Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The NPXXY motif (X represents any amino acid) in the seventh transmembrane domain of the chemotactic formyl peptide receptor (FPR) is highly conserved among G protein-coupled receptors. Recent work suggested that this motif contributes to G protein-coupled receptor internalization and signal transduction; however, its role in FPR signaling remains unclear. In this study we replaced Asn(297) and Tyr(301) in the NPXXY motif of the human FPR with Ala (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effects of the substitutions on FPR functions in transfected rat basophilic leukemia cells. Whereas all the mutant receptors were expressed on the cell surface, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-beta and the p42/44 mitogen-activated protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, suggesting that the hydrogen bonding at Tyr(301) is critical for these functions. The Y301F receptor showed a chemotactic response similar to that of wild-type FPR, indicating that cell chemotaxis does not require receptor internalization and hydrogen bonding at the Tyr(301) position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis response that peaked at 0.1-1 nM. Finally, using a specific MAP kinase kinase inhibitor, we found that activation of MAP kinase is required for efficient FPR internalization, but is not essential for chemotaxis. These findings demonstrate that residues within the NPXXY motif differentially regulate the functions of FPR.
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PMID:Differential roles of the NPXXY motif in formyl peptide receptor signaling. 1123 59

Cotreatment with a minimally toxic concentration of the protein kinase C (PKC) activator (and down-regulator) bryostatin 1 (BRY) induced a marked increase in mitochondrial dysfunction and apoptosis in U937 monocytic leukemia cells exposed to the proteasome inhibitor lactacystin (LC). This effect was blocked by cycloheximide, but not by alpha-amanitin or actinomycin D. Qualitatively similar interactions were observed with other PKC activators (eg, phorbol 12-myristate 13-acetate and mezerein), but not phospholipase C, which does not down-regulate the enzyme. These events were examined in relationship to functional alterations in stress (eg, SAPK, JNK) and survival (eg, MAPK, ERK) signaling pathways. The observations that LC/BRY treatment failed to trigger JNK activation and that cell death was unaffected by a dominant-interfering form of c-JUN (TAM67) or by pretreatment with either curcumin or the p38/RK inhibitor, SB203580, suggested that the SAPK pathway was not involved in potentiation of apoptosis. In marked contrast, perturbations in the PKC/Raf/MAPK pathway played an integral role in LC/BRY-mediated cell death based on evidence that pretreatment of cells with bisindolylmaleimide I, a selective PKC inhibitor, or geldanamycin, a benzoquinone ansamycin, which destabilizes and depletes Raf-1, markedly suppressed apoptosis. Furthermore, ERK phosphorylation was substantially prolonged in LC/BRY-treated cells compared to those exposed to BRY alone, and pretreatment with the highly specific MEK inhibitors, PD98059, U0126, and SL327, opposed ERK activation while protecting cells from LC/BRY-induced lethality. Together, these findings suggest a role for activation and/or dysregulation of the PKC/MAPK cascade in modulation of leukemic cell apoptosis following exposure to the proteasome inhibitor LC. (Blood. 2001;97:2105-2114)
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PMID:Synergistic induction of apoptosis in human leukemia cells (U937) exposed to bryostatin 1 and the proteasome inhibitor lactacystin involves dysregulation of the PKC/MAPK cascade. 1126 78

The Ca(2+)-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A(2) (PLA(2)) by the Ca(2+)-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA(2) activity was attributable to cytosolic PLA(2) (cPLA(2)) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA(2). No CaR-stimulated cPLA(2) activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca(2+) (Ca(2+)(i)), whereas inhibition of phospholipase C (PLC) with completely inhibited CaR-stimulated PLC and cPLA(2) activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of G protein Signaling) protein that inhibits Galpha(q) activity. CaR-stimulated cPLA(2) activity was inhibited 80% by chelation of extracellular Ca(2+) and depletion of intracellular Ca(2+) with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca(2+), calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEK(K97R), had no effect on CaR-stimulated cPLA(2) activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA(2) via a Galpha(q), PLC, Ca(2+)-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.
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PMID:The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gqalpha -dependent ERK-independent pathway. 1127 41

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) beta1, 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 beta1, which was completely blocked by the MEK inhibitor PD 98059. Coimmunoprecipitation detected a specific association between the activated ERK and PLC beta1 within the nucleus. In vitro studies revealed that recombinant PLC beta1 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 beta1. In cells overexpressing a PLC beta1 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 beta1. This result suggests that ERK-evoked phosphorylation of PLC beta1 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.
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PMID:Phosphorylation of nuclear phospholipase C beta1 by extracellular signal-regulated kinase mediates the mitogenic action of insulin-like growth factor I. 1128 4

Microtubule-associated protein MAP-2 is a neuronal phosphoprotein which modulates microtubule stability and spatial organization of signal transduction pathways. The functions of MAP-2 are modulated by phosphorylation. We studied the modulation of MAP-2 phosphorylation using the N-methyl- D-aspartate (NMDA) type of glutamate receptors and the signal transduction pathways mediating this modulation in primary cultures of rat cerebellar neurons. NMDA induced a rapid increase (330% of basal at 5 min) in MAP-2 phosphorylation which was not prevented by KN-62, indicating that it is not mediated by activation of Ca-calmodulin-dependent protein kinase. NMDA-induced phosphorylation of MAP-2 was inhibited by the nitric oxide synthase inhibitors nitroarginine and 7-nitroindazole and by PD098059 (an inhibitor of MAP kinase kinase), but was only slightly reduced by calphostin C or U-73122, inhibitors of protein kinase C and of phospholipase C, respectively. This indicates that the main pathway mediating NMDA-induced phosphorylation of MAP-2 is activation of nitric oxide synthase and subsequent activation of MAP kinase. We show that activation of NMDA receptors induces an activation of MAP kinase which is prevented by nitroarginine. The nitric oxide-generating agent (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) also induced activation of MAP kinase and increased phosphorylation of MAP-2. Other nitric oxide-generating agents (NOC-18 and NOR-3) also increased MAP-2 phosphorylation. The interplay between NMDA receptors-associated signal transduction pathways and MAP-2 may be involved in the modulation of neuronal responses to extracellular signals and in the regulation of neuronal function.
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PMID:NMDA-induced phosphorylation of the microtubule-associated protein MAP-2 is mediated by activation of nitric oxide synthase and MAP kinase. 1129 88

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.
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PMID:Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells. 1130 43

This study investigated the effects of C7 and C9 aliphatic (n-heptane, n-nonane), naphthenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)) and aromatic (toluene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat brain synaptosome fraction. Methyl mercury (MeHg) was included as a positive control. Exposure of the synaptosomes to the hydrocarbons produced a concentration-dependent linear increase in the formation of the fluorescence of 2',7'-dichlorofluorescein (DCF) as a measure of the production of ROS and RNS. Formation of RNS was demonstrated by preincubation of the synaptosome fraction with the neuronal nitric oxide synthase (nNOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME), which reduced the MeHg and TMCH-stimulated fluorescence by 51% and 65%, respectively. The naphthenic hydrocarbon TMCH showed the strongest potential for ROS and RNS formation in rat brain synaptosomes, followed by TMB, toluene, n-nonane, n-heptane, and methylcyclohexane, respectively. TMCH was selected for mechanistic studies of the formation of ROS. Both MeHg and TMCH induced an increase in intracellular calcium concentration [Ca(2+)]i as measured with Fura-2. Blockade of voltage-dependent Ca(2+) channels with lanthanum prior to stimulation with MeHg and TMCH led to a reduction in the ROS/RNS formation of 72% and 70%, respectively. Furthermore, addition of cyclosporin A (CSA), a blocker of the mitochondrial permeability transition pore (MTP), lowered both the MeHg and TMCH-elevated DCF fluorescence by 72% and 59%. Preincubation of the synaptosome fraction with the protein tyrosine kinase inhibitor genistein lowered the MeHg and TMCH-stimulated fluorescence by 85% and 91%, respectively. Addition of the extracellular signal-regulated protein kinase (MEK)-1 and -2 inhibitor U0126 reduced the fluorescence stimulated by MeHg and TMCH by 62% and 63%. Furthermore, the protein kinase C inhibitor bisindolylmaleimide reduced the fluorescence stimulated by MeHg and TMCH by 52% and 56%. The compound 1-(6-[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-aminohexyl)-1H-pyrrole-2,5-dione (U73122), which inhibits phospholipase C, was shown to decrease the ROS and RNS formation induced by MeHg and TMCH by 49% and 64%, respectively. The phospholipase A2 (PLA2) inhibitor 7,7-dimethyl eicosadienoic acid (DEDA) reduced fluorescence in response to MeHg and TMCH by 49% and 54%. Simultaneous addition of L-NAME, CSA, and DEDA to the synaptosome fraction totally abolished the DCF fluorescence. In conclusion, C7 and C9 aliphatic, naphthenic, and aromatic hydrocarbons stimulated formation of ROS and RNS in rat brain synaptosomes. The naphthenic hydrocarbon TMCH stimulated formation of ROS and RNS in the synaptosomes through Ca(2+)-dependent activation of PLA2 and nNOS, and through increased transition permeability of the MTP. Exposure of humans to the naphthenic hydrocarbon TMCH may stimulate formation of free radicals in the brain, which may be a key factor leading to neurotoxicity.
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PMID:The effect of aliphatic, naphthenic, and aromatic hydrocarbons on production of reactive oxygen species and reactive nitrogen species in rat brain synaptosome fraction: the involvement of calcium, nitric oxide synthase, mitochondria, and phospholipase A. 1137 3

1. Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (L(p)) in vivo. To determine the signal transduction cascade through which this is mediated, we measured the effect of inhibition of various signalling pathways on VEGF-mediated acute increases in L(p) in individually perfused frog mesenteric microvessels. 2. VEGF receptors have previously been shown to activate phospholipase C-gamma (PLCgamma), protein kinase C (PKC) and MEK, the mitogen-activated and extracellular signal-related kinase (ERK) kinase. To determine the role of these signalling pathways we measured the effects of inhibitors of each on the VEGF-mediated increase in L(p). 3. VEGF-mediated increases in L(p) were attenuated by pre-treatment with the PLC inhibitor U73122, but not affected by treatment with the inactive enantiomer U73343. The PLC inhibitor was also able to attenuate the increase in L(p) mediated by the inflammatory mediator ATP. 4. Inhibition of either PKC or MEK activation using the selective inhibitors bisindolylmaleimide (BIM, 1 microM) and PD98059 (30 microM), respectively, did not change the VEGF-mediated increase in L(p). However, PD98059, BIM and U73122 all reduced phosphorylation of ERK1/2 determined by Western blot analysis with anti-phospho-ERK1/2 antibodies. 5. Furthermore, inhibition of the conversion of diacyl glycerol (DAG) to arachidonic acid, by perfusion with the DAG lipase inhibitor RHC80267 (50 microM), did not attenuate the increase in L(p) brought about by VEGF. 6. These data suggest that VEGF acutely increases microvascular permeability in vivo through a mechanism that is dependent on PLC stimulation, but is independent of PKC or MEK activation or production of arachidonic acid from DAG. We therefore propose that VEGF acutely acts to increase L(p) through the direct actions of DAG, independently of PKC or arachidonic acid.
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PMID:In vivo mechanisms of vascular endothelial growth factor-mediated increased hydraulic conductivity of Rana capillaries. 1145 65

TNF-alpha induced an increase in intercellular adhesion molecule-1 (ICAM-1) expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. The enhanced ICAM-1 expression was shown to increase the adhesion of U937 cells to A549 cells. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D 609) attenuated TNF-alpha-induced ICAM-1 expression. TNF-alpha produced an increase in protein kinase C (PKC) activity and this effect was inhibited by D 609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited TNF-alpha-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression, this effect was inhibited by genistein or tyrphostin 23. Treatment of cells with TNF-alpha resulted in stimulation of p44/42 MAPK, p38, and JNK. However, TNF-alpha-induced ICAM-1 expression was not affected by either MEK inhibitor, PD 98059, or p38 inhibitor, SB 203580. A cell-permeable ceramide analog, C(2) ceramide, also stimulated the activation of these three MAPKs, but had no effect on ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by TNF-alpha and these effects were inhibited by D 609, calphostin C, or tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity, these effects being inhibited by genistein or tyrphostin 23. TNF-alpha- or TPA-induced ICAM-1 promoter activity was inhibited by dominant negative PKCalpha or IKK2, but not IKK1 mutant. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. These data suggest that, in A549 cells, TNF-alpha activates PC-PLC to induce activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression and neutrophil adhesion. However, activation of p44/42 MAPK, p38, and JNK is not involved in this event.
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PMID:Tumor necrosis factor alpha-induced activation of downstream NF-kappaB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion. Involvement of PKCalpha, tyrosine kinase, and IKK2, but not MAPKs, pathway. 1148 7

In Jurkat T lymphocytes, hydrogen peroxide (H(2)O(2)) potentiates the phosphorylation level of extracellular signal-regulated kinase 1 and 2 (ERK1/2) caused by T cell receptor (TCR) stimulation with anti-CD3 and anti-CD28 or anti-CD3 alone. Submillimolar concentrations of H(2)O(2)-induced phosphorylation of ERK1/2 and MAP/ERK kinase 1 and 2 (MEK1/2) without antigenic stimulation. H(2)O(2) also induced the electrophoretic mobility shift of Lck from 56 to 60 kDa. The MEK inhibitor, PD98059 attenuated ERK1/2 and MEK1/2 phosphorylation, as well as the migration shift of Lck induced by H(2)O(2). The phospholipase C (PLC) inhibitor, U73122, and EGTA reduced the phosphorylation of both ERK1/2 and MEK1/2 induced by H(2)O(2). Interestingly, an increase of intracellular cAMP level with forskolin or 8-(4-chlorophenylthio)-cAMP augmented ERK1/2 phosphorylation by H(2)O(2), while inhibiting MEK1/2 phosphorylation by H(2)O(2). These results demonstrate an alternative pathway that results in augmentation of ERK1/2 phosphorylation without concomitant MEK1/2 phosphorylation in T cells.
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PMID:cAMP potentiates H(2)O(2)-induced ERK1/2 phosphorylation without the requirement for MEK1/2 phosphorylation. 1149 22


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