Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary astrocytic cultures derived from day-15 chick embryo (E15) cerebral hemispheres (CH) or cerebellum (CB) express a calcium/phospholipid-dependent isoform as the major protein kinase C (PKC-alpha/beta). PKC was activated (translocation of activity from cytosol to membrane) following stimulation with carbachol, so we tested for activation of phospholipase C (PLC) as the source of diacylglycerol released from polyphosphoinositide (PIP2) hydrolysis. Carbachol activated PLC (inositol phosphate release) 4-fold in a time- and dose-dependent manner in cortical (CH) astrocytes, but there was no activation of PLC in astrocytes from cerebellum (CB). Pirenzepine, but not gallamine, attenuated both carbachol-induced PKC translocation and PIP2 hydrolysis in E15CH astrocytes, arguing for contribution of M1 subtype. The phorbol ester TPA completely inhibited PIP2 hydrolysis, both basal and carbachol-stimulated, and elicited a stronger, but shorter (10 min) activation of PKC than that observed with carbachol. We investigated phospholipase D (PLD) activation as an alternate source of diacylglycerol in astrocytes, since the ratio of PLC to PKC activation by carbachol was lower in astrocytes than observed in neurons. We observed a dramatic (10-fold) time- and dose-dependent activation of PLD by TPA in CH and a 3-fold increase in CB. The duration of TPA-dependent PLD activation correlated well with increased cell proliferation and changes in astrocytic phenotype markers. Carbachol-stimulated PLD activation was observed in CH but not in CB astrocytes, being mostly dependent on the M3 receptor subtype in the former. In contrast, glutamate elicited a greater PLD activation in CB astrocytes, than in CH astrocytes. TPA activation of PLD was totally blocked by staurosporine (PKC inhibitor) and genistein (a tyrosine kinase inhibitor) in cerebellar (CB) astrocytes; however, total inhibition of TPA-dependent PLD activation was only achieved in cortical (CH) astrocytes after addition of EGTA. Thapsigargin activated PLD in both populations, further emphasizing the PLD activation dependency on [Ca2+]i. Taken together with our previous observations that TPA induces proliferation, cytoskeleton changes, and decreases of glutamine synthetase activity, these data suggest that phospholipase D is a differential but important participant in the regulation of the signalling of mitosis and differentiation in astrocytes during their development.
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PMID:Differential regulation of phospholipases C and D by phorbol esters and the physiological activators carbachol and glutamate in astrocytes from chicken embryo cerebrum and cerebellum. 755 28

Protein kinase C (PKC) isoenzymes are essential components of cell signaling. In this study, we investigated the regulation of PKC-alpha in murine B16 amelanotic melanoma (B16a) cells by the monohydroxy fatty acids 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE]. 12(S)-HETE induced a translocation of PKC-alpha to the plasma membrane and focal adhesion plaques, leading to enhanced adhesion of B16a cells to the matrix protein fibronectin. However, 13(S)-HODE inhibited these 12(S)-HETE effects on PKC-alpha. A receptor-mediated mechanism of action for 12(S)-HETE and 13(S)-HODE is supported by the following findings. First, 12(S)-HETE triggered a rapid increase in cellular levels of diacylglycerol and inositol trisphosphate in B16a cells. 13(S)-HODE blocked the 12(S)-HETE-induced bursts of both second messengers. Second, the 12(S)-HETE-increased adhesion of B16a cells to fibronectin was sensitive to inhibition by a phospholipase C inhibitor and pertussis toxin. Finally, a high-affinity binding site (Kd = 1 nM) for 12(S)-HETE was detected in B16a cells, and binding of 12(S)-HETE to B16a cells was effectively inhibited by 13(S)-HODE (IC50 = 4 nM). In summary, our data provide evidence that regulation of PKC-alpha by 12(S)-HETE and 13(S)-HODE may be through a guanine nucleotide-binding protein-linked receptor-mediated hydrolysis of inositol phospholipids.
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PMID:12(S)-hydroxyeicosatetraenoic acid and 13(S)-hydroxyoctadecadienoic acid regulation of protein kinase C-alpha in melanoma cells: role of receptor-mediated hydrolysis of inositol phospholipids. 756 26

Intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma R is known to be a phospholipase C (PLC)-dependent process. Activation of PLC leads to the formation of second messengers that synergistically activate protein kinase C (PKC). The aim of this study was to obtain more insight into the role of PKC in Fc gamma R-mediated killing process. PKC inhibitors H-7 and staurosporine markedly suppressed the killing of S. aureus by monocytes stimulated by cross-linking Fc gamma RI or -II. Cross-linking Fc gamma R caused a transient increase in PKC activity in the membranes of monocytes, as measured by Ca2+/phospholipid-dependent phosphorylation of histone. Western blot analysis revealed that cross-linking Fc gamma R stimulated a transient increase in PKC-beta in the membranes of monocytes with kinetics that correlated closely with the translocation of PKC activity. Cross-linking Fc gamma R on monocytes also stimulated the translocation of PKC-epsilon but not PKC-alpha. PMA and 1-oleoyl-2-acetylglycerol (OAG), which caused translocation of PKC-alpha, -beta, and -epsilon, did not stimulate the killing process. Incubation with these PKC activators for 10 min rendered monocytes unresponsive to stimulation of killing of S. aureus via Fc gamma R. It could be that activation of certain PKC isozymes, probably PKC-alpha and -epsilon, by these activators causes feedback inhibition of PLC and, consequently, the killing in monocytes, because PMA blocks the Fc gamma R-mediated intracellular inositol(1,4,5)P3 formation and PKC translocation. Together, our results indicate that PKC isozymes play an important role in both stimulation and inhibition of the Fc gamma R-mediated intracellular killing of bacteria by monocytes.
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PMID:Role of protein kinase C isozymes in Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes. 760 54

Cultured astrocytes express bradykinin (BK) receptors coupled to phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis. Short term (10- or 90-min) treatment of cells with 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) decreased BK-induced PI breakdown, but this inhibitory action was lost after 3-hr TPA treatment. Extended (6- or 24-hr) pretreatment resulted in marked potentiation of the BK response. Western blot analysis using protein kinase C (PKC) isozyme-specific antibodies indicated that astrocytes express PKC-alpha, PKC-delta, and PKC-zeta. With TPA treatment of the cells for various times (10 min, 90 min, 3 hr, 6 hr, or 24 hr), translocation of PKC-alpha and PKC-delta from the cytosol to the membrane was seen after 10- or 90-min treatment and restoration to basal levels in the membrane fraction was seen after 3-hr treatment. However, partial or complete down-regulation of PKC-alpha and PKC-delta was seen after 6- or 24-hr treatment, respectively. No translocation or down-regulation of PKC-zeta was seen after either short term or long term TPA treatment. The inactive phorbol ester alpha-TPA had no effect on BK-induced PI hydrolysis or on the translocation or down-regulation of PKC-alpha and PKC-delta. These results suggest that, in unstimulated astrocytes, both PKC-alpha and PKC-delta, but not PKC-zeta, may exert tonic inhibition of BK-mediated PI turnover. After 10- or 90-min TPA treatment, AIF4(-)--but not Ca2+ ionophore-induced PI hydrolysis was inhibited, whereas [3H]BK binding was unaffected, indicating that the site of action of PKC-alpha and PKC-delta in the BK receptor/G protein/PLC pathway is after the receptor and before PLC, i.e., the G protein. After down-regulation of PKC-alpha and -delta, increases in both AIF4(-)-induced inositol phosphate formation and [3H]BK binding contributed to marked potentiation of BK-induced PI responses. Scatchard plot analysis showed an increase in both the maximal number of binding sites and the binding affinity. Both the up-regulation of [3H]BK binding and the subsequent BK-induced PI turnover were blocked by 0.5 microM cycloheximide, a protein synthesis inhibitor. The increase in AIF4(-)-induced PI hydrolysis after 24-hr TPA treatment was also inhibited by cycloheximide, indicating that new synthesis of BK receptors and G proteins was required after down-regulation of PKC-alpha and PKC-delta.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of protein kinase C subtypes alpha and delta in the regulation of bradykinin-stimulated phosphoinositide breakdown in astrocytes. 762 73

Rapid in vitro effects of aldosterone (ALDO) on intracellular sodium, potassium and calcium, cell volume and the sodium-proton-antiport have been described in human mononuclear leukocytes and rat vascular smooth muscle cells (VSMC). These nongenomic effects are signaled through membrane receptors with a high affinity for aldosterone, but not for hydrocortisone. Effects of ALDO on the production of diacylglycerol (DAG) and protein kinase C alpha (PKC) were measured in VSCM by enzymatic assay and immunoblotting. DAG production was stimulated twofold by ALDO (> or = 1 nM) within 30 sec while hydrocortisone was inactive at concentrations of up to 1 microM. The inhibitors of phospholipase C, neomycin and U-73122 completely blocked this effect. PKC translocation from cytosol to membranes by ALDO occurred within 5 min, the extent of this effect was comparable to that of angiotensin II. These data demonstrate rapid intracellular signaling for ALDO in VSMC through phospholipase C, DAG and PKC in addition to calcium and inositol-1,4,5-trisphosphate as determined earlier.
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PMID:Rapid aldosterone signaling in vascular smooth muscle cells: involvement of phospholipase C, diacylglycerol and protein kinase C alpha. 763 25

Rapid and long term effects of protein kinase C alpha activation on receptor tyrosine kinase signaling parameters were investigated in human 293 embryonic fibroblasts and mouse NIH 3T3 cells. Within minutes of phorbol 12-myristate 13-acetate treatment, epidermal growth factor receptor and HER2 tyrosine phosphorylation was decreased, while platelet-derived growth factor receptor and insulin receptor autophosphorylation was upregulated. These effects are not mediated by protein kinase C-dependent receptor tyrosine kinase phosphorylation but apparently by activation or inactivation of receptor tyrosine kinase-specific phosphatases, as indicated by neutralization of these phenomena upon treatment of cells with sodium orthovanadate. In contrast to these short term effects, sustained activation of protein kinase C alpha by phorbol 12-myristate 13-acetate results in translocation of protein kinase C from the cytosol to the membrane fraction where it forms stable complexes with all receptor tyrosine kinases investigated. Ligand-induced receptor tyrosine kinase/protein kinase C association in NIH 3T3 fibroblasts is accompanied by a mobility shift of the receptor, indicating phosphorylation by activated protein kinase C. This phenomenon correlates with the disappearance of receptor tyrosine kinases from the cell surface, implying that this interaction plays a role in the process of receptor internalization and degradation. Interestingly, ligand-stimulated receptor down-regulation is also enhanced by overexpression of phospholipase C gamma, which strongly indicates a role for this common receptor tyrosine kinase substrate in negative regulation of growth factor signals.
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PMID:Rapid and long-term effects on protein kinase C on receptor tyrosine kinase phosphorylation and degradation. 764 54

Transforming growth factor beta (TGF-beta) regulates the proliferation and differentiation of chondrocytes; however, the mechanism of TGF-beta signal transduction remains unclear. We examined whether the response to TGF-beta is mediated by protein kinase C activity in chondrocytes at different stages of maturation. The aims were to examine the effect of recombinant human TGF-beta 1 (rhTGF-beta 1) on protein kinase C in rat costochondral chondrocyte cultures; determine the major isoform present; assess the involvement of phospholipase C or tyrosine kinases; determine whether genomic or nongenomic pathways are involved; and test whether these mechanisms differ as a function of the stage of cell maturation. Dose-dependent increases in protein kinase C activity were observed in confluent, fourth-passage cultures of rat costochondral growth zone and resting zone chondrocytes treated with rhTGF-beta 1. In growth zone cells, elevated activity was observed at 12 h and decreased markedly by 24 h. In resting zone cells, elevated activity was observed at 9 h, maximum stimulation occurred at 12 h, and activity returned to baseline levels after 48 h. Immunoprecipitation studies showed protein kinase C alpha is the major isoform present in both untreated and treated cells. Neither the phospholipase C inhibitor, U73122, nor the tyrosine kinase inhibitor, genistein, significantly reduced the protein kinase C response to rhTGF-beta 1. Actinomycin D and cycloheximide, inhibitors of transcription and translation, produced dose-dependent inhibition of rhTGF-beta 1 stimulated protein kinase C activity in both resting zone and growth zone chondrocytes. The time course of activation and insensitivity to U73122 suggest that phospholipase C-mediated events are not involved in rhTGF-beta 1 stimulation of protein kinase C in costochondral chondrocytes. Similarly, because genistein had no effect, tyrosine kinases are not implicated. Rather, the reduction in protein kinase C activity observed when rhTGF-beta 1 is administered along with actinomycin D or cycloheximide indicates that new gene expression and protein synthesis are required for the response. These results indicate that the effect of rhTGF-beta 1 is mediated by protein kinase C; however, it is very slow and may require new protein kinase C production, perhaps via a cytokine cascade. Moreover, the classic mechanism of activation of protein kinase C by phospholipase C was not found, suggesting a novel mechanism of activation. Finally, the effects of rhTGF-beta 1 on protein kinase C are dependent on the state of cell maturation with respect to onset and duration of response.
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PMID:Regulation of protein kinase C by transforming growth factor beta 1 in rat costochondral chondrocyte cultures. 781 33

Our laboratory recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly increases the breakdown of membrane phosphoinositides, raises intracellular calcium concentration ([Ca2+]i), and translocates protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells. In the present experiments, we found that Caco-2 cells contained predominantly the alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)2D3 and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-alpha, but not PKC-zeta. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-alpha, but not PKC-zeta. Since PKC inhibits phospholipase C-induced mobilization of Ca2+ in other cells, we examined the effects of staurosporine and H-7, PKC inhibitors, and TPA on 1,25(OH)2D3-stimulated increase in [Ca2+]i. As previously demonstrated by our laboratory, 1,25(OH)2D3 caused a biphasic increase in [Ca2+]i, with an initial elevation (transient phase) followed by a sustained increase (plateau phase). We previously demonstrated that the transient phase is mediated, at least in part, by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] stimulated by the secosteroid. Acute pretreatment with staurosporine or H-7 caused a significant stimulation, whereas acute TPA pretreatment caused a significant inhibition of the 1,25(OH)2D3-induced increase in the transient phase of [Ca2+]i. Preincubation of Caco-2 cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (BAPTA-AM) abolished both the rise in [Ca2+]i and the increase in particulate-associated PKC-alpha stimulated by 1,25(OH)2D3. Moreover, downregulation of PKC-alpha by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)2D3-stimulated rise in [Ca2+]i but had no effect on the 1,25(OH)2D3-induced change in Ins(1,4,5)P3 concentration. Furthermore, in these PKC-alpha downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca2+]i. These results indicate that 1,25(OH)2D3, which increases [Ca2+]i and diacylglycerol, activates PKC-alpha, but not PKC-zeta. The alpha-isoform, in turn, limits the secosteroid-stimulated rise in [Ca2+]i, at a step distal to Ins(1,4,5)P3 accumulation in Caco-2 cells.
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PMID:1,25(OH)2 vitamin D3 activates PKC-alpha in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i. 794 45

Prompted by the reversal of skin hyperproliferation to normal by 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid, we investigated a possible mechanism for this antiproliferative action. To address this we first demonstrated that 13-HODE is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns4,5-P2) and released as 13-HODE-containing diacylglycerol by epidermal phospholipase C. Secondly, we tested the possibility whether this putative 13-HODE-containing DAG (13HODE-DAG) could exert a modulatory effect on epidermal protein kinase C (PKC) activity which previously has been associated with skin hyperproliferation. Unlabeled 13HODE-DAG was generated from 13-HODE-containing phosphatidylcholine after phospholipase C hydrolytic cleavage. The effects of the 13HODE-DAG were determined on: i) total epidermal PKC activity; ii) diolein-activated PKC activity; and iii) the two identified epidermal PKC-isozymes (PKC-beta and PKC-alpha). Our data revealed over a twofold activation of total basal PKC activity by diolein. In contrast, replacement of diolein (1,2-dioleoylglycerol) with 13HODE-DAG (1-palmitoyl,2-13HODE-glycerol) in the incubation mixture exerted no effect on total basal PKC activity. In an another experiment, 13HODE-DAG inhibited diolein-activated PKC activity in a dose-dependent manner. To determine whether the effects of 13HODE-DAG are selective, we tested its effects on DEAE-Sephacel-purified and Western blot-confirmed PKC isozymes. Our data revealed that 13HODE-DAG selectively inhibited the activity of PKC-beta isozyme, while exerting negligible effect on the PKC-alpha isozyme. This selective inhibitory effect of 13HODE-DAG on a major epidermal PKC isozyme activity suggests that 13HODE-containing DAG seemingly can modulate epidermal PKC activity, which purportedly is associated with epidermal hyperproliferation.
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PMID:Expression of protein kinase C isozymes in guinea pig epidermis: selective inhibition of PKC-beta activity by 13-hydroxyoctadecadienoic acid-containing diacylglycerol. 807 13

The effects of a series of protein kinase C (PKC) activators with different spectra of biological activities and reportedly different patterns of PKC isoenzyme activation were examined in renal mesangial cells. Treatment of mesangial cells with the tumor promoters phorbol 12-myristate 13-acetate (PMA), debromoaplysiatoxin, dihydroteleocidin and thymeleatoxin, as well as with the marine natural product bryostatin 1, caused translocation and at least partial down-regulation of the PKC-alpha, -delta and -epsilon isoenzymes as assessed by immunoblot analysis. Bryostatin 1 mediates a faster depletion of PKC-alpha isoform than any of the other PKC activators. Thymeleatoxin, which has been reported to selectively activate PKC-alpha, -beta and -gamma, but not PKC-delta or -epsilon isoenzymes in vitro, turned out to exert the most potent effect on PKC-delta and -epsilon in mesangial cells and down-regulated these isotypes within 8-24 hr. None of the compounds tested affected cellular distribution or amount of PKC-zeta in mesangial cells. Thus, all of the PKC activators tested are able to translocate and down-regulate three of the four PKC isoenzymes present in mesangial cells although with different kinetics. All PKC activators stimulated a phospholipase A2-mediated arachidonic acid release, a phospholipase D-mediated phosphatidylcholine hydrolysis, a comparable small proliferative response and an inhibition of phospholipase C-mediated inositol trisphosphate generation. These results suggest: (i) that the PKC activators investigated in this study do not display any type of isotype-specificity that could be used to selectively activate or down-regulate PKC isoenzymes in intact cell-systems; (ii) that thymeleatoxin has a different isoenzyme selectivity in intact cells as compared to in vitro enzyme inhibition data; and (iii) PKC-zeta is resistant to all PKC activators investigated in this study.
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PMID:Comparison of different tumour promoters and bryostatin 1 on protein kinase C activation and down-regulation in rat renal mesangial cells. 808 Apr 41


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