multiple CpG doublets, and contains multiple binding sites for Sp1. Lacking typical transcriptional regulatory sequences such as TATA and CAAT boxes, the putative promoter region conforms to the group of housekeeping promoters. 28 refs., 4 figs., 1 tab.

  3. The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin.

    PubMed Central

    Si-Tahar, M; Renesto, P; Falet, H; Rendu, F; Chignard, M

    1996-01-01

    Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 microM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 microM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses. PMID:8573071

  4. Brain phospholipase C-diacylglycerol lipase pathway is involved in vasopressin-induced release of noradrenaline and adrenaline from adrenal medulla in rats.

    PubMed

    Shimizu, Takahiro; Okada, Shoshiro; Yamaguchi-Shima, Naoko; Yokotani, Kunihiko

    2004-09-19

    Recently, we reported that intracerebroventricularly (i.c.v.) administered arginine-vasopressin evokes the release of noradrenaline and adrenaline from adrenal medulla by brain thromboxane A2-mediated mechanisms in rats. These results suggest the involvement of brain arachidonic acid in the vasopressin-induced activation of the central adrenomedullary outflow. Arachidonic acid is released mainly by two pathways: phospholipase A2 (PLA2)-dependent pathway; phospholipase C (PLC)- and diacylglycerol lipase-dependent pathway. In the present study, therefore, we attempted to identify which pathway is involved in the vasopressin-induced release of both catecholamines from adrenal medulla using urethane-anesthetized rats. Vasopressin (0.2 nmol/animal, i.c.v.)-induced elevation of plasma noradrenaline and adrenaline was dose-dependently reduced by neomycin [0.28 and 0.55 micromol (250 and 500 microg)/animal, i.c.v.] and 1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) [5 and 10 nmol (2.3 and 4.6 microg)/animal, i.c.v.] (inhibitors of PLC), and also by 1,6-bis(cyclohexyloximinocarbonylamino)hexane (RHC-80267) [1.3 and 2.6 micromol (500 and 1000 microg)/animal, i.c.v.] (an inhibitor of diacylglycerol lipase). On the other hand, mepacrine [1.1 and 2.2 micromol (500 and 1000 microg)/animal, i.c.v.] (an inhibitor of PLA2) was largely ineffective on the vasopressin-induced elevation of plasma catecholamines. These results suggest that vasopressin evokes the release of noradrenaline and adrenaline from adrenal medulla by the brain PLC- and diacylglycerol lipase-dependent mechanisms in rats.

  5. L-Theanine Improves Immunity by Altering TH2/TH1 Cytokine Balance, Brain Neurotransmitters, and Expression of Phospholipase C in Rat Hearts.

    PubMed

    Li, Chengjian; Tong, Haiou; Yan, Qiongxian; Tang, Shaoxun; Han, Xuefeng; Xiao, Wenjun; Tan, Zhiliang

    2016-02-28

    BACKGROUND This study aimed to investigate the regulatory effects of L-theanine on secretion of immune cytokines, hormones, and neurotransmitters, and mRNA expression of phospholipase C (PLC) in rats, and to explore its regulatory mechanism in immune function. MATERIAL AND METHODS Sixty-four Sprague-Dawley rats received daily intragastric infusion of different doses of L-theanine solution [0, 50 (LT), 200 (MT), and 400 (HT) mg/kg BW]. Cytokines, immunoglobulins, and hormones in the serum, neurotransmitters, and mRNA expression of PLC in the relevant tissues were assayed. RESULTS L-theanine administration increased the splenic organ index and decreased the contents of ILs-4/6/10 and the ratio of IL-4/IFN-γ in the serum. High-dose L-theanine administration increased the levels of dopamine and 5-hydroxytryptamine in the pituitary and hippocampus, resulting in decrease in corticosterone level in the serum. L-theanine administration decreased the mRNA expressions of PLC isomers in the liver and PLC-γ1 and PLC-δ1 in the spleen. Interestingly, mRNA expressions of PLC-β1 in the spleen and PLC isomers mRNA in the heart were up-regulated by L-theanine administration. CONCLUSIONS Administration of 400 mg/kg BWL-theanine improved immune function of the rats by increasing the splenic weight, altering the Th2/Th1 cytokine balance, decreasing the corticosterone level in the serum, elevating dopamine and 5-hydroxytryptamine in the brain, and regulating the mRNA expression of PLC isomers in the heart.

  6. L-Theanine Improves Immunity by Altering TH2/TH1 Cytokine Balance, Brain Neurotransmitters, and Expression of Phospholipase C in Rat Hearts

    PubMed Central

    Li, Chengjian; Tong, Haiou; Yan, Qiongxian; Tang, Shaoxun; Han, Xuefeng; Xiao, Wenjun; Tan, Zhiliang

    2016-01-01

    Background This study aimed to investigate the regulatory effects of L-theanine on secretion of immune cytokines, hormones, and neurotransmitters, and mRNA expression of phospholipase C (PLC) in rats, and to explore its regulatory mechanism in immune function. Material/Methods Sixty-four Sprague-Dawley rats received daily intragastric infusion of different doses of L-theanine solution [0, 50 (LT), 200 (MT), and 400 (HT) mg/kg BW]. Cytokines, immunoglobulins, and hormones in the serum, neurotransmitters, and mRNA expression of PLC in the relevant tissues were assayed. Results L-theanine administration increased the splenic organ index and decreased the contents of ILs-4/6/10 and the ratio of IL-4/IFN-γ in the serum. High-dose L-theanine administration increased the levels of dopamine and 5-hydroxytryptamine in the pituitary and hippocampus, resulting in decrease in corticosterone level in the serum. L-theanine administration decreased the mRNA expressions of PLC isomers in the liver and PLC-γ1 and PLC-δ1 in the spleen. Interestingly, mRNA expressions of PLC-βf1 in the spleen and PLC isomers mRNA in the heart were up-regulated by L-theanine administration. Conclusions Administration of 400 mg/kg BWL-theanine improved immune function of the rats by increasing the splenic weight, altering the Th2/Th1 cytokine balance, decreasing the corticosterone level in the serum, elevating dopamine and 5-hydroxytryptamine in the brain, and regulating the mRNA expression of PLC isomers in the heart. PMID:26922362

  7. Activation of Phosphatidylcholine-Specific Phospholipase C in Breast and Ovarian Cancer: Impact on MRS-Detected Choline Metabolic Profile and Perspectives for Targeted Therapy

    PubMed Central

    Podo, Franca; Paris, Luisa; Cecchetti, Serena; Spadaro, Francesca; Abalsamo, Laura; Ramoni, Carlo; Ricci, Alessandro; Pisanu, Maria Elena; Sardanelli, Francesco; Canese, Rossella; Iorio, Egidio

    2016-01-01

    Elucidation of molecular mechanisms underlying the aberrant phosphatidylcholine cycle in cancer cells plays in favor of the use of metabolic imaging in oncology and opens the way for designing new targeted therapies. The anomalous choline metabolic profile detected in cancer by magnetic resonance spectroscopy and spectroscopic imaging provides molecular signatures of tumor progression and response to therapy. The increased level of intracellular phosphocholine (PCho) typically detected in cancer cells is mainly attributed to upregulation of choline kinase, responsible for choline phosphorylation in the biosynthetic Kennedy pathway, but can also be partly produced by activation of phosphatidylcholine-specific phospholipase C (PC-PLC). This hydrolytic enzyme, known for implications in bacterial infection and in plant survival to hostile environmental conditions, is reported to be activated in mitogen- and oncogene-induced phosphatidylcholine cycles in mammalian cells, with effects on cell signaling, cell cycle regulation, and cell proliferation. Recent investigations showed that PC-PLC activation could account for 20–50% of the intracellular PCho production in ovarian and breast cancer cells of different subtypes. Enzyme activation was associated with PC-PLC protein overexpression and subcellular redistribution in these cancer cells compared with non-tumoral counterparts. Moreover, PC-PLC coimmunoprecipitated with the human epidermal growth factor receptor-2 (HER2) and EGFR in HER2-overexpressing breast and ovarian cancer cells, while pharmacological PC-PLC inhibition resulted into long-lasting HER2 downregulation, retarded receptor re-expression on plasma membrane and antiproliferative effects. This body of evidence points to PC-PLC as a potential target for newly designed therapies, whose effects can be preclinically and clinically monitored by metabolic imaging methods. PMID:27532027

  8. Angiotensin II-induced delayed stimulation of phospholipase C gamma1 requires activation of both phosphatidylinositol 3-kinase gamma and tyrosine kinase in vascular myocytes.

    PubMed

    Rakotoarisoa, Lala; Carricaburu, Valérie; Leblanc, Catherine; Mironneau, Chantal; Mironneau, Jean; Macrez, Nathalie

    2006-01-01

    In vascular smooth muscles, angiotensin II (AII) has been reported to activate phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K). We investigated the time-dependent effects of AII on both phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) and inositol phosphates (InsPs) accumulation in permeabilized microsomes from rat portal vein smooth muscle in comparison with those of noradrenaline (NA). AII stimulated an early production of PtdInsP3 (within 30 s) followed by a delayed production of InsPs (within 3-5 min), in contrast to NA which activated only a fast production of InsPs. The use of pharmacological inhibitors and antibodies raised against the PI3K and PLC isoforms expressed in portal vein smooth muscle showed that AII specifically activated PI3Kgamma and that this isoform was involved in the AII-induced stimulation of InsPs accumulation. NA-induced InsPs accumulation depended on PLCbeta1 activation whereas AII-induced InsPs accumulation depended on PLCgamma1 activation. AII-induced PLCgamma1 activation required both tyrosine kinase and PI3Kgamma since genistein and tyrphostin B48 (inhibitors of tyrosine kinase), LY294002 and wortmannin (inhibitors of PI3K) and anti-PI3Kgamma antibody abolished AII-induced stimulation of InsPs accumulation. Increased tyrosine phosphorylation of PLCgamma1 was only detected for long-lasting applications of AII and was suppressed by genistein. These data indicate that activation of both PI3Kgamma and tyrosine kinase is a prerequisite for AII-induced stimulation of PLCgamma1 in vascular smooth muscle and suggest that the sequential activation of the three enzymes may be responsible for the slow and long-lasting contraction induced by AII.

  9. Angiotensin II-induced delayed stimulation of phospholipase C γ1 requires activation of both phosphatidiylinositol 3-kinase γ and tyrosine kinase in vascular myocytes

    PubMed Central

    Rakotoarisoa, Lala; Carricaburu, Valérie; Leblanc, Catherine; Mironneau, Chantal; Mironneau, Jean; Macrez, Nathalie

    2006-01-01

    In vascular smooth muscles, angiotensin II (AII) has been reported to activate phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K). We investigated the time-dependent effects of AII on both phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) and inositol phosphates (InsPs) accumulation in permeabilized microsomes from rat portal vein smooth muscle in comparison with those of noradrenaline (NA). AII stimulated an early production of PtdInsP3 (within 30 s) followed by a delayed production of InsPs (within 3-5 min), in contrast to NA which activated only a fast production of InsPs. The use of pharmacological inhibitors and antibodies raised against the PI3K and PLC isoforms expressed in portal vein smooth muscle showed that AII specifically activated PI3Kδ and that this isoform was involved in the AII-induced stimulation of InsPs accumulation. NA-induced InsPs accumulation depended on PLCβ1 activation whereas AII-induced InsPs accumulation depended on PLCγ1 activation. AII-induced PLCδ1 activation required both tyrosine kinase and PI3Kδ since genistein and tyrphostin B48 (inhibitors of tyrosine kinase), LY294002 and wortmannin (inhibitors of PI3K) and anti-PI3Kδ antibody abolished AII-induced stimulation of InsPs accumulation. Increased tyrosine phosphorylation of PLCβ1 was only detected for long-lasting applications of AII and was suppressed by genistein. These data indicate that activation of both PI3Kβ and tyrosine kinase is a prerequisite for AII-induced stimulation of PLCβ1 in vascular smooth muscle and suggest that the sequential activation of the three enzymes may be responsible for the slow and long-lasting contraction induced by AII. PMID:16989733

  10. Interaction of phospholipase C with liposome: A conformation transition of the enzyme is critical and specific to liposome composition for burst hydrolysis and fusion in concert

    NASA Astrophysics Data System (ADS)

    Patra, Samir Kumar; Sengupta, Dipta; Deb, Moonmoon; Kar, Swayamsiddha; Kausar, Chahat

    2017-02-01

    Phospholipase C (PLC)1 is known to help the pathogen B. cereus entry to the host cell and human PLC is over expressed in multiple cancers. Knowledge of dynamic activity of the enzyme PLC while in action on membrane lipids is essential and helpful to drug design and delivery. In view of this, interactions of PLC with liposome of various lipid compositions have been visualized by testing enzyme activity and microenvironments around the intrinsic fluorophores of the enzyme. Overall change of the protein's conformation has been monitored by fluorescence spectroscopy and circular dichroism (CD). Liposome aggregation and fusion were predicted by increase in turbidity and vesicle size. PLC in solution has high fluorescence and exhibit appreciable shift in its emission maxima, upon gradual change in excitation wavelength towards the red edge of the absorption band. REES fluorescence studies indicated that certain Trp fluorophores of inactive PLC are in motionally restricted compact/rigid environments in solution conformation. PLC fluorescence decreased in association with liposome and Trps loosed rigidity where liposome aggregation and fusion occurred. We argue that the structural flexibility is the cause of decrease of fluorescence, mostly to gain optimum conformation for maximum activity of the enzyme PLC. Further studies deciphered that the enzyme PLC undergoes change of conformation when mixed to LUVs prepared with specific lipids. CD data at the far-UV and near-UV regions of PLC in solution are in excellent agreement with the previous reports. CD analyses of PLC with LUVs, showed significant reduction of α-helices, increase of β-sheets; and confirmed dramatic change of orientations of Trps. In case of liposome composed of lipid raft like composition, the enzyme binds very fast, hydrolyze PC with higher rate, exhibit highest structural flexibility and promote vesicle fusion. These data strongly suggest marked differences in conformation transition induced PLC

  11. Phosphorylation of Nuclear Phospholipase C β1 by Extracellular Signal-Regulated Kinase Mediates the Mitogenic Action of Insulin-Like Growth Factor I

    PubMed Central

    Xu, Aimin; Suh, Pann-Ghill; Marmy-Conus, Nelly; Pearson, Richard B.; Seok, Oh Yong; Cocco, Lucio; Gilmour, R. Stewart

    2001-01-01

    It is well established that a phosphoinositide (PI) cycle which is operationally distinct from the classical plasma membrane PI cycle exists within the nucleus, where it is involved in both cell proliferation and differentiation. However, little is known about the regulation of the nuclear PI cycle. Here, we report that nucleus-localized phospholipase C (PLC) β1, the key enzyme for the initiation of this cycle, is a physiological target of extracellular signal-regulated kinase (ERK). Stimulation of Swiss 3T3 cells with insulin-like growth factor I (IGF-I) caused rapid nuclear translocation of activated ERK and concurrently induced phosphorylation of nuclear PLC β1, which was completely blocked by the MEK inhibitor PD 98059. Coimmunoprecipitation detected a specific association between the activated ERK and PLC β1 within the nucleus. In vitro studies revealed that recombinant PLC β1 could be efficiently phosphorylated by activated mitogen-activated protein kinase but not by PKA. The ERK phosphorylation site was mapped to serine 982, which lies within a PSSP motif located in the characteristic carboxy-terminal tail of PLC β1. In cells overexpressing a PLC β1 mutant in which serine 982 is replaced by glycine (S982G), IGF-I failed to activate the nuclear PI cycle, and its mitogenic effect was also markedly attenuated. Expression of S982G was found to inhibit ERK-mediated phosphorylation of endogenous PLC β1. This result suggests that ERK-evoked phosphorylation of PLC β1 at serine 982 plays a critical role in the activation of the nuclear PI cycle and is also crucial to the mitogenic action of IGF-I. PMID:11287604

  12. Norepinephrine Causes a Biphasic Change in Mammalian Pinealocye Membrane Potential: Role of α1B-Adrenoreceptors, Phospholipase C, and Ca2+

    PubMed Central

    Zemkova, Hana; Stojilkovic, Stanko S.

    2011-01-01

    Perforated patch clamp recording was used to study the control of membrane potential (Vm) and spontaneous electrical activity in the rat pinealocyte by norepinephrine. Norepinephrine did not alter spiking frequency. However, it was found to act through α1B-adrenoreceptors in a concentration-dependent manner (0.1–10 μm) to produce a biphasic change in Vm. The initial response was a hyperpolarization (∼13 mV from a resting potential of −46 mV) due to a transient (∼5 sec) outward K+ current (∼50 pA). This current appears to be triggered by Ca2+ released from intracellular stores, based on the observation that it was also seen in cells bathed in Ca2+-deficient medium. In addition, pharmacological studies indicate that this current was dependent on phospholipase C (PLC) activation and was in part mediated by bicuculline methiodide and apamin-sensitive Ca2+-controlled K+ channels. The initial transient hyperpolarization was followed by a sustained depolarization (∼4 mV) due to an inward current (∼10 pA). This response was dependent on PLC-dependent activation of Na+/Ca2+ influx but did not involve nifedipine-sensitive voltage-gated Ca2+ channels. Together, these results indicate for the first time that activation of α1B-adrenoreceptors initiates a PLC-dependent biphasic change in pinealocyte Vm characterized by an initial transient hyperpolarization mediated by a mixture of Ca2+-activated K+ channels followed by a sustained depolarization mediated by a Ca2+-conducting nonselective cation channel. These observations indicate that both continuous elevation of intracellular Ca2+ and sustained depolarization at approximately −40 mV are associated with and are likely to be required for activation of the pinealocyte. PMID:21828176

  13. Norepinephrine causes a biphasic change in mammalian pinealocye membrane potential: role of alpha1B-adrenoreceptors, phospholipase C, and Ca2+.

    PubMed

    Zemkova, Hana; Stojilkovic, Stanko S; Klein, David C

    2011-10-01

    Perforated patch clamp recording was used to study the control of membrane potential (V(m)) and spontaneous electrical activity in the rat pinealocyte by norepinephrine. Norepinephrine did not alter spiking frequency. However, it was found to act through α(1B)-adrenoreceptors in a concentration-dependent manner (0.1-10 μM) to produce a biphasic change in V(m). The initial response was a hyperpolarization (∼13 mV from a resting potential of -46 mV) due to a transient (∼5 sec) outward K(+) current (∼50 pA). This current appears to be triggered by Ca(2+) released from intracellular stores, based on the observation that it was also seen in cells bathed in Ca(2+)-deficient medium. In addition, pharmacological studies indicate that this current was dependent on phospholipase C (PLC) activation and was in part mediated by bicuculline methiodide and apamin-sensitive Ca(2+)-controlled K(+) channels. The initial transient hyperpolarization was followed by a sustained depolarization (∼4 mV) due to an inward current (∼10 pA). This response was dependent on PLC-dependent activation of Na(+)/Ca(2+) influx but did not involve nifedipine-sensitive voltage-gated Ca(2+) channels. Together, these results indicate for the first time that activation of α(1B)-adrenoreceptors initiates a PLC-dependent biphasic change in pinealocyte V(m) characterized by an initial transient hyperpolarization mediated by a mixture of Ca(2+)-activated K(+) channels followed by a sustained depolarization mediated by a Ca(2+)-conducting nonselective cation channel. These observations indicate that both continuous elevation of intracellular Ca(2+) and sustained depolarization at approximately -40 mV are associated with and are likely to be required for activation of the pinealocyte.

  14. Knockdown of phospholipase C-β1 in the medial prefrontal cortex of male mice impairs working memory among multiple schizophrenia endophenotypes

    PubMed Central

    Kim, Seong-Wook; Seo, Misun; Kim, Duk-Soo; Kang, Moonkyung; Kim, Yeon-Soo; Koh, Hae-Young; Shin, Hee-Sup

    2015-01-01

    Background Decreased expression of phospholipase C-β1 (PLC-β1) has been observed in the brains of patients with schizophrenia, but, to our knowledge, no studies have shown a possible association between this altered PLC-β1 expression and the pathogenesis of schizophrenia. Although PLC-β1-null (PLC-β1−/−) mice exhibit multiple endophenotypes of schizophrenia, it remains unclear how regional decreases in PLC-β1 expression in the brain contribute to specific behavioural defects. Methods We selectively knocked down PLC-β1 in the medial prefrontal cortex (mPFC) using a small hairpin RNA strategy in mice. Results Silencing PLC-β1 in the mPFC resulted in working memory deficits, as assayed using the delayed non-match-to-sample T-maze task. Notably, however, other schizophrenia- related behaviours observed in PLC-β1−/− mice, including phenotypes related to locomotor activity, sociability and sensorimotor gating, were normal in PLC-β1 knockdown mice. Limitations Phenotypes of PLC-β1 knockdown mice, such as locomotion, anxiety and sensorimotor gating, have already been published in our previous studies. Further, the neural mechanisms underlying the working memory deficit in mice may be different from those in human schizophrenia. Conclusion These results indicate that PLC-β1 signalling in the mPFC is required for working memory. Importantly, these results support the notion that the decrease in PLC-β1 expression in the brains of patients with schizophrenia is a pathogenically relevant molecular marker of the disorder. PMID:25268789

  15. Effect of acute acid-base disturbances on the phosphorylation of phospholipase C-γ1 and Erk1/2 in the renal proximal tubule

    PubMed Central

    Skelton, Lara A; Boron, Walter F

    2015-01-01

    The renal proximal tubule (PT) plays a major role in whole-body pH homeostasis by secreting H+ into the tubule lumen. Previous work demonstrated that PTs respond to basolateral changes in [CO2] and [] by appropriately altering H+ secretion—responses blocked by the ErbB inhibitor PD168393, or by eliminating signaling through AT1 angiotensin receptors. In the present study, we analyze phosphorylation of three downstream targets of both ErbBs and AT1: phospholipase C-γ1 (PLC-γ1), extracellular-regulated kinase 1 (Erk1), and Erk2. We expose rabbit PT suspensions for 5 and 20 min to our control (Ctrl) condition (5% CO2, 22 mmol/L , pH 7.40) or one of several conditions that mimic acid-base disturbances. We found that each disturbance produces characteristic phosphorylation patterns in the three enzymes. For example, respiratory acidosis (elevated [CO2], normal []) at 20 min decreases PLC-γ1 phosphorylation at tyrosine-783 (relative to Ctrl). Metabolic acidosis (normal [CO2], decreased []) for 5 min increases Erk1 phosphorylation (p-Erk1) but not p-Erk2, whereas metabolic alkalosis (normal [CO2], elevated []) for 5 min decreases p-Erk1 and p-Erk2. In the presence of CO2/, PD168393 blocks only two of eight induced decreases in phosphorylation. In two cases in which disturbances have no remarkable effects on phosphorylation, PD168393 unmasks decreases and in two others, increases. These drug effects provide insight into the roles of PD168393-sensitive kinases. Our results indicate that PLC-γ1.pY783, p-Erk1, and p-Erk2 in the PT change in characteristic ways in response to acute acid-base disturbances, and thus presumably contribute to the transduction of acid-base signals. PMID:25780091

  16. Anti-neuroinflammatory efficacy of the aldose reductase inhibitor FMHM via phospholipase C/protein kinase C-dependent NF-κB and MAPK pathways

    SciTech Connect

    Zeng, Ke-Wu; Li, Jun; Dong, Xin; Wang, Ying-Hong; Ma, Zhi-Zhong; Jiang, Yong; Jin, Hong-Wei; Tu, Peng-Fei

    2013-11-15

    Aldose reductase (AR) has a key role in several inflammatory diseases: diabetes, cancer and cardiovascular diseases. Therefore, AR inhibition seems to be a useful strategy for anti-inflammation therapy. In the central nervous system (CNS), microglial over-activation is considered to be a central event in neuroinflammation. However, the effects of AR inhibition in CNS inflammation and its underlying mechanism of action remain unknown. In the present study, we found that FMHM (a naturally derived AR inhibitor from the roots of Polygala tricornis Gagnep.) showed potent anti-neuroinflammatory effects in vivo and in vitro by inhibiting microglial activation and expression of inflammatory mediators. Mechanistic studies showed that FMHM suppressed the activity of AR-dependent phospholipase C/protein kinase C signaling, which further resulted in downstream inactivation of the IκB kinase/IκB/nuclear factor-kappa B (NF-κB) inflammatory pathway. Therefore, AR inhibition-dependent NF-κB inactivation negatively regulated the transcription and expression of various inflammatory genes. AR inhibition by FMHM exerted neuroprotective effects in lipopolysaccharide-induced neuron–microglia co-cultures. These findings suggested that AR is a potential target for neuroinflammation inhibition and that FMHM could be an effective agent for treating or preventing neuroinflammatory diseases. - Highlights: • FMHM is a natural-derived aldose reductase (AR) inhibitor. • FMHM inhibits various neuroinflammatory mediator productions in vitro and in vivo. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent NF-κB pathway. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent MAPK pathway. • FMHM protects neurons against inflammatory injury in microglia-neuron co-cultures.

  17. Phospholipase C-zeta deficiency as a cause for repetitive oocyte fertilization failure during ovarian stimulation for in vitro fertilization with ICSI: a case report.

    PubMed

    Chithiwala, Zahabiya H; Lee, Hoi Chang; Hill, David L; Jellerette-Nolan, Teru; Fissore, Rafael; Grow, Daniel; Dumesic, Daniel A

    2015-09-01

    The purpose of this study is to describe impaired oocyte fertilization from phospholipase C-zeta (PLC-ζ) deficiency in normal-appearing sperm that was successfully treated using calcium (Ca(2+)) ionophore with intracytoplasmic sperm injection (ICSI) of oocytes matured in vitro. An infertile couple undergoing in vitro fertilization (IVF) experienced failed oocyte fertilization following ICSI with normal-appearing sperm. A semen sample collected from the patient was used to assess the expression of sperm PLC- ζ protein by Western blot analysis and immunofluorescence and PLC-ζ bioactivity by an in vitro model of Ca(2+) release. A second IVF cycle was performed using Ca(2+) ionophore with ICSI to enhance Ca(2+)-induced oocyte activation of oocytes matured in vitro. Sperm PLC-ζ protein deficiency was demonstrated by Western blot analysis and immunofluorescence and confirmed by reduced PLC-ζ bioactivity using an in vitro model of Ca(2+) release. Nevertheless, with this sperm and supplementation of Ca(2+) ionophore following ICSI, fertilization of four of six oocytes matured in vitro was obtained. In addition, four embryos underwent cleavage and two of them reached the blastocyst stage. Transfer of these blastocysts into the uterus led to a single pregnancy and live birth. Deficiency of PLC-ζ in normal-appearing human sperm is associated with impaired Ca(2+)-dependent oocyte activation during ICSI. Under this condition, use of Ca(2+) ionophore following ICSI of oocytes matured in vitro improves embryo developmental competence, possibly through the activation of Ca(2+)-dependent mechanisms governing fertilization and preimplantation embryogenesis.

  18. Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: Studies with genistein and monoclonal antibody to phosphotyrosine

    SciTech Connect

    Dhar, A.; Paul, A.K.; Shukla, S.D. )

    1990-04-01

    Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-(2-3H)inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity (( 3H)inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated (3H)inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation.

  19. Phospholipase C δ-4 overexpression upregulates ErbB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells

    PubMed Central

    Leung, David W; Tompkins, Chris; Brewer, Jim; Ball, Alexey; Coon, Mike; Morris, Valerie; Waggoner, David; Singer, Jack W

    2004-01-01

    Background The expression of the rodent phosphoinositide-specific phospholipase C δ-4 (PLCδ4) has been found to be elevated upon mitogenic stimulation and expression analysis have linked the upregulation of PLCδ4 expression with rapid proliferation in certain rat transformed cell lines. The human homologue of PLCδ4 has not been extensively characterized. Accordingly, we investigate the effects of overexpression of human PLCδ4 on cell signaling and proliferation in this study. Results The cDNA for human PLCδ4 has been isolated and expressed ectopically in breast cancer MCF-7 cells. Overexpression of PLCδ4 selectively activates protein kinase C-φ and upregulates the expression of epidermal growth factor receptors EGFR/erbB1 and HER2/erbB2, leading to constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in MCF-7 cells. MCF-7 cells stably expressing PLCδ4 demonstrates several phenotypes of transformation, such as rapid proliferation in low serum, formation of colonies in soft agar, and capacity to form densely packed spheroids in low-attachment plates. The growth signaling responses induced by PLCδ4 are not reversible by siRNA. Conclusion Overexpression or dysregulated expression of PLCδ4 may initiate oncogenesis in certain tissues through upregulation of ErbB expression and activation of ERK pathway. Since the growth responses induced by PLCδ4 are not reversible, PLCδ4 itself is not a suitable drug target, but enzymes in pathways activated by PLCδ4 are potential therapeutic targets for oncogenic intervention. PMID:15140260

  20. Phospholipase C-related catalytically inactive protein (PRIP) regulates lipolysis in adipose tissue by modulating the phosphorylation of hormone-sensitive lipase.

    PubMed

    Okumura, Toshiya; Harada, Kae; Oue, Kana; Zhang, Jun; Asano, Satoshi; Hayashiuchi, Masaki; Mizokami, Akiko; Tanaka, Hiroto; Irifune, Masahiro; Kamata, Nobuyuki; Hirata, Masato; Kanematsu, Takashi

    2014-01-01

    Phosphorylation of hormone-sensitive lipase (HSL) and perilipin by protein kinase A (PKA) promotes the hydrolysis of lipids in adipocytes. Although activation of lipolysis by PKA has been well studied, inactivation via protein phosphatases is poorly understood. Here, we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a binding partner for protein phosphatase 1 and protein phosphatase 2A (PP2A), is involved in lipolysis by regulating phosphatase activity. PRIP knockout (PRIP-KO) mice displayed reduced body-fat mass as compared with wild-type mice fed with standard chow ad libitum. Most other organs appeared normal, suggesting that mutant mice had aberrant fat metabolism in adipocytes. HSL in PRIP-KO adipose tissue was highly phosphorylated compared to that in wild-type mice. Starvation of wild-type mice or stimulation of adipose tissue explants with the catabolic hormone, adrenaline, translocated both PRIP and PP2A from the cytosol to lipid droplets, but the translocation of PP2A was significantly reduced in PRIP-KO adipocytes. Consistently, the phosphatase activity associated with lipid droplet fraction in PRIP-KO adipocytes was significantly reduced and was independent of adrenaline stimulation. Lipolysis activity, as assessed by measurement of non-esterified fatty acids and glycerol, was higher in PRIP-KO adipocytes. When wild-type adipocytes were treated with a phosphatase inhibitor, they showed a high lipolysis activity at the similar level to PRIP-KO adipocytes. Collectively, these results suggest that PRIP promotes the translocation of phosphatases to lipid droplets to trigger the dephosphorylation of HSL and perilipin A, thus reducing PKA-mediated lipolysis.

  1. Contrasting role of phospholipase C-{gamma}1 in the expression of immediate early genes induced by epidermal or platelet-derived growth factors

    SciTech Connect

    Liao Hongjun; Santos, Josue de los; Carpenter, Graham . E-mail: graham.carpenter@vanderbilt.edu

    2006-04-01

    While significant progress has been achieved in identifying the signal transduction elements that operate downstream of activated receptor tyrosine kinases, it remains unclear how different receptors utilize these signaling elements to achieve a common response. This study compares the capacity of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) to elicit the induction of immediate early gene (IEG) mRNAs in the presence or absence of phospholipase C-{gamma}1 (PLC-{gamma}1). The results show that while PDGF induction of nearly all IEG mRNAs is abrogated in plcg1 null cells, EGF induction of the same genes is variable in the null cells and exhibits three distinct responses. Five IEG mRNAs (Nup475, Cyr61, TF, Gly, TS7) are completely inducible by EGF in the presence or absence of PLC-{gamma}1, while three others (JE, KC, FIC) exhibit a stringent requirement for the presence of PLC-{gamma}1. The third type of response is exhibited by c-fos and COX-2. While these mRNAs are completely induced by EGF in the absence of PLC-{gamma}1, the time course of their accumulation is significantly delayed. No IEG was identified as completely inducible by EGF and PDGF in the absence of PLC-{gamma}1. Electrophoretic mobility shift assays (EMSA) demonstrate that PLC-{gamma}1 is necessary for nuclear extracts from PDGF-treated cells, but not EGF-treated cells, to interact with probes for AP-1 or NF-{kappa}B.

  2. Nerve growth factor rapidly stimulates tyrosine phosphorylation of phospholipase C-gamma 1 by a kinase activity associated with the product of the trk protooncogene.

    PubMed

    Vetter, M L; Martin-Zanca, D; Parada, L F; Bishop, J M; Kaplan, D R

    1991-07-01

    Nerve growth factor (NGF) promotes the survival and differentiation of specific populations of neurons. The molecular mechanisms by which cells respond to NGF are poorly understood, but two clues have emerged recently. First, NGF rapidly stimulates tyrosine phosphorylation of several unidentified proteins in the NGF-responsive pheochromocytoma cell line PC12 [Maher, P. (1988) Proc. Natl. Acad. Sci. USA 85, 6788-6791]. Second, the protein-tyrosine kinase encoded by the protooncogene trk (p140trk), a member of the receptor class of tyrosine kinases, becomes activated and phosphorylated on tyrosine after NGF treatment of PC12 cells [Kaplan, D. R., Martin-Zanca, D. & Parada, L. F. (1991) Nature (London) 350, 158-160]. We now report that NGF rapidly induces tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), and we present evidence that the responsible tyrosine kinase is either p140trk or a closely associated protein. Treatment of responsive cells with NGF elicited phosphorylation of PLC-gamma 1 on tyrosine and serine. PLC-gamma 1 immunoprecipitated from NGF-stimulated cells was phosphorylated in vitro by coprecipitating protein kinase activity, and the phosphorylations occurred principally on tyrosine. The responsible kinase could be depleted from cellular lysates by antibodies specific for p140trk. This procedure also depleted a 140-kDa protein that normally coprecipitated with PLC-gamma 1 and became phosphorylated on tyrosine in vivo in response to NGF. Analysis of tryptic peptides from PLC-gamma 1 indicated that the residues phosphorylated in vitro by p140trk-associated kinase activity were largely congruent with those phosphorylated in vivo after NGF treatment. Our findings identify PLC-gamma 1 as a likely substrate for the trk-encoded tyrosine kinase, and they provide a link between NGF-dependent activation of p140trk and the stimulation of intracellular second messenger pathways.

  3. Genome-Wide Association Study Identifies Phospholipase C zeta 1 (PLCz1) as a Stallion Fertility Locus in Hanoverian Warmblood Horses

    PubMed Central

    Schrimpf, Rahel; Dierks, Claudia; Martinsson, Gunilla; Sieme, Harald; Distl, Ottmar

    2014-01-01

    A consistently high level of stallion fertility plays an economically important role in modern horse breeding. We performed a genome-wide association study for estimated breeding values of the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) in Hanoverian stallions. A total of 228 Hanoverian stallions were genotyped using the Equine SNP50 Beadchip. The most significant association was found on horse chromosome 6 for a single nucleotide polymorphism (SNP) within phospholipase C zeta 1 (PLCz1). In the close neighbourhood to PLCz1 is located CAPZA3 (capping protein (actin filament) muscle Z-line, alpha 3). The gene PLCz1 encodes a protein essential for spermatogenesis and oocyte activation through sperm induced Ca2+-oscillation during fertilization. We derived equine gene models for PLCz1 and CAPZA3 based on cDNA and genomic DNA sequences. The equine PLCz1 had four different transcripts of which two contained a premature termination codon. Sequencing all exons and their flanking sequences using genomic DNA samples from 19 Hanoverian stallions revealed 47 polymorphisms within PLCz1 and one SNP within CAPZA3. Validation of these 48 polymorphisms in 237 Hanoverian stallions identified three intronic SNPs within PLCz1 as significantly associated with EBV-PAT. Bioinformatic analysis suggested regulatory effects for these SNPs via transcription factor binding sites or microRNAs. In conclusion, non-coding polymorphisms within PLCz1 were identified as conferring stallion fertility and PLCz1 as candidate locus for male fertility in Hanoverian warmblood. CAPZA3 could be eliminated as candidate gene for fertility in Hanoverian stallions. PMID:25354211

  4. Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC-ɛ to RACK2 in adult rat and mouse

    PubMed Central

    Fenton, Richard A.; Komatsu, Satoshi; Ikebe, Mitsuo; Shea, Lynne G.; Dobson, James G.

    2009-01-01

    Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A1 receptors (A1R) on the adrenergic signaling pathway. An indirect A1R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-ɛ to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A1R activation of PKC-ɛ induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A1R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-ɛ and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-ɛ to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A1R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-ɛ co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-ɛ with RACK2 by 61%. With rat cardiomyocytes, the β-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A1R stimulation of the heart is associated with PLC-initiated PKC-ɛ translocation and association with RACK2. PMID:19525381

  5. Phospholipase C-β4 Is Essential for the Progression of the Normal Sleep Sequence and Ultradian Body Temperature Rhythms in Mice

    PubMed Central

    Ikeda, Masayuki; Hirono, Moritoshi; Sugiyama, Takashi; Moriya, Takahiro; Ikeda-Sagara, Masami; Eguchi, Naomi; Urade, Yoshihiro; Yoshioka, Tohru

    2009-01-01

    Background The sleep sequence: i) non-REM sleep, ii) REM sleep, and iii) wakefulness, is stable and widely preserved in mammals, but the underlying mechanisms are unknown. It has been shown that this sequence is disrupted by sudden REM sleep onset during active wakefulness (i.e., narcolepsy) in orexin-deficient mutant animals. Phospholipase C (PLC) mediates the signaling of numerous metabotropic receptors, including orexin receptors. Among the several PLC subtypes, the β4 subtype is uniquely localized in the geniculate nucleus of thalamus which is hypothesized to have a critical role in the transition and maintenance of sleep stages. In fact, we have reported irregular theta wave frequency during REM sleep in PLC-β4-deficient mutant (PLC-β4−/−) mice. Daily behavioral phenotypes and metabotropic receptors involved have not been analyzed in detail in PLC-β4−/− mice, however. Methodology/Principal Findings Therefore, we analyzed 24-h sleep electroencephalogram in PLC-β4−/− mice. PLC-β4−/− mice exhibited normal non-REM sleep both during the day and nighttime. PLC-β4−/− mice, however, exhibited increased REM sleep during the night, their active period. Also, their sleep was fragmented with unusual wake-to-REM sleep transitions, both during the day and nighttime. In addition, PLC-β4−/− mice reduced ultradian body temperature rhythms and elevated body temperatures during the daytime, but had normal homeothermal response to acute shifts in ambient temperatures (22°C–4°C). Within the most likely brain areas to produce these behavioral phenotypes, we found that, not orexin, but group-1 metabotropic glutamate receptor (mGluR)-mediated Ca2+ mobilization was significantly reduced in the dorsal lateral geniculate nucleus (LGNd) of PLC-β4−/− mice. Voltage clamp recordings revealed that group-1 mGluR-mediated currents in LGNd relay neurons (inward in wild-type mice) were outward in PLC-β4−/− mice. Conclusions/Significance These lines

  6. Effect of acute acid-base disturbances on the phosphorylation of phospholipase C-γ1 and Erk1/2 in the renal proximal tubule.

    PubMed

    Skelton, Lara A; Boron, Walter F

    2015-03-01

    The renal proximal tubule (PT) plays a major role in whole-body pH homeostasis by secreting H(+) into the tubule lumen. Previous work demonstrated that PTs respond to basolateral changes in [CO2] and [HCO3-] by appropriately altering H(+) secretion-responses blocked by the ErbB inhibitor PD168393, or by eliminating signaling through AT1 angiotensin receptors. In the present study, we analyze phosphorylation of three downstream targets of both ErbBs and AT1: phospholipase C-γ1 (PLC-γ1), extracellular-regulated kinase 1 (Erk1), and Erk2. We expose rabbit PT suspensions for 5 and 20 min to our control (Ctrl) condition (5% CO2, 22 mmol/L HCO3-, pH 7.40) or one of several conditions that mimic acid-base disturbances. We found that each disturbance produces characteristic phosphorylation patterns in the three enzymes. For example, respiratory acidosis (elevated [CO2], normal [HCO3-]) at 20 min decreases PLC-γ1 phosphorylation at tyrosine-783 (relative to Ctrl). Metabolic acidosis (normal [CO2], decreased [HCO3-]) for 5 min increases Erk1 phosphorylation (p-Erk1) but not p-Erk2, whereas metabolic alkalosis (normal [CO2], elevated [HCO3-]) for 5 min decreases p-Erk1 and p-Erk2. In the presence of CO2/HCO3-, PD168393 blocks only two of eight induced decreases in phosphorylation. In two cases in which disturbances have no remarkable effects on phosphorylation, PD168393 unmasks decreases and in two others, increases. These drug effects provide insight into the roles of PD168393-sensitive kinases. Our results indicate that PLC-γ1.pY783, p-Erk1, and p-Erk2 in the PT change in characteristic ways in response to acute acid-base disturbances, and thus presumably contribute to the transduction of acid-base signals. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  7. ADP-Induced Ca(2+) Signaling and Proliferation of Rat Ventricular Myofibroblasts Depend on Phospholipase C-Linked TRP Channels Activation Within Lipid Rafts.

    PubMed

    Certal, Mariana; Vinhas, Adriana; Barros-Barbosa, Aurora; Ferreirinha, Fátima; Costa, Maria Adelina; Correia-de-Sá, Paulo

    2017-06-01

    Nucleotides released during heart injury affect myocardium electrophysiology and remodeling through P2 purinoceptors activation in cardiac myofibroblasts. ATP and UTP endorse [Ca(2+) ]i accumulation and growth of DDR-2/α-SMA-expressing myofibroblasts from adult rat ventricles via P2Y4 and P2Y2 receptors activation, respectively. Ventricular myofibroblasts also express ADP-sensitive P2Y1 , P2Y12 , and P2Y13 receptors as demonstrated by immunofluorescence confocal microscopy and western blot analysis, but little information exists on ADP effects in these cells. ADP (0.003-3 mM) and its stable analogue, ADPßS (100 μM), caused fast [Ca(2+) ]i transients originated from thapsigargin-sensitive internal stores, which partially declined to a plateau sustained by capacitative Ca(2+) entry through transient receptor potential (TRP) channels inhibited by 2-APB (50 μM) and flufenamic acid (100 μM). Hydrophobic interactions between Gq/11 -coupled P2Y purinoceptors and TRP channels were suggested by prevention of the ADP-induced [Ca(2+) ]i plateau following PIP2 depletion with LiCl (10 mM) and cholesterol removal from lipid rafts with methyl-ß-cyclodextrin (2 mM). ADP [Ca(2+) ]i transients were insensitive to P2Y1 , P2Y12 , and P2Y13 receptor antagonists, MRS2179 (10μM), AR-C66096 (0.1 μM), and MRS2211 (10μM), respectively, but were attenuated by suramin and reactive blue-2 (100 μM) which also blocked P2Y4 receptors activation by UTP. Cardiac myofibroblasts growth and type I collagen production were favored upon activation of MRS2179-sensitive P2Y1 receptors with ADP or ADPßS (30 μM). In conclusion, ADP exerts a dual role on ventricular myofibroblasts: [Ca(2+) ]i transients are mediated by fast-desensitizing P2Y4 receptors, whereas the pro-fibrotic effect of ADP involves the P2Y1 receptor activation. Data also show that ADP-induced capacitative Ca(2+) influx depends on phospholipase C-linked TRP channels opening in lipid raft microdomains. J. Cell

  8. Pasteurella multocida toxin activates the inositol triphosphate signaling pathway in Xenopus oocytes via G(q)alpha-coupled phospholipase C-beta1.

    PubMed

    Wilson, B A; Zhu, X; Ho, M; Lu, L

    1997-01-10

    Pasteurella multocida toxin (PMT) has been hypothesized to cause activation of a GTP-binding protein (G-protein)-coupled phosphatidylinositol-specific phospholipase C (PLC) in intact cells. We used voltage-clamped Xenopus oocytes to test for direct PMT-mediated stimulation of PLC by monitoring the endogenous Ca2+-dependent C1- current. Injection of PMT induced an inward, two-component Cl- current, similar to that evoked by injection of IP3 through intracellular Ca2+ mobilization and Ca2+ influx through voltage-gated Ca2+ channels. These PMT-induced currents were blocked by specific inhibitors of Ca2+ and Cl- channels, removal of extracellular Ca2+, or chelation of intracellular Ca2+. Specific antibodies directed against an N-terminal, but not a C-terminal, peptide of PMT inhibited the toxin-induced currents, implicating that the N terminus of PMT is important for toxin activity. Injection with specific antibodies against PLCbeta1, PLCbeta2, PLCbeta3, or PLCgamma1 identified PLCbeta1 as the primary mediator of the PMT-induced Cl- currents. Injection with guanosine 5'-O-(2-(thio)diphosphate), antibodies to the common GTP-binding region of G-protein alpha subunits, or antibodies to different regions of G-protein beta subunits established the involvement of a G-protein alpha subunit in PMT-activation of PLCbeta1. Injection with specific antibodies against the alpha-subunits of G(q/11), G(s/olf), G(i/o/t/z), or G(i-1/i-2/i-3) isoforms confirmed the involvement of Gq/11alpha. Preinjection of oocytes with pertussis toxin enhanced the PMT response. Overexpression of G(q)alpha in oocytes could enhance the PMT response by 30-fold to more than 300-fold, whereas introduction of antisense G(q)alpha cRNA reduced the response by 7-fold. The effects of various specific antibodies on the PMT response were reproduced in oocytes overexpressing G(q)alpha.

  9. Phospholipase C-beta4 is essential for the progression of the normal sleep sequence and ultradian body temperature rhythms in mice.

    PubMed

    Ikeda, Masayuki; Hirono, Moritoshi; Sugiyama, Takashi; Moriya, Takahiro; Ikeda-Sagara, Masami; Eguchi, Naomi; Urade, Yoshihiro; Yoshioka, Tohru

    2009-11-09

    THE SLEEP SEQUENCE: i) non-REM sleep, ii) REM sleep, and iii) wakefulness, is stable and widely preserved in mammals, but the underlying mechanisms are unknown. It has been shown that this sequence is disrupted by sudden REM sleep onset during active wakefulness (i.e., narcolepsy) in orexin-deficient mutant animals. Phospholipase C (PLC) mediates the signaling of numerous metabotropic receptors, including orexin receptors. Among the several PLC subtypes, the beta4 subtype is uniquely localized in the geniculate nucleus of thalamus which is hypothesized to have a critical role in the transition and maintenance of sleep stages. In fact, we have reported irregular theta wave frequency during REM sleep in PLC-beta4-deficient mutant (PLC-beta4-/-) mice. Daily behavioral phenotypes and metabotropic receptors involved have not been analyzed in detail in PLC-beta4-/- mice, however. Therefore, we analyzed 24-h sleep electroencephalogram in PLC-beta4-/- mice. PLC-beta4-/- mice exhibited normal non-REM sleep both during the day and nighttime. PLC-beta4-/- mice, however, exhibited increased REM sleep during the night, their active period. Also, their sleep was fragmented with unusual wake-to-REM sleep transitions, both during the day and nighttime. In addition, PLC-beta4-/- mice reduced ultradian body temperature rhythms and elevated body temperatures during the daytime, but had normal homeothermal response to acute shifts in ambient temperatures (22 degrees C-4 degrees C). Within the most likely brain areas to produce these behavioral phenotypes, we found that, not orexin, but group-1 metabotropic glutamate receptor (mGluR)-mediated Ca(2+) mobilization was significantly reduced in the dorsal lateral geniculate nucleus (LGNd) of PLC-beta4-/- mice. Voltage clamp recordings revealed that group-1 mGluR-mediated currents in LGNd relay neurons (inward in wild-type mice) were outward in PLC-beta4-/- mice. These lines of evidence indicate that impaired LGNd relay, possibly mediated

  10. The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells.

    PubMed Central

    Batty, I H; Downes, C P

    1994-01-01

    Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively

  11. Soluble low-Km 5'-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage.

    PubMed

    Vogel, M; Kowalewski, H; Zimmermann, H; Hooper, N M; Turner, A J

    1992-06-15

    Soluble and membrane-bound low-Km 5'-nucleotidase was isolated from high-speed supernatants and membrane fractions derived from the electric organ of the electric ray (Torpedo marmorata) or from bovine brain cerebral cortex. Purification of both enzymes included chromatography on concanavalin A-Sepharose and AMP-Sepharose. The contribution to the total of soluble enzyme activity was lower in electric organ (1.6%) than in bovine cerebral cortex (27.9%). Membrane-bound and soluble forms have very similar Km values for AMP and are inhibited by micromolar concentrations of ATP. Both forms cross-react with, and are inhibited by, an antibody against the membrane-bound surface-located (ecto-) 5'-nucleotidase from electric organ. The HNK-1 carbohydrate epitope is present on both forms of the Torpedo enzyme, but is entirely absent from bovine cerebral-cortex 5'-nucleotidase. An antibody specific for the inositol 1,2-(cyclic)monophosphate that is formed on phospholipase C cleavage of an intact glycosyl-phosphatidylinositol (GPI) anchor binds to the soluble, but not to the membrane-bound, form of the enzyme from both sources. Our results suggest that soluble low-Km 5'-nucleotidase in both electric organ and bovine brain is derived from the membrane-bound GPI-anchored form of the enzyme by the action of a phospholipase C and is not a soluble cytoplasmic enzyme.

  12. Molecular Characteristics of the Mycobacterium tuberculosis LAM-RUS Family Prevalent in Central Russia▿ †

    PubMed Central

    Dubiley, Svetlana; Kirillov, Eugene; Ignatova, Anna; Stepanshina, Valentina; Shemyakin, Igor

    2007-01-01

    We analyzed IS6110-associated polymorphisms in the phospholipase C genes of 107 isolates of Mycobacterium tuberculosis selected to be representative of isolates circulating in central Russia. We found that the majority of Latin American-Mediterranean family strains contained an insertion in a unique position in the plcA gene, suggesting a common ancestor. This insertion can serve as a specific genetic marker for this group, which we designate the LAM-RUS family. PMID:17942651

  13. Stimulation of phosphatidylcholine breakdown by thrombin and carbachol but not by tyrosine kinase receptor ligands in cells transfected with M1 muscarinic receptors. Rapid desensitization of phosphocholine-specific (PC) phospholipase D but sustained activity of PC-phospholipase C.

    PubMed

    McKenzie, F R; Seuwen, K; Pouysségur, J

    1992-11-15

    In order to evaluate the possible contribution of phospholipase D (PLD) stimulation to the mitogenic response, a screening of a variety of different compounds, some of which are known to be potent mitogens, was performed using the well characterized Chinese hamster lung fibroblast (CCL39) cell line. In wild type CCL39 cells, or derivatives expressing high levels of either the human M1 muscarinic receptor (Hm1) or the human epidermal growth factor (EGF) receptor (39M1-81 and 39ER22 clones, respectively), thrombin, a potent mitogen for all three cell types, elicited the rapid activation of PLD (t1/2 activation, 30 s). Carbachol-mediated activation of the Hm1 receptor in the 39M1-81 clone, which is not a mitogenic signal, produced a similarly rapid although greater activation of PLD. Addition of EGF to the 39ER22 clone was able to provoke both a mitogenic response and stimulate PLD, albeit a comparatively small effect. In each case, the stimulation of PLD correlated closely with the ability to stimulate inositol phospholipid breakdown and was entirely dependent on the activation of protein kinase C. Moreover, the ability of both thrombin and carbachol to stimulate PLD was found to be rapidly desensitized, with a similar time course of desensitization (t1/2 desensitization, 90 s). It has recently been reported that an increase in phospholipase C (PLC)-mediated phosphocholine (PC) hydrolysis by either addition of agonist or by extracellular addition of PC-specific PLC enzyme constitutes a mitogenic signal. In this regard, in addition to stimulation of PLD, thrombin and carbachol were both able to stimulate the activity of a phosphocholine-specific phospholipase C (PC-PLC), which did not appear to desensitize within the time course employed. By contrast, EGF was unable to elicit the stimulation of PC-PLC. Ligands such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), which bind to and activate receptors with intrinsic tyrosine kinase activity

  14. Diacylglycerol generated by exogenous phospholipase C activates the mitogen-activated protein kinase pathway independent of Ras- and phorbol ester-sensitive protein kinase C: dependence on protein kinase C-zeta.

    PubMed Central

    van Dijk, M; Muriana, F J; van Der Hoeven, P C; de Widt, J; Schaap, D; Moolenaar, W H; van Blitterswijk, W J

    1997-01-01

    The role of diacylglycerol (DG) formation from phosphatidylcholine in mitogenic signal transduction is poorly understood. We have generated this lipid at the plasma membrane by treating Rat-1 fibroblasts with bacterial phosphatidylcholine-specific phospholipase C (PC-PLC). This treatment leads to activation of mitogen-activated protein kinase (MAPK). However, unlike platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), PC-PLC fails to activate Ras and to induce DNA synthesis, and activates MAPK only transiently (<45 min). Down-regulation of protein kinase C (PKC) -alpha, -delta and -epsilon isotypes has little or no effect on MAPK activation by either PC-PLC or growth factors. However, Ro 31-8220, a highly selective inhibitor of all PKC isotypes, including atypical PKC-zeta but not Raf-1, blocks MAPK activation by PDGF and PC-PLC, but not that by EGF, suggesting that atypical PKC mediates the PDGF and PC-PLC signal. In line with this, PKC-zeta is activated by PC-PLC and PDGF, but not by EGF, as shown by a kinase assay in vitro, using biotinylated epsilon-peptide as a substrate. Furthermore, dominant-negative PKC-zeta inhibits, while (wild-type) PKC-zeta overexpression enhances MAPK activation by PDGF and PC-PLC. The results suggest that DG generated by PC-PLC can activate the MAPK pathway independent of Ras and phorbol-ester-sensitive PKC but, instead, via PKC-zeta. PMID:9169602

  15. Basic fibroblast growth factor-induced translocation of p21-activated kinase to the membrane is independent of phospholipase C-gamma1 in the differentiation of PC12 cells.

    PubMed

    Shin, Kyung-Sun; Shin, Eun-Young; Lee, Chan-Soo; Quan, Song-Hua; Woo, Kyung-Nam; Soung, Nak-Kyun; Kwak, Sahng-June; Kim, Seung Ryul; Kim, Eung-Gook

    2002-05-31

    p21-activated kinase (PAK) targeting to the plasma membrane is essential for PC12 cell neurite outgrowth. Phospholipase C-gamma1 (PLC-gamma1) can mediate the PAK translocation in response to growth factors, since PLC-gamma1 binds to both tyrosine-phosphorylated receptor tyrosine kinases and PAK through its SH2 and SH3 domain, respectively. In the present study, we examined a potential role for PLC-gamma1 in the basic fibroblast growth factor (bFGF)-induced PAK translocation using stable PC12 cell lines that overexpress in a tetracycline-inducible manner either the wild-type FGFR-1 or the Y766F FGFR-1 mutant. Phosphatidylinositol hydrolysis was increased 6.5-fold in response to bFGF in the wild type cells but negligible in the mutant cells. The recombinant GST-PLC-gamma1 SH3 was able to bind to PAK1 but not GST alone. However, examination of PLC-gamma1 as an adaptor for translocation of PAK1 in cells showed that both cells transfected with pEGFP-PAK1 was able to differentiate for 24 h, as visualized by laser confocal microscopy. Translocation of PAK1 to growth cones occurs at similar levels in both wild and mutant cells. These results suggest that a protein(s) other than PLC-gamma1 is functionally relevant for PAK targeting.

  16. Over-expression of Brassica napus phosphatidylinositol-phospholipase C2 in canola induces significant changes in gene expression and phytohormone distribution patterns, enhances drought tolerance and promotes early flowering and maturation.

    PubMed

    Georges, Fawzy; DAS, Shankar; Ray, Heather; Bock, Cheryl; Nokhrina, Kateryna; Kolla, Venkat Apparao; Keller, Wilf

    2009-12-01

    Phosphatidylinositol-specific phospholipase C (PtdIns-PLC2) plays a central role in the phosphatidylinositol-specific signal transduction pathway. It catalyses the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to produce two second messengers, sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate. The former is a membrane activator of protein kinase C in mammalian systems, and the latter is a Ca(2+) modulator which induces distinctive oscillating bursts of cytosolic Ca(2+), resulting in regulation of gene expression and activation of proteins. Sustained over-expression of BnPtdIns-PLC2 in transgenic Brassica napus lines brought about an early shift from vegetative to reproductive phases, and shorter maturation periods, accompanied by notable alterations in hormonal distribution patterns in various tissues. The photosynthetic rate increased, while stomata were partly closed. Numerous gene expression changes that included induction of stress-related genes such as glutathione S-transferase, hormone-regulated and regulatory genes, in addition to a number of kinases, calcium-regulated factors and transcription factors, were observed. Other changes included increased phytic acid levels and phytohormone organization patterns. These results suggest the importance of PtdIns-PLC2 as an elicitor of a battery of events that systematically control hormone regulation, and plant growth and development in what may be a preprogrammed mode.

  17. Orofacial movements in phospholipase C-related catalytically inactive protein-1/2 double knockout mice: Effect of the GABAergic agent diazepam and the D(1) dopamine receptor agonist SKF 83959.

    PubMed

    Tomiyama, Katsunori; Song, Liqiu; Kobayashi, Masayuki; Kinsella, Anthony; Kanematsu, Takashi; Hirata, Masato; Koshikawa, Noriaki; Waddington, John L

    2010-09-01

    Orofacial movements are regulated by D(1)-like dopamine receptors interacting with additional mechanisms. Phospholipase C-related catalytically inactive protein (PRIP) regulates cell surface expression of GABA(A) receptors containing a gamma2 subunit. Mutant mice with double knockout of PRIP-1 and PRIP-2 were used to investigate aspects of GABAergic regulation of orofacial movements and interactions with D(1) mechanisms. Vertical jaw movements, tongue protrusions and movements of the head and vibrissae were reduced in PRIP-1/2 double knockouts. The GABA(A)ergic agent diazepam reduced movements of the head and vibrissae; these effects were unaltered in PRIP-1/2 double knockouts. The D(1)-like agonist SKF 83959 induced vertical jaw movements, incisor chattering, and movements of the head and vibrissae that were unaltered in PRIP-1/2 double knockouts. However, SKF 83959-induced tongue protrusions were reduced in PRIP-1/2 double knockouts. PRIP-mediated regulation of GABA(A)ergic receptor mechanisms influences topographically distinct aspects of orofacial movement and interacts with D(1) receptor systems.

  18. Phosphatidylcholine-specific phospholipase C/heat shock protein 70 (Hsp70)/transcription factor B-cell translocation gene 2 signaling in rat bone marrow stromal cell differentiation to cholinergic neuron-like cells.

    PubMed

    Shao, Jing; Sun, Chunhui; Su, Le; Zhao, Jing; Zhang, Shangli; Miao, Junying

    2012-12-01

    Although bone marrow stromal cells (BMSCs) can differentiate into neuron-like cells, the mechanisms underlying neuronal differentiation are not well understood. We recently found that inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC) by its inhibitor D609 promoted BMSCs' differentiation into cholinergic neuron-like cells. Using the effective small molecule D609 and gene microarray technology, we investigated the change of gene expression profile to identify key mediators involved in the neuronal differentiation. We selected heat shock protein 70 (Hsp70) and transcription factor B-cell translocation gene 2 (Btg2) that were maximally up-regulated for further study. We found that functional suppression of Hsp70 blocked D609-induced increase of Btg2 expression and cholinergic neuronal differentiation of BMSCs. These results demonstrated that Hsp70 was the pivotal factor in PC-PLC-medicated neuronal differentiation of BMSCs, and Btg2 might be its downstream target. Our findings provide new clues for controlling BMSCs' differentiation into cholinergic neuron-like cells and provide a putative strategy for neurodegenerative diseases therapies. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  19. Synthesis of 2-deoxy-2-fluoro-phosphatidylinositol-4,5-bisphosphate and analogues: probes and modulators of the mammalian PI-PLCS.

    PubMed

    Aneja, S G; Ivanova, P T; Aneja, R

    1998-05-05

    An approach to synthesis of 2-modified phosphatidylinositol-4,5-bisphosphates, which are substrate analogues useful as probes and modulators of the PI-PLC enzyme family, is described and illustrated for the dibutyl-2-deoxy-2-fluoro analogue, a probe designed for delineating substrate and PI-PLC interactions by X-ray crystallography.

  20. Impairments in brain-derived neurotrophic factor-induced glutamate release in cultured cortical neurons derived from rats with intrauterine growth retardation: possible involvement of suppression of TrkB/phospholipase C-γ activation.

    PubMed

    Numakawa, Tadahiro; Matsumoto, Tomoya; Ooshima, Yoshiko; Chiba, Shuichi; Furuta, Miyako; Izumi, Aiko; Ninomiya-Baba, Midori; Odaka, Haruki; Hashido, Kazuo; Adachi, Naoki; Kunugi, Hiroshi

    2014-04-01

    Low birth weight due to intrauterine growth retardation (IUGR) is suggested to be a risk factor for various psychiatric disorders such as schizophrenia. It has been reported that developmental cortical dysfunction and neurocognitive deficits are observed in individuals with IUGR, however, the underlying molecular mechanisms have yet to be elucidated. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are associated with schizophrenia and play a role in cortical development. We previously demonstrated that BDNF induced glutamate release through activation of the TrkB/phospholipase C-γ (PLC-γ) pathway in developing cultured cortical neurons, and that, using a rat model for IUGR caused by maternal administration of thromboxane A2, cortical levels of TrkB were significantly reduced in IUGR rats at birth. These studies prompted us to hypothesize that TrkB reduction in IUGR cortex led to impairment of BDNF-dependent glutamatergic neurotransmission. In the present study, we found that BDNF-induced glutamate release was strongly impaired in cultured IUGR cortical neurons where TrkB reduction was maintained. Impairment of BDNF-induced glutamate release in IUGR neurons was ameliorated by transfection of human TrkB (hTrkB). Although BDNF-stimulated phosphorylation of TrkB and of PLC-γ was decreased in IUGR neurons, the hTrkB transfection recovered the deficits in their phosphorylation. These results suggest that TrkB reduction causes impairment of BDNF-stimulated glutamatergic function via suppression of TrkB/PLC-γ activation in IUGR cortical neurons. Our findings provide molecular insights into how IUGR links to downregulation of BDNF function in the cortex, which might be involved in the development of IUGR-related diseases such as schizophrenia.

  1. Phospholipase C mediates (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-, but not lysergic acid diethylamide (LSD)-elicited head bobs in rabbit medial prefrontal cortex.

    PubMed

    Schindler, Emmanuelle A D; Harvey, John A; Aloyo, Vincent J

    2013-01-23

    The phenethylamine and indoleamine classes of hallucinogens demonstrate distinct pharmacological properties, although they share a serotonin(2A) (5-HT(2A)) receptor mechanism of action (MOA). The 5-HT(2A) receptor signals through phosphatidylinositol (PI) hydrolysis, which is initiated upon activation of phospholipase C (PLC). The role of PI hydrolysis in the effects of hallucinogens remains unclear. In order to better understand the role of PI hydrolysis in the MOA of hallucinogens, the PLC inhibitor, 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), was used to study the effects of two hallucinogens, the phenethylamine, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and the indoleamine, lysergic acid diethylamide (LSD). PI hydrolysis was quantified through release of [3H]inositol-4-phosphate from living rabbit frontocortical tissue prisms. Head bobs were counted after hallucinogens were infused into the medial prefrontal cortex (mPFC) of rabbits. Both DOI and LSD stimulated PI hydrolysis in frontocortical tissue through activation of PLC. DOI-stimulated PI hydrolysis was blocked by 5-HT(2A/2C) receptor antagonist, ketanserin, whereas the LSD signal was blocked by 5-HT(2B/2C) receptor antagonist, SB206553. When infused into the mPFC, both DOI- and LSD-elicited head bobs. Pretreatment with U73122 blocked DOI-, but not LSD-elicited head bobs. The two hallucinogens investigated were distinct in their activation of the PI hydrolysis signaling pathway. The serotonergic receptors involved with DOI and LSD signals in frontocortical tissue were different. Furthermore, PLC activation in mPFC was necessary for DOI-elicited head bobs, whereas LSD-elicited head bobs were independent of this pathway. These novel findings urge closer investigation into the intracellular mechanism of action of these unique compounds. Published by Elsevier B.V.

  2. Identification of a phospholipase C-gamma1 (PLC-gamma1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-gamma1 and NFAT.

    PubMed

    Yablonski, D; Kadlecek, T; Weiss, A

    2001-07-01

    SLP-76 is an adapter protein required for T-cell receptor (TCR) signaling. In particular, TCR-induced tyrosine phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1), and the resultant TCR-inducible gene expression, depend on SLP-76. Nonetheless, the mechanisms by which SLP-76 mediates PLC-gamma1 activation are not well understood. We now demonstrate that SLP-76 directly interacts with the Src homology 3 (SH3) domain of PLC-gamma1. Structure-function analysis of SLP-76 revealed that each of the previously defined protein-protein interaction domains can be individually deleted without completely disrupting SLP-76 function. Additional deletion mutations revealed a new, 67-amino-acid functional domain within the proline-rich region of SLP-76, which we have termed the P-1 domain. The P-1 domain mediates a constitutive interaction of SLP-76 with the SH3 domain of PLC-gamma1 and is required for TCR-mediated activation of Erk, PLC-gamma1, and NFAT (nuclear factor of activated T cells). The adjacent Gads-binding domain of SLP-76, also within the proline-rich region, mediates inducible recruitment of SLP-76 to a PLC-gamma1-containing complex via the recruitment of both PLC-gamma1 and Gads to another cell-type-specific adapter, LAT. Thus, TCR-induced activation of PLC-gamma1 entails the binding of PLC-gamma1 to both LAT and SLP-76, a finding that may underlie the requirement for both LAT and SLP-76 to mediate the optimal activation of PLC-gamma1.

  3. Phospholipase C-β1 and β4 contribute to non-genetic cell-to-cell variability in histamine-induced calcium signals in HeLa cells.

    PubMed

    Ishida, Sachiko; Matsu-Ura, Toru; Fukami, Kiyoko; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2014-01-01

    A uniform extracellular stimulus triggers cell-specific patterns of Ca(2+) signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca(2+) oscillations in terms of the time constant of Ca(2+) spike amplitude decay and the Ca(2+) oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca(2+) signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca(2+) signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca(2+) oscillations, such as the time constant of the temporal changes in the Ca(2+) spike amplitude and the Ca(2+) oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca(2+) release, can cause cell-to-cell variability in the patterns of Ca(2+) signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca(2+) signals evoked by G protein-coupled receptor stimulation.

  4. PDZ domain-containing 1 (PDZK1) protein regulates phospholipase C-β3 (PLC-β3)-specific activation of somatostatin by forming a ternary complex with PLC-β3 and somatostatin receptors.

    PubMed

    Kim, Jung Kuk; Kwon, Ohman; Kim, Jinho; Kim, Eung-Kyun; Park, Hye Kyung; Lee, Ji Eun; Kim, Kyung Lock; Choi, Jung Woong; Lim, Seyoung; Seok, Heon; Lee-Kwon, Whaseon; Choi, Jang Hyun; Kang, Byoung Heon; Kim, Sanguk; Ryu, Sung Ho; Suh, Pann-Ghill

    2012-06-15

    Phospholipase C-β (PLC-β) is a key molecule in G protein-coupled receptor (GPCR)-mediated signaling. Many studies have shown that the four PLC-β subtypes have different physiological functions despite their similar structures. Because the PLC-β subtypes possess different PDZ-binding motifs, they have the potential to interact with different PDZ proteins. In this study, we identified PDZ domain-containing 1 (PDZK1) as a PDZ protein that specifically interacts with PLC-β3. To elucidate the functional roles of PDZK1, we next screened for potential interacting proteins of PDZK1 and identified the somatostatin receptors (SSTRs) as another protein that interacts with PDZK1. Through these interactions, PDZK1 assembles as a ternary complex with PLC-β3 and SSTRs. Interestingly, the expression of PDZK1 and PLC-β3, but not PLC-β1, markedly potentiated SST-induced PLC activation. However, disruption of the ternary complex inhibited SST-induced PLC activation, which suggests that PDZK1-mediated complex formation is required for the specific activation of PLC-β3 by SST. Consistent with this observation, the knockdown of PDZK1 or PLC-β3, but not that of PLC-β1, significantly inhibited SST-induced intracellular Ca(2+) mobilization, which further attenuated subsequent ERK1/2 phosphorylation. Taken together, our results strongly suggest that the formation of a complex between SSTRs, PDZK1, and PLC-β3 is essential for the specific activation of PLC-β3 and the subsequent physiologic responses by SST.

  5. Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

    PubMed Central

    Ishida, Sachiko; Matsu-ura, Toru; Fukami, Kiyoko; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2014-01-01

    A uniform extracellular stimulus triggers cell-specific patterns of Ca2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca2+ oscillations in terms of the time constant of Ca2+ spike amplitude decay and the Ca2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca2+ oscillations, such as the time constant of the temporal changes in the Ca2+ spike amplitude and the Ca2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca2+ release, can cause cell-to-cell variability in the patterns of Ca2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca2+ signals evoked by G protein-coupled receptor stimulation. PMID:24475116

  6. PDZ Domain-containing 1 (PDZK1) Protein Regulates Phospholipase C-β3 (PLC-β3)-specific Activation of Somatostatin by Forming a Ternary Complex with PLC-β3 and Somatostatin Receptors*

    PubMed Central

    Kim, Jung Kuk; Kwon, Ohman; Kim, Jinho; Kim, Eung-Kyun; Park, Hye Kyung; Lee, Ji Eun; Kim, Kyung Lock; Choi, Jung Woong; Lim, Seyoung; Seok, Heon; Lee-Kwon, Whaseon; Choi, Jang Hyun; Kang, Byoung Heon; Kim, Sanguk; Ryu, Sung Ho; Suh, Pann-Ghill

    2012-01-01

    Phospholipase C-β (PLC-β) is a key molecule in G protein-coupled receptor (GPCR)-mediated signaling. Many studies have shown that the four PLC-β subtypes have different physiological functions despite their similar structures. Because the PLC-β subtypes possess different PDZ-binding motifs, they have the potential to interact with different PDZ proteins. In this study, we identified PDZ domain-containing 1 (PDZK1) as a PDZ protein that specifically interacts with PLC-β3. To elucidate the functional roles of PDZK1, we next screened for potential interacting proteins of PDZK1 and identified the somatostatin receptors (SSTRs) as another protein that interacts with PDZK1. Through these interactions, PDZK1 assembles as a ternary complex with PLC-β3 and SSTRs. Interestingly, the expression of PDZK1 and PLC-β3, but not PLC-β1, markedly potentiated SST-induced PLC activation. However, disruption of the ternary complex inhibited SST-induced PLC activation, which suggests that PDZK1-mediated complex formation is required for the specific activation of PLC-β3 by SST. Consistent with this observation, the knockdown of PDZK1 or PLC-β3, but not that of PLC-β1, significantly inhibited SST-induced intracellular Ca2+ mobilization, which further attenuated subsequent ERK1/2 phosphorylation. Taken together, our results strongly suggest that the formation of a complex between SSTRs, PDZK1, and PLC-β3 is essential for the specific activation of PLC-β3 and the subsequent physiologic responses by SST. PMID:22528496

  7. Variance in total levels of phospholipase C zeta (PLC-ζ) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability.

    PubMed

    Kashir, Junaid; Jones, Celine; Mounce, Ginny; Ramadan, Walaa M; Lemmon, Bernadette; Heindryckx, Bjorn; de Sutter, Petra; Parrington, John; Turner, Karen; Child, Tim; McVeigh, Enda; Coward, Kevin

    2013-01-01

    To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability. Laboratory study. University laboratory. Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5). Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm. Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men. Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern. The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  8. The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.

    PubMed Central

    Fazioli, F; Kim, U H; Rhee, S G; Molloy, C J; Segatto, O; Di Fiore, P P

    1991-01-01

    The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts. Images PMID:1672440

  9. Evidence that a lipolytic enzyme--hematopoietic-specific phospholipase C-β2--promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes.

    PubMed

    Adamiak, M; Poniewierska-Baran, A; Borkowska, S; Schneider, G; Abdelbaset-Ismail, A; Suszynska, M; Abdel-Latif, A; Kucia, M; Ratajczak, J; Ratajczak, M Z

    2016-04-01

    Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4β1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-β2 (PLC-β2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-β2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs.

  10. Evidence that a lipolytic enzyme—hematopoietic-specific phospholipase C-β2—promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes

    PubMed Central

    Adamiak, M; Poniewierska-Baran, A; Borkowska, S; Schneider, G; Abdelbaset-Ismail, A; Suszynska, M; Abdel-Latif, A; Kucia, M; Ratajczak, J; Ratajczak, M Z

    2016-01-01

    Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4β1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-β2 (PLC-β2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-β2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs. PMID:26582648

  11. Membrane associated phospholipase C from bovine brain

    SciTech Connect

    Lee, K.; Ryu, S.H.; Suh, P.; Choi, W.C.; Rhee, S.G.

    1987-05-01

    Cytosolic fractions of bovine brain contain 2 immunologically distinct phosphoinositide-specific phospholipase (PLC), PLC-I and PLC-II, whose MW are 150,000 and 145,000 respectively, under a denaturing condition. Monoclonal antibodies were derived against each form and specific radioimmunoassays were developed. Distribution of PLC-I and PLC-II in cytosolic and particulate fractions was measured using the radioimmunoassay. More than 90% of PLC-II was found in the cytosolic fraction, while the anti-PLC-I antibody cross-reacting protein was distributed nearly equally between the soluble fraction and the 2 M KCl extract of particulate fraction. The PLC enzyme in the particulate fraction was purified to homogeneity, yielding 2 proteins of 140 KDa and 150 KDa when analyzed on SDS-PAGE. Neither of the 2 enzymes cross-reacted with anti-PLC-II antibodies, but both could be immunoblotted by all 4 different anti-PLC-I antibodies. This suggests that the 140 KDa PLC was derived from the 150 KDa form. The 150 Kda form from particulate fraction was indistinguishable from the cytosolic PLC-I when their mixture was analyzed on SDS-PAGE. In addition, the elution profile of tryptic peptides derived from the 150 KDa particulate form was identical to that of cytosolic PLC-I. This result indicates that PLC-I is reversibly associated to membranes.

  12. Parathyroid Hormone Activates Phospholipase C (PLC)-Independent Protein Kinase C Signaling Pathway via Protein Kinase A (PKA)-Dependent Mechanism: A New Defined Signaling Route Would Induce Alternative Consideration to Previous Conceptions.

    PubMed

    Tong, Guojun; Meng, Yue; Hao, Song; Hu, Shaoyu; He, Youhua; Yan, Wenjuan; Yang, Dehong

    2017-04-20

    BACKGROUND Parathyroid hormone (PTH) is an effective anti-osteoporosis agent, after binding to its receptor PTHR1, several signaling pathways, including cAMP/protein kinase A (PKA) and phospholipase C (PLC)/protein kinase C (PKC), are initiated through G proteins; with the cAMP/PKA pathway as the major pathway. Earlier studies have reported that PTHR1 might also activate PKC via a PLC-independent mechanism, but this pathway remains unclear. MATERIAL AND METHODS In HEK293 cells, cAMP accumulation was measured with ELISA and PKC was measured with fluorescence resonance energy transfer (FRET) analysis using CKAR plasmid. In MC3T3-E1 cells, real-time PCR was performed to examine gene expressions. Then assays for cell apoptosis, cell differentiation, alkaline phosphatase activity, and mineralization were performed. RESULTS The FRET analysis found that PTH(1-34), [G1,R19]PTH(1-34) (GR(1-34), and [G1,R19]PTH(1-28) (GR(1-28) were all activated by PKC. The PKC activation ability of GR(1-28) was blocked by cAMP inhibitor (Rp-cAMP) and rescued with the addition of active PKA-α and PKA-β. The PKC activation ability of GR(1-34) was partially inhibited by Rp-cAMP. In MC3T3-E1 cells, gene expressions of ALP, CITED1, NR4a2, and OSX that was regulated by GR(1-28) were significantly changed by the pan-PKC inhibitor Go6983. After pretreatment with Rp-cAMP, the gene expressions of ALP, CITED1, and OPG were differentially regulated by GR(1-28) or GR(1-34), and the difference was blunted by Go6983. PTH(1-34), GR(1-28), and GR(1-34) significantly decreased early apoptosis and augmented osteoblastic differentiation in accordance with the activities of PKA and PKC. CONCLUSIONS PLC-independent PKC activation induced by PTH could be divided into two potential mechanisms: one was PKA-dependent and associated with PTH(1-28); the other was PKA-independent and associated with PTH(29-34). We also found that PTH could activate PLC-independent PKC via PKA-dependent mechanisms.

  13. Diacylglycerol induces fusion of nuclear envelope membrane precursor vesicles.

    PubMed

    Barona, Teresa; Byrne, Richard D; Pettitt, Trevor R; Wakelam, Michael J O; Larijani, Banafshe; Poccia, Dominic L

    2005-12-16

    Purified membrane vesicles isolated from sea urchin eggs form nuclear envelopes around sperm nuclei following GTP hydrolysis in the presence of cytosol. A low density subfraction of these vesicles (MV1), highly enriched in phosphatidylinositol (PtdIns), is required for nuclear envelope formation. Membrane fusion of MV1 with a second fraction that contributes most of the nuclear envelope can be initiated without GTP by an exogenous bacterial PtdIns-specific phospholipase C (PI-PLC) which hydrolyzes PtdIns to form diacylglycerides and inositol 1-phosphate. This PI-PLC hydrolyzes a subset of sea urchin membrane vesicle PtdIns into diglycerides enriched in long chain, polyunsaturated species as revealed by a novel liquid chromatography-mass spectrometry analysis. Large unilammelar vesicles (LUVs) enriched in PtdIns can substitute for MV1 in PI-PLC induced nuclear envelope formation. Moreover, MV1 prehydrolyzed with PI-PLC and washed to remove inositols leads to spontaneous nuclear envelope formation with MV2 without further PI-PLC treatment. LUVs enriched in diacylglycerol mimic prehydrolyzed MV1. These results indicate that production of membrane-destabilizing diglycerides in membranes enriched in PtdIns may facilitate membrane fusion in a natural membrane system and suggest that MV1, which binds only to two places on the sperm nucleus, may initiate fusion locally.

  14. Photodynamic triggering of calcium oscillation in the isolated rat pancreatic acini.

    PubMed Central

    Cui, Z J; Kanno, T

    1997-01-01

    1. Photodynamic agents, due to their photon-dependent selective activation, can selectively activate a number of physiological processes and may directly modulate signal transduction in a number of cells including pancreatic acinar cells. 2. Activation of the photodynamic agent sulphonated aluminium phthalocyanine (SALPC) triggered recurrent cytosolic calcium ([Ca2+]i) spiking in pancreatic acinar cells. 3. The photodynamically triggered calcium spiking could be blocked by phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor U73122, but not by phosphatidylcholine-specific phospholipase C inhibitor D609. 4. Removal of extracellular Ca2+ abolished spiking, as did 2-aminoethoxydiphenylborate (2-APB), an inhibitory modulator of IP3-mediated Ca2+ release from intracellular stores. 5. These data suggest that SALPC photodynamic action may permanently fix PI-PLC in an active conformation, and this produced recurrent [Ca2+]i spiking. PMID:9350616

  15. Fine mapping of Co-x, an anthracnose resistance gene to a highly virulent strain of Colletotrichum lindemuthianum in common bean.

    PubMed

    Richard, Manon M S; Pflieger, Stéphanie; Sévignac, Mireille; Thareau, Vincent; Blanchet, Sophie; Li, Yupeng; Jackson, Scott A; Geffroy, Valérie

    2014-07-01

    The Co - x anthracnose R gene of common bean was fine-mapped into a 58 kb region at one end of chromosome 1, where no canonical NB-LRR-encoding genes are present in G19833 genome sequence. Anthracnose, caused by the phytopathogenic fungus Colletotrichum lindemuthianum, is one of the most damaging diseases of common bean, Phaseolus vulgaris. Various resistance (R) genes, named Co-, conferring race-specific resistance to different strains of C. lindemuthianum have been identified. The Andean cultivar JaloEEP558 was reported to carry Co-x on chromosome 1, conferring resistance to the highly virulent strain 100. To fine map Co-x, 181 recombinant inbred lines derived from the cross between JaloEEP558 and BAT93 were genotyped with polymerase chain reaction (PCR)-based markers developed using the genome sequence of the Andean genotype G19833. Analysis of RILs carrying key recombination events positioned Co-x at one end of chromosome 1 to a 58 kb region of the G19833 genome sequence. Annotation of this target region revealed eight genes: three phosphoinositide-specific phospholipases C (PI-PLC), one zinc finger protein and four kinases, suggesting that Co-x is not a classical nucleotide-binding leucine-rich encoding gene. In addition, we identified and characterized the seven members of common bean PI-PLC gene family distributed into two clusters located at the ends of chromosomes 1 and 8. Co-x is not a member of Co-1 allelic series since these two genes are separated by at least 190 kb. Comparative analysis between soybean and common bean revealed that the Co-x syntenic region, located at one end of Glycine max chromosome 18, carries Rhg1, a major QTL contributing to soybean cyst nematode resistance. The PCR-based markers generated in this study should be useful in marker-assisted selection for pyramiding Co-x with other R genes.

  16. Activation of the sigma receptor 1 modulates AMPA receptor-mediated light-evoked excitatory postsynaptic currents in rat retinal ganglion cells.

    PubMed

    Liu, Lei-Lei; Deng, Qin-Qin; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-09-22

    Sigma receptor (σR), a unique receptor family, is classified into three subtypes: σR1, σR2 and σR3. It was previously shown that σR1 activation induced by 1μM SKF10047 (SKF) suppressed N-methyl-d-aspartate (NMDA) receptor-mediated responses of rat retinal ganglion cells (GCs) and the suppression was mediated by a distinct Ca(2+)-dependent phospholipase C (PLC)-protein kinase C (PKC) pathway. In the present work, using whole-cell patch-clamp techniques in rat retinal slice preparations, we further demonstrate that SKF of higher dosage (50μM) significantly suppressed AMPA receptor (AMPAR)-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) of retinal ON-type GCs (ON GCs), and the effect was reversed by the σR1 antagonist BD1047, suggesting the involvement of σR1. The SKF (50μM) effect was unlikely due to a change in glutamate release from bipolar cells, as suggested by the unaltered paired-pulse ratio (PPR) of AMPAR-mediated EPSCs of ON GCs. SKF (50μM) did not change L-EPSCs of ON GCs when the G protein inhibitor GDP-β-S or the protein kinase G (PKG) inhibitor KT5823 was intracellularly infused. Calcium imaging further revealed that SKF (50μM) did not change intracellular calcium concentration in GCs and persisted to suppress L-EPSCs when intracellular calcium was chelated by BAPTA. The SKF (50μM) effect was intact when protein kinase A (PKA) and phosphatidylinostiol (PI)-PLC signaling pathways were both blocked. We conclude that the SKF (50μM) effect is Ca(2+)-independent, PKG-dependent, but not involving PKA, PI-PLC pathways.

  17. Null mutation in PGAP1 impairing Gpi-anchor maturation in patients with intellectual disability and encephalopathy.

    PubMed

    Murakami, Yoshiko; Tawamie, Hasan; Maeda, Yusuke; Büttner, Christian; Buchert, Rebecca; Radwan, Farah; Schaffer, Stefanie; Sticht, Heinrich; Aigner, Michael; Reis, André; Kinoshita, Taroh; Jamra, Rami Abou

    2014-05-01

    Many eukaryotic cell-surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). There are at least 26 genes involved in biosynthesis and remodeling of GPI anchors. Hypomorphic coding mutations in seven of these genes have been reported to cause decreased expression of GPI anchored proteins (GPI-APs) on the cell surface and to cause autosomal-recessive forms of intellectual disability (ARID). We performed homozygosity mapping and exome sequencing in a family with encephalopathy and non-specific ARID and identified a homozygous 3 bp deletion (p.Leu197del) in the GPI remodeling gene PGAP1. PGAP1 was not described in association with a human phenotype before. PGAP1 is a deacylase that removes an acyl-chain from the inositol of GPI anchors in the endoplasmic reticulum immediately after attachment of GPI to proteins. In silico prediction and molecular modeling strongly suggested a pathogenic effect of the identified deletion. The expression levels of GPI-APs on B lymphoblastoid cells derived from an affected person were normal. However, when those cells were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC), GPI-APs were cleaved and released from B lymphoblastoid cells from healthy individuals whereas GPI-APs on the cells from the affected person were totally resistant. Transfection with wild type PGAP1 cDNA restored the PI-PLC sensitivity. These results indicate that GPI-APs were expressed with abnormal GPI structure due to a null mutation in the remodeling gene PGAP1. Our results add PGAP1 to the growing list of GPI abnormalities and indicate that not only the cell surface expression levels of GPI-APs but also the fine structure of GPI-anchors is important for the normal neurological development.

  18. 1alpha,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid.

    PubMed

    Schwartz, Z; Shaked, D; Hardin, R R; Gruwell, S; Dean, D D; Sylvia, V L; Boyan, B D

    2003-05-01

    1alpha,25(OH)(2)D(3) activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A(2) (PLA(2)). The purpose of this study was to determine if 1alpha,25(OH)(2)D(3) activates PI-PLC directly or through a PLA(2)-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by 1alpha,25(OH)(2)D(3). Inhibitors and activators of PLA(2) were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1alpha,25(OH)(2)D(3), was increased within 30s with peak activity at 1-3 min. PI-PLC activity in resting zone cells was unaffected by 1alpha,25(OH)(2)D(3). 1beta,25(OH)(2)D(3), 24R,25(OH)(2)D(3), actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1alpha,25(OH)(2)D(3) regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA(2)-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1alpha,25(OH)(2)D(3); PLA(2)-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF(3)) reduced the effect of 1alpha,25(OH)(2)D(3). Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1alpha,25(OH)(2)D(3) stimulated PI-PLC and PKC activities via Gq; GDPbetaS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-beta1a, PLC-beta1b, PLC-beta3 and PLC-gamma1 mRNA. Antibodies to PLC-beta1 and PLC-beta3 blocked the 1alpha,25(OH)(2)D(3) effect; antibodies to PLC-delta and PLC-gamma did not. Thus, 1alpha,25(OH)(2)D(3) regulates PLC-beta through PLA(2)-dependent production of

  19. Dimer Structure of an Interfacially Impaired Phosphatidylinositol-Specific Pholpholipase C

    SciTech Connect

    Shao,C.; Shi, X.; Wehbi, H.; Zambonelli, C.; Head, J.; Seaton, B.; Roberts, M,.

    2007-01-01

    The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8{angstrom} resolution. The W47A/W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp {yields} Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membrane-partitioning residues.

  20. Deletion of PLCB1 gene in schizophrenia-affected patients.

    PubMed

    Lo Vasco, Vincenza Rita; Cardinale, Giuseppina; Polonia, Patrizia

    2012-04-01

    A prevalence of 1% in the general population and approximately 50% concordance rate in monozygotic twins was reported for schizophrenia, suggesting that genetic predisposition affecting neurodevelopmental processes might combine with environmental risk factors. A multitude of pathways seems to be involved in the aetiology and/or pathogenesis of schizophrenia, including dopaminergic, serotoninergic, muscarinic and glutamatergic signalling. The phosphoinositide signal transduction system and related phosphoinositide-specific phospholipase C (PI-PLC) enzymes seem to represent a point of convergence in these networking pathways during the development of selected brain regions. The existence of a susceptibility locus on the short arm of chromosome 20 moved us to analyse PLCB1, the gene codifying for PI-PLC β1 enzyme, which maps on 20p12. By using interphase fluorescent in situ hybridization methodology, we found deletions of PLCB1 in orbito-frontal cortex samples of schizophrenia-affected patients.

  1. Deletion of PLCB1 gene in schizophrenia-affected patients

    PubMed Central

    Vasco, Vincenza Rita Lo; Cardinale, Giuseppina; Polonia, Patrizia

    2012-01-01

    Abstract A prevalence of 1% in the general population and approximately 50% concordance rate in monozygotic twins was reported for schizophrenia, suggesting that genetic predisposition affecting neurodevelopmental processes might combine with environmental risk factors. A multitude of pathways seems to be involved in the aetiology and/or pathogenesis of schizophrenia, including dopaminergic, serotoninergic, muscarinic and glutamatergic signalling. The phosphoinositide signal transduction system and related phosphoinositide-specific phospholipase C (PI-PLC) enzymes seem to represent a point of convergence in these networking pathways during the development of selected brain regions. The existence of a susceptibility locus on the short arm of chromosome 20 moved us to analyse PLCB1, the gene codifying for PI-PLC β1 enzyme, which maps on 20p12. By using interphase fluorescent in situ hybridization methodology, we found deletions of PLCB1 in orbito-frontal cortex samples of schizophrenia-affected patients. PMID:22507702

  2. Parkin deficiency disrupts calcium homeostasis by modulating phospholipase C signaling

    PubMed Central

    Sandebring, Anna; Dehvari, Nodi; Perez-Manso, Monica; Thomas, Kelly Jean; Karpilovski, Elena; Cookson, Mark R.; Cowburn, Richard F.; Cedazo-Mínguez, Angel

    2010-01-01

    Mutations in the E3 ubiquitin ligase parkin cause early onset autosomal recessive juvenile Parkinsonism (ARJP) presumably by having lack of function that alter the level, activity, aggregation or localization of its substrates. We recently reported that phospholipase Cγ1 (PLCγ1) is a substrate for parkin. Here, we show that parkin mutants and siRNA parkin knockdown cells have enhanced levels of PLCγ1 phosphorylation, basal phosphoinositide hydrolysis and intracellular Ca2+ ([Ca2+]i). The protein levels of Ca2+ regulated Protein Kinase C α (PKCα) were decreased in ARJP parkin mutant cells. Neomycin and dantrolene decreased [Ca2+]i levels in parkin mutants to those seen in wild-type (WT) parkin cells, suggesting that differences were a consequence of altered PLC activity. The protection of WT parkin against 6-hydroxydopamine (6OHDA) toxicity could also be established in ARJP mutants when pretreating with dantrolene, implying that balancing Ca2+ release from ryanodine-sensitive stores is decreasing the toxic effects from 6OHDA. Our findings suggests parkin as an important factor for maintaining Ca2+ homeostasis and that parkin deficiency leads to a PLC-dependent increase in [Ca2+]i levels that makes cells more vulnerable to neurotoxins such as 6OHDA. PMID:19663908

  3. Effects of carbocisteine on sialyl-Lewis x expression in an airway carcinoma cell line stimulated with tumor necrosis factor-alpha.

    PubMed

    Ishibashi, Yuji; Imai, Shigeru; Inouye, Yoshio; Okano, Teruo; Taniguchi, Akiyoshi

    2006-01-20

    Carbocisteine is a mucoregulatory drug normalizing sialic acid and fucose contents in mucins through the regulation of glycosyltransferase activities. Tumor necrosis factor (TNF)-alpha-induced overexpression of sialyl-Lewis x epitopes, containing sialic acid and fucose, in mucins were previously reported to be regulated by glycosyltransferase mRNAs expression through phosphatidyl inositol-specific phospholipase C (PI-PLC) signaling pathways [Ishibashi, Y., Inouye, Y., Okano, T., Taniguchi, A., 2005. Regulation of sialyl-Lewis x epitope expression by TNF-alpha and EGF in an airway carcinoma cell line. Glycoconj. J. 22, 53-62]. To investigate the mechanism behind the mucoregulatory action of carbocisteine, the present study evaluated the effects of carbocisteine on TNF-alpha-induced overexpression of sialyl-Lewis x epitopes in NCI-H292 cells. 100 mug/ml of carbocisteine was able to inhibit the TNF-alpha-induced expression of hST3GallV mRNA, FUT3 mRNA, C2/4GnT mRNA and sialyl-Lewis x epitopes as well as the TNF-alpha-induced activity of PI-PLC in NCI-H292 cells. These findings suggest that carbocisteine may normalize the sialyl-Lewis x epitopes expression in mucins through the inhibition of cellular PI-PLC activity in vivo.

  4. Major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid

    SciTech Connect

    Nagel, S.D.; Boothroyd, J.C.

    1989-04-05

    P30, the major surface antigen of the parasitic protozoan Toxoplasma gondii, can be specifically labeled with (/sup 3/H)palmitic acid and with myo-(2-/sup 3/H)inositol. The fatty acid label can be released by treatment of P30 with phosphatidylinositol-specific phospholipase C (PI-PLC). Such treatment exposes an immunological cross-reacting determinant first described on Trypanosoma brucei variant surface glycoprotein. PI-PLC cleavage of intact parasites metabolically labeled with (/sup 35/S)methionine results in the release of intact P30 polypeptide in a form which migrates faster in polyacrylamide gel electrophoresis. These results argue that P30 is anchored by a glycolipid. Results from thin layer chromatography analysis of purified (/sup 3/H) palmitate-labeled P30 treated with PI-PLC, together with susceptibility to mild alkali hydrolysis and to cleavage with phospholipase A2, suggest that the glycolipid anchor of T. gondii P30 includes a 1,2-diacylglycerol moiety.

  5. Signal transduction by the formyl peptide receptor. Studies using chimeric receptors and site-directed mutagenesis define a novel domain for interaction with G-proteins.

    PubMed

    Amatruda, T T; Dragas-Graonic, S; Holmes, R; Perez, H D

    1995-11-24

    The binding of small peptide ligands to high affinity chemoattractant receptors on the surface of neutrophils and monocytes leads to activation of heterotrimeric G-proteins, stimulation of phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the inflammatory response. It was recently shown (Amatruda, T. T., Gerard, N. P., Gerard, C., and Simon, M. I. (1993) J. Biol. Chem. 268, 10139-10144) that the receptor for the chemoattractant peptide C5a specifically interacts with G alpha 16, a G-protein alpha subunit of the Gq class, to trigger ligand-dependent stimulation of PI-PLC in transfected cells. In order to further characterize this chemoattractant peptide signal transduction pathway, we transfected cDNAs encoding the formylmethionylleucylphenylalanine receptor (fMLPR) into COS cells and measured the production of inositol phosphates. Ligand-dependent activation of PI-PLC was seen in COS cells transfected with the fMLPR and G alpha 16 and stimulated with fMLP but not in cells transfected with receptor alone or with receptor plus G alpha q. Chimeric receptors in which the N-terminal extracellular domain, the second intracellular domain, or the intracellular C-terminal tail of the fMLP receptor was replaced with C5a receptor domains (Perez, H. D., Holmes, R., Vilander, L. R., Adams, R. R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295) were capable of ligand-dependent activation of PI-PLC when co-transfected with G alpha 16. A chimeric receptor exchanging the first intracellular domain of the fMLPR was constitutively activated, stimulating PI-PLC in the absence of ligand. Constitutive activation of PI-PLC, to a level 233% of that seen in cells transfected with wild-type fMLP receptors, was dependent on G alpha 16. Site-directed mutagenesis of the first intracellular domain of the fMLPR (amino acids 54-62) reveals this to be a domain necessary for ligand-dependent activation of G alpha 16. These results suggest that

  6. PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries.

    PubMed

    Mauban, Joseph R H; Zacharia, Joseph; Fairfax, Seth; Wier, Withrow Gil

    2015-06-15

    Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K(+) concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca(2+) release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca(2+) waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca(2+) concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca(2+) entry and promote vasoconstriction.

  7. Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes.

    PubMed

    Bonilla, Julia B; Cid, M Belén; Contreras, F-Xabier; Goñi, Félix M; Martín-Lomas, Manuel

    2006-02-01

    The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs.

  8. Transient receptor potential melastatin 3 is a phosphoinositide-dependent ion channel.

    PubMed

    Badheka, Doreen; Borbiro, Istvan; Rohacs, Tibor

    2015-07-01

    Phosphoinositides are emerging as general regulators of the functionally diverse transient receptor potential (TRP) ion channel family. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been reported to positively regulate many TRP channels, but in several cases phosphoinositide regulation is controversial. TRP melastatin 3 (TRPM3) is a heat-activated ion channel that is also stimulated by chemical agonists, such as pregnenolone sulfate. Here, we used a wide array of approaches to determine the effects of phosphoinositides on TRPM3. We found that channel activity in excised inside-out patches decreased over time (rundown), an attribute of PI(4,5)P2-dependent ion channels. Channel activity could be restored by application of either synthetic dioctanoyl (diC8) or natural arachidonyl stearyl (AASt) PI(4,5)P2. The PI(4,5)P2 precursor phosphatidylinositol 4-phosphate (PI(4)P) was less effective at restoring channel activity. TRPM3 currents were also restored by MgATP, an effect which was inhibited by two different phosphatidylinositol 4-kinase inhibitors, or by pretreatment with a phosphatidylinositol-specific phospholipase C (PI-PLC) enzyme, indicating that MgATP acted by generating phosphoinositides. In intact cells, reduction of PI(4,5)P2 levels by chemically inducible phosphoinositide phosphatases or a voltage-sensitive 5'-phosphatase inhibited channel activity. Activation of PLC via muscarinic receptors also inhibited TRPM3 channel activity. Overall, our data indicate that TRPM3 is a phosphoinositide-dependent ion channel and that decreasing PI(4,5)P2 abundance limits its activity. As all other members of the TRPM family have also been shown to require PI(4,5)P2 for activity, our data establish PI(4,5)P2 as a general positive cofactor of this ion channel subfamily.

  9. New chromogenic plating media for detection and enumeration of pathogenic Listeria spp.--an overview.

    PubMed

    Reissbrodt, Rolf

    2004-08-15

    In recent years a number of selective chromogenic plating media for pathogenic Listeria spp. have been developed and marketed. Their advantages are direct detection and enumeration of pathogenic Listeria spp. utilizing cleavage of substrates by the virulence factor phosphatidylinositol-phospholipase C (PI-PLC) and, to a lesser extent, by phosphatidylcholin-phospholipase C (PC-PLC). There are two groups of such media: the first utilizes cleavage by PI-PLC of L-alpha-phosphatidyl-inositol, forming a white precipitation zone around the colony, combined with the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside for detection of beta-d-glucosidase, which occurs in all Listeria spp. All Listeria spp. produce turquoise colonies on these media which include ALOA , CHROMagar Listeria, BBL CHROMagar Listeria, and OCLA. The second group of media utilizes 5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate, forming blue-turquoise colonies of pathogenic Listeria spp. and white colonies of non-pathogenic Listeria spp. BCM trade mark Listeria monocytogenes plating medium, Rapid'L.mono and LIMONO-Ident-Agar belong to this group. Selective chromogenic L. monocytogenes plating media offer the attraction of rapid economic detection and enumeration of pathogenic Listeria spp. within 24 or 48 h of incubation at 36+/-1 degrees C. This overview summarises the characteristics of these chromogenic plating media, reviews important evaluations, and focuses on replacement of conventional by these chromogenic plating media, particularly for applications in the food industry.

  10. Stalling autophagy: a new function for Listeria phospholipases

    PubMed Central

    Tattoli, Ivan; Sorbara, Matthew T.; Philpott, Dana J.; Girardin, Stephen E.

    2014-01-01

    Listeria monocytogenes is a Gram-positive bacterial pathogen that induces its own uptake in non-phagocytic cells. Following invasion, Listeria escapes from the entry vacuole through the secretion of a pore-forming toxin, listeriolysin O (LLO) that acts to damage and disrupt the vacuole membrane. Listeria then replicates in the cytosol and is able to spread from cell-to-cell using actin-based motility. In addition to LLO, Listeria produces two phospholipase toxins, a phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB) and a broad-range phospholipase C (PC-PLC, encoded by plcA), which contribute to bacterial virulence. It has long been recognized that secretion of PI- and PC-PLC enables the disruption of the double membrane vacuole during cell-to-cell spread, and those phospholipases have also been shown to augment LLO-dependent escape from the entry endosome. However, a specific role for Listeria phospholipases during the cytosolic stage of infection has not been previously reported. In a recent study, we demonstrated that Listeria PI-PLC and PC-PLC contribute to the bacterial escape from autophagy through a mechanism that involves direct inhibition of the autophagic flux in the infected cells [Tattoli et al. EMBO J (2013), 32, 3066-3078].

  11. A Role for the Tec Family Tyrosine Kinase Txk in T Cell Activation and Thymocyte Selection

    PubMed Central

    Sommers, Connie L.; Rabin, Ronald L.; Grinberg, Alexander; Tsay, Henry C.; Farber, Joshua; Love, Paul E.

    1999-01-01

    Recent data indicate that several members of the Tec family of protein tyrosine kinases function in antigen receptor signal transduction. Txk, a Tec family protein tyrosine kinase, is expressed in both immature and mature T cells and in mast cells. By overexpressing Txk in T cells throughout development, we found that Txk specifically augments the phospholipase C (PLC)-γ1–mediated calcium signal transduction pathway upon T cell antigen receptor (TCR) engagement. Although Txk is structurally different from inducible T cell kinase (Itk), another Tec family member expressed in T cells, expression of the Txk transgene could partially rescue defects in positive selection and signaling in itk−/− mice. Conversely, in the itk+/+ (wild-type) background, overexpression of Txk inhibited positive selection of TCR transgenic thymocytes, presumably due to induction of cell death. These results identify a role for Txk in TCR signal transduction, T cell development, and selection and suggest that the Tec family kinases Itk and Txk perform analogous functions. PMID:10562318

  12. Mechanisms of Inhibition and Potentiation of α4β2 Nicotinic Acetylcholine Receptors by Members of the Ly6 Protein Family*

    PubMed Central

    Wu, Meilin; Puddifoot, Clare A.; Taylor, Palmer; Joiner, William J.

    2015-01-01

    α4β2 nicotinic acetylcholine receptors (nAChRs) are abundantly expressed throughout the central nervous system and are thought to be the primary target of nicotine, the main addictive substance in cigarette smoking. Understanding the mechanisms by which these receptors are regulated may assist in developing compounds to selectively interfere with nicotine addiction. Here we report previously unrecognized modulatory properties of members of the Ly6 protein family on α4β2 nAChRs. Using a FRET-based Ca2+ flux assay, we found that the maximum response of α4β2 receptors to agonist was strongly inhibited by Ly6h and Lynx2 but potentiated by Ly6g6e. The mechanisms underlying these opposing effects appear to be fundamentally distinct. Receptor inhibition by Lynx2 was accompanied by suppression of α4β2 expression at the cell surface, even when assays were preceded by chronic exposure of cells to an established chaperone, nicotine. Receptor inhibition by Lynx2 also was resistant to pretreatment with extracellular phospholipase C, which cleaves lipid moieties like those that attach Ly6 proteins to the plasma membrane. In contrast, potentiation of α4β2 activity by Ly6g6e was readily reversible by pretreatment with phospholipase C. Potentiation was also accompanied by slowing of receptor desensitization and an increase in peak currents. Collectively our data support roles for Lynx2 and Ly6g6e in intracellular trafficking and allosteric potentiation of α4β2 nAChRs, respectively. PMID:26276394

  13. On the Emerging Role of the Taste Receptor Type 1 (T1R) Family of Nutrient-Sensors in the Musculoskeletal System.

    PubMed

    Kokabu, Shoichiro; Lowery, Jonathan W; Toyono, Takashi; Sato, Tsuyoshi; Yoda, Tetsuya

    2017-03-15

    The special sense of taste guides and guards food intake and is essential for body maintenance. Salty and sour tastes are sensed via ion channels or gated ion channels while G protein-coupled receptors (GPCRs) of the taste receptor type 1 (T1R) family sense sweet and umami tastes and GPCRs of the taste receptor type 2 (T2R) family sense bitter tastes. T1R and T2R receptors share similar downstream signaling pathways that result in the stimulation of phospholipase-C-β2. The T1R family includes three members that form heterodimeric complexes to recognize either amino acids or sweet molecules such as glucose. Although these functions were originally described in gustatory tissue, T1R family members are expressed in numerous non-gustatory tissues and are now viewed as nutrient sensors that play important roles in monitoring global glucose and amino acid status. Here, we highlight emerging evidence detailing the function of T1R family members in the musculoskeletal system and review these findings in the context of the musculoskeletal diseases sarcopenia and osteoporosis, which are major public health problems among the elderly that affect locomotion, activities of daily living, and quality of life. These studies raise the possibility that T1R family member function may be modulated for therapeutic benefit.

  14. Family Therapy

    MedlinePlus

    ... Family therapy is a type of psychological counseling (psychotherapy) that helps family members improve communication and resolve ... organizations? Do you have specialty training in family psychotherapy? What is your experience with my family's type ...

  15. Family History

    MedlinePlus

    Your family history includes health information about you and your close relatives. Families have many factors in common, including their genes, ... as heart disease, stroke, and cancer. Having a family member with a disease raises your risk, but ...

  16. Family Folklore

    ERIC Educational Resources Information Center

    Kotkin, Amy J.; Baker, Holly C.

    1977-01-01

    Discusses the Family Folklore Program of the Smithsonian Institution's annual Festival of American Folklife, in which the whole family can be involved in tracing family history through story telling, photographs, etc. (MS)

  17. Family History

    MedlinePlus

    ... Aneurysms » Diagnosis » Family History A- A A+ Family History Familial intracranial aneurysms are generally defined as the ... patients with an Intracranial Aneurysm (IA) have a history of smoking at some time in their life. ...

  18. Family Folklore

    ERIC Educational Resources Information Center

    Kotkin, Amy J.; Baker, Holly C.

    1977-01-01

    Discusses the Family Folklore Program of the Smithsonian Institution's annual Festival of American Folklife, in which the whole family can be involved in tracing family history through story telling, photographs, etc. (MS)

  19. Family Literacy

    ERIC Educational Resources Information Center

    Holloway, John H.

    2004-01-01

    Rese