Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cascade of transmembrane signaling events that follow the occupancy of the interleukin 1 receptor remain poorly defined. We examined potential postreceptor transduction systems involved in human recombinant interleukin 1-beta-stimulated prostacyclin synthesis in human umbilical vein endothelium. Challenge of human umbilical vein endothelium monolayers with recombinant interleukin 1-beta resulted in dose- and time-dependent tritiated arachidonate release and prostacyclin synthesis consistent with phospholipase A2 activation. Prostacyclin synthesis after interleukin 1-beta (10 ng/ml) was detected 4 hours after stimulation and peaked at 16 to 24 hours. To examine whether interleukin 1-beta produced early activation of a phosphoinositide-specific phospholipase C, human umbilical vein endothelium monolayers were labeled with tritiated-2-myoinositol and inositol polyphosphates recovered after interleukin 1-beta stimulation. In contrast to the potent agonist, alpha-thrombin, interleukin 1-beta failed to significantly increase inositol phosphate production when examined for up to 4 hours. The absence of a significant increase in the Cai++ secretagogue, IP3, was confirmed in human umbilical vein endothelium monolayers loaded with the Ca++ photoprotein probe aequorin. Basal aequorin luminescence was unaltered after interleukin 1-beta (0 to 2 hours), whereas both alpha-thrombin and Ca++ ionophore A23187 produced rapid rises in Cai++. The intracellular Ca++ antagonist BAPTA and the extracellular Ca++ chelator EGTA produced significant inhibition of interleukin 1-beta-stimulated prostacyclin generation at 4 to 8 hours, suggesting either an indirect inhibitory effect of these agents on phospholipase A2 activity or that an increase in Ca++ may be a late event in the transduction scheme after interleukin 1 stimulation. Interleukin 1-beta-stimulated protein kinase C, phospholipase D, and adenylyl cyclase activities (0 to 4 hours) were unchanged from controls. Despite the absence of increased plasma membrane protein kinase C activity up to 4 hours after interleukin 1, pretreatment of human umbilical vein endothelium monolayers with staurosporine or phorbol myristate acetate (18 hours) to reduce protein kinase C activities, significantly attenuated the interleukin 1-stimulated prostanoid responses at 16 hours but not at 4 hours. Furthermore, short (5 minute) pretreatment with phorbol myristate acetate dramatically augmented interleukin 1-mediated prostacyclin responses in synergistic fashion, suggesting that protein kinase C may modulate interleukin 1 signal transducing pathways. In summary, these studies suggest that interleukin 1-beta-mediated endothelial cell phospholipase A2 activity and prostacyclin synthesis occur via a novel transducing pathway that does not involve early activation of phospholipase C, phospholipase D, or adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Interleukin 1-stimulated prostacyclin synthesis in endothelium: lack of phospholipase C, phospholipase D, or protein kinase C involvement in early signal transduction. 133 14

Stimulation of phospholipase D (PLD) by cell surface receptors has been observed in many cell types. We have investigated the mechanism of activation of this enzyme in undifferentiated HL60 cells. GTP analogues and Ca2+ (buffered in the nanomolar to micromolar range) were introduced into HL60 cells in the presence of the permeabilizing agent, streptolysin O. We report that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is a potent activator of phospholipase D when Ca2+ is available at micromolar levels. Phorbol 12-myristate 13-acetate or Ca2+ alone can also stimulate PLD, but to a limited extent. The activation of PLD by GTP[S] can be partially dissociated from GTP[S]-stimulated phosphoinositide-specific phospholipase C, suggesting that a G-protein may be directly involved in regulating PLD. However, maximal activation of PLD only occurs under conditions that are permissive to phospholipase C stimulation. We conclude that PLD activation is under dual control, i.e. protein kinase C- as well as G-protein-mediated regulation. Synergistic activation occurs when both pathways are simultaneously stimulated. We conclude that full activation of PLD requires protein kinase C, increased Ca2+ and a GTP-binding protein. Evidence for cytosolic components that may also be involved in obtaining full activation of PLD is also presented.
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PMID:Synergistic activation of phospholipase D by protein kinase C- and G-protein-mediated pathways in streptolysin O-permeabilized HL60 cells. 159 36

Ligation of the antigen receptors on both T and B lymphocytes induces phosphoinositide (PI) hydrolysis, Ca(2+)-mobilization and protein kinase C activation. The activation of the phosphoinositide-specific phosphodiesterase (PPI-PDE) following crosslinking of surface Ig receptors on B cells is controlled by an uncharacterized guanine nucleotide-regulatory (G) protein. Here we have used permeabilized murine T cells (both resting T cells and a conalbumin-specific CD4-positive T cell clone) to investigate a role for G protein(s) in coupling the TCR to the PPI-PDE. We found that anti-TCR McAb (or processed antigen)-induced PI hydrolysis cannot be uncoupled by permeabilizing T cells, as occurs with classical G protein-linked receptors. Furthermore, the TCR-mediated release of inositol phosphates in permeabilized T cells was not enhanced by non-hydrolyzable analogs of GTP, nor inhibited by GDP analogs. These findings therefore argue strongly against the concept that TCR-mediated PI hydrolysis is G-protein controlled.
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PMID:Antigen receptor-mediated phosphoinositide hydrolysis in murine T cells is not initiated via G-protein activation. 165 2

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. The inhibition of PLC by Genistein raised the possibility of involvement of tyrosine kinase in PAF receptor-coupled PLC activation. Western blot analysis using monoclonal antibody to phosphotyrosine demonstrated that PAF stimulated the tyrosine phosphorylation of two major proteins of 50,000 and 60,000 molecular weight. When platelets were challenged with PAF after treatment with either Genistein or CV-6209 (a PAF receptor antagonist), the reactivity of these proteins to monoclonal antibody was inhibited. Phosphoamino acid analysis of Mr 50,000 and 60,000 proteins confirmed that PAF increased the phosphorylation of tyrosine residues in both Mr 50,000 and 60,000 proteins and that this was inhibited by Genistein. Thus, PAF caused a receptor-dependent phosphorylation of tyrosine residues on Mr 50,000 and 60,000 proteins. Based on these observations, it is concluded that tyrosine kinase is involved in the PAF receptor-coupled PLC activation and signal transduction mechanism.
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PMID:Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: studies with genistein and monoclonal antibody to phosphotyrosine. 169 37

Complement receptor (CR)-mediated phagocytosis is associated with an increased accumulation of diglyceride (sn-1,2-diacylglycerol and/or 1-O-alkyl-2-acyl-glycerol) in human neutrophils. The C3bi-mediated increase in diglyceride (5-20 min) was only partially impaired when phosphoinositide-specific phospholipase C (PLC) activity was abolished by reduction of cytosolic free Ca2+. At an early time point (1 min), however, diglyceride production was barely detectable in control cells, whereas production was considerable in cells with a reduced cytosolic free Ca2+ concentration. C3bi stimulation of 32P-labeled neutrophils caused a rapid and significant breakdown of [32P]phosphatidylcholine (PC) which was not affected by inhibition of Ca(2+)-dependent phosphoinositide-specific PLC. Thus, PC hydrolysis could be involved in C3bi-induced diglyceride formation. Stimulation of cells labeled with [3H]1-O-alkyl-lyso-PC ([3H]alkyl-lyso-PC), resulted in an increased formation of [3H]1-O-alkyl-phosphatidic acid ([3H]alkyl-PA) and a later and slower formation of [3H]1-O-alkyl-diglyceride ([3H]alkyl-diglyceride); this suggests activation of phospholipase D (PLD). When these labeled cells were stimulated in the presence of 0.5% ethanol a marked accumulation of [3H]1-O-alkyl-phosphatidylethanol ([3H]alkyl-PEt) was observed in both controls and calcium-reduced cells, further strengthening the suggested involvement of PLD activity. In parallel with the sustained increase in diglyceride formation, CR-mediated phagocytosis was also associated with phosphorylation of a cellular protein kinase C substrate (MARCKS). Therefore it seems reasonable to suggest a causal relationship between C3bi-induced PLD activation, which results in diglyceride formation, and activation of protein kinase C. In electropermeabilized cells which were incapable of ingesting particles, C3bi particles were still able to activate PLD and induce formation of diglyceride. This signaling event must therefore be triggered by binding of particles to the cell and not by the engulfment process. Most importantly, introduction of the protein kinase C inhibitor peptides, PKC(19-36) and PKC(19-31), into these permeabilized cells resulted in a clear reduction of the C3bi-induced production of diglyceride, indicating that CR-mediated activation of protein kinase C directly triggers a positive feedback mechanism for additional diglyceride formation. Taken together, these data further clarify the mechanisms of CR-mediated diglyceride formation and give added support to the concept that protein kinase C plays an important role in the phagocytic process.
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PMID:Complement receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. Involvement of phospholipase D and direct evidence for a positive feedback signal of protein kinase. 173 62

We showed previously that glomerular mesangial cells displayed increased fibronectin, laminin, and type IV collagen synthesis and mRNA levels when grown in medium containing 30 mM glucose compared with those cells grown in 10 mM glucose [S. H. Ayo, R. A. Radnik, W. F. Glass II, J. A. Garoni, E. R. Rampt, D. R. Appling, and J. I. Kreisberg. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F185-F191, 1990]. However, total protein synthesis and actin mRNA were unchanged. In this report, we show that an increase in medium glucose concentration resulted in an increase in diacylglycerol (DAG) mass and transiently increased protein kinase C (PKC) activity as assessed by the translocation of PKC from the soluble to the particulate fraction. Effects of increased glucose on DAG were evident at 30 min and were maintained through 1 wk of growth in medium containing 30 mM glucose. Although total PKC activity (i.e., soluble plus particulate fractions) did not change with high-glucose treatment, the percent activity associated with the particulate fraction (i.e., activated PKC) increased significantly after 60 min in RPMI 1640 medium with 30 mM glucose. The distribution of PKC returned to control values by 24 h. High glucose did not stimulate phosphoinositide hydrolysis, as evidenced by the absence of an increase in the water-soluble inositol phosphates, indicating that DAG was not generated through the action of a phosphoinositide-specific phospholipase C. Cells treated with the cell-permeable DAG analogue 1-oleoyl-2-acetyl glycerol to activate PKC displayed approximately two-fold increases of fibronectin, laminin, and type IV collagen mRNA levels after normalization against actin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:High glucose increases diacylglycerol mass and activates protein kinase C in mesangial cell cultures. 192 72

Langendorff perfusion of guinea pig hearts with phorbol 12-myristate, 13-acetate or 1,2-dioctanoylglycerol caused a progressive impairment of contraction and relaxation of the left ventricle. Exposure of the hearts to 4 microM phorbol 12-myristate, 13-acetate or 200 microM 1,2-dioctanoylglycerol for 3 minutes resulted in a redistribution of protein kinase C activity and increased phosphorylation of a 28 kDa cytosolic protein. Examination of the incorporation of [32P]Pi into phosphatidylinositols and inositoltrisphosphates, under identical conditions, revealed that the degree of 32P-labeling of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4.5-bisphosphate was significantly increased. However, the degree of phosphate labeling of inositol trisphosphates was decreased. The effects of phorbol 12-myristate, 13-acetate and 1,2-dioctanoylglycerol on the intermediates of the phosphatidylinositol cycle were observed in the presence of prazosin, propranolol and atropine. Examination of the activity of phosphoinositide-specific phospholipase C in the perfused guinea pig hearts revealed that treatment with phorbol 12-myristate, 13-acetate was associated with a decrease in the membrane-associated enzymatic activity, assayed at low concentrations of calcium. Control hearts, perfused with a phorbol ester (4 alpha-phorbol 12,13-didecanoate) which does not activate protein kinase C, did not show any changes in cardiac contraction and relaxation or in the intermediates of the phosphatidylinositol cycle. The findings suggest that the basal production of inositol phosphates may be down-regulated by agents which activate protein kinase C in guinea pig hearts.
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PMID:The effect of phorbol esters and diacylglycerol analogues on the basal phosphoinositide turnover in isolated guinea pig hearts. 196 45

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.
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PMID:Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S. 196 91

We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.
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PMID:Stimulation of phospholipase D activity in human neuroblastoma (LA-N-2) cells by activation of muscarinic acetylcholine receptors or by phorbol esters: relationship to phosphoinositide turnover. 200 44

A major biochemical pathway that has been implicated in the control of normal and malignant growth involves phosphoinositide metabolism. In this pathway, receptor-mediated activation of a phosphoinositide-specific phospholipase C causes the hydrolysis of phosphatidylinositol-4,5-bisphosphate which generates two putative second messengers, inositol-1,4,5-trisphosphate and diacylglycerol (DAG). Since DAG has been shown to be elevated in many transformed cells, we sought to determine if the levels of PKC isoenzymes are also increased. Northern blot analysis of mRNAs from 46 human tumour cell lines was performed using probes for the human PKC-I (gamma), PKC-II (beta) and PKC-III (alpha) genes. PKC-II mRNAs were significantly increased in 4 out of 12 sarcoma lines and 1 malignant melanoma cell line. PKC-III was increased in 2 out of 12 sarcoma cell lines and 1 kidney carcinoma cell line. In contrast, in the majority of carcinoma-derived cell lines tested, there was a decreased or moderate expression of either PKC-II or PKC-III mRNAs or both. It is interesting that tumour cell lines which overexpressed one isoenzyme (e.g. PKC-II), did not contain detectable levels of another isoenzyme (e.g. PKC-III), as determined by Northern blotting. Altogether, these results suggest that the overexpression of distinct PKC isoenzymes may be involved in abnormal growth regulation in some human tumours, especially in sarcomas.
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PMID:[Overexpression of protein kinase-C-isoenzymes in human tumor cell lines]. 203 50


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