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)

In response to epidermal growth factor (EGF), HeLa cells and A431 cells rapidly accumulate substantial amounts of phosphatidic acid (up to 0.16 and 0.2 micrograms/10(6) cells respectively), which represents approx. 0.17% of total phospholipid. Phosphatidic acid may be a potential product of diacylglycerol kinase and/or of phospholipase D. To evaluate the contribution of phospholipase D, the phosphatidyl-transfer reaction to a primary alcohol (mostly butan-1-ol; 0.2%) was measured; this reaction is known to be mediated exclusively by phospholipase D in intact cells. In HeLa and in A431 cells prelabelled with [1-14C]oleic acid, EGF (10 and 100 nM respectively) caused a 3-fold increase in radioactive phosphatidylbutanol within 5 min at the expense of labelled phosphatidic acid. Dose-response relationships showed 10 nM- and 100 nM-EGF to be maximally effective in HeLa cells and A431 cells respectively. Mass determinations showed that the phosphatidylbutanol formed within 5 min represented only part of the phosphatidic acid. Depletion of protein kinase C by pretreatment of A431 cells for 17 h with the phorbol ester phorbol 12-myristate 13-acetate (0.1 microM) did not impair EGF-induced formation of phosphatidylbutanol, thus indicating that the reaction was independent of this enzyme. Since phosphatidic acid is suggested to exert second-messenger functions as well as to induce biophysical changes in cellular membranes, its formation, including that via the phospholipase D pathway, may represent an important link between extracellular signals and intracellular targets.
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PMID:Epidermal-growth-factor-induced production of phosphatidylalcohols by HeLa cells and A431 cells through activation of phospholipase D. 141 90

Lysophosphatidic acid (LPA) is a simple phospholipid that possesses hormone- and growth-factor-like properties. LPA initiates its action by inducing GTP-dependent phosphoinositide hydrolysis and inhibiting adenylate cyclase [van Corven, Groenink, Jalink, Eichholtz & Moolenaar (1989) Cell 59, 45-54]. Here we show that LPA stimulates rapid breakdown of phosphatidylcholine (PC) in Rat-1 fibroblasts. LPA-induced PC breakdown occurs through activation of phospholipase D (PLD), as measured by the formation of free choline and phosphatidic acid and by transphosphatidylation in the presence of butan-1-ol. LPA also stimulates generation of diacylglycerol, but there is no detectable formation of phosphocholine, suggesting that a PC-specific phospholipase C (PLC) is not involved. The response to LPA was compared with that to endothelin, a potent inducer of phospholipid hydrolysis but a poor mitogen for Rat-1 cells. Our results indicate that: (1) LPA is less efficient than endothelin in inducing phosphoinositide and PC breakdown; (2) LPA-induced PLD activation is short-lived, levelling off after 2 min, whereas the endothelin-stimulated increase in PLD activity persists for at least 1 h; (3) the effect of LPA on PLD, like that of endothelin, is blocked by long-term pretreatment of the cells with phorbol ester, suggesting that PLD activation occurs through a protein kinase C-dependent mechanism. Furthermore, our results support the notion that there is no simple causal relationship between the degree of agonist-induced phospholipid hydrolysis and the magnitude of the mitogenic response.
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PMID:The biologically active phospholipid, lysophosphatidic acid, induces phosphatidylcholine breakdown in fibroblasts via activation of phospholipase D. Comparison with the response to endothelin. 163 5

Addition of epidermal growth factor (EGF) to quiescent Swiss 3T3 cells resulted in a sustained increase in cellular diacylglycerol (DG) content in the absence of inositol-lipid hydrolysis. In the presence of non-cytotoxic concentrations of butan-1-ol, EGF stimulated the formation of phosphatidylbutanol, indicating that the EGF receptor was able to couple to the activation of phospholipase D (PLD). EGF-stimulated release of choline from Swiss 3T3 cells suggested that the major substrate for this PLD was phosphatidylcholine. Unlike bombesin-stimulated PLD activity, the response to EGF was not inhibited by a selective protein kinase C (PKC) inhibitor (Ro-31-8220), suggesting that it was not dependent on PKC activation. Pre-treatment of Swiss 3T3 cells with the EGF-receptor tyrosine kinase inhibitor AG18 selectively inhibited EGF-stimulated PLD activity; bombesin-stimulated PLD activity was unaffected. Butan-1-ol inhibited phorbol ester- and bombesin-stimulated DG formation suggesting a role for a coupled PLD/phosphatidate phosphohydrolase pathway; in contrast, EGF-stimulated DG formation was unaffected.
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PMID:Epidermal growth factor increases sn-1,2-diacylglycerol levels and activates phospholipase D-catalysed phosphatidylcholine breakdown in Swiss 3T3 cells in the absence of inositol-lipid hydrolysis. 163 7

Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) to quiescent Swiss 3T3 cells resulted in a sustained increase in sn-1,2-diradylglycerol (DG) mass and [3H]DG in [3H]palmitate-labelled cells where phosphatidylcholine was the major labelled phospholipid. This occurred in the absence of inositol phosphate accumulation. In [3H]palmitate-labelled cells both bombesin and PMA stimulated the formation of phosphatidylbutanol ([3H]PtdBut) in the presence of 0.3% (v/v) butan-1-ol. The kinetics of [3H]PtdBut formation were consistent with phospholipase D (PLD) activation preceding sustained DG formation. The inclusion of butan-1-ol inhibited 70% of PMA-stimulated DG formation but only 30% of the bombesin response. The ability of bombesin and PMA to stimulate the accumulation of [3H]PtdBut was completely abolished in Swiss 3T3 cells which had been pre-treated with 400 nM-PMA for 48 h to down-regulate protein kinase C activity. PMA-stimulated [3H]PtdBut formation was inhibited by 90% by the protein kinase C inhibitor Ro-31-8220 (10 microM), but bombesin-stimulated PtdBut accumulation was inhibited by at most 50% by the same concentration of inhibitor. Cyclic AMP-elevating agents, i.e. forskolin, dibutyryl cyclic AMP and isobutylmethylxanthine, did not inhibit bombesin stimulation of PLD activity. Bombesin-stimulated PLD activity was inhibited by 50% by buffering of the extracellular Ca2+ concentration to 150 nM, but combination of this treatment with Ro-31-8220 addition was less than additive. Ionophore A23187 alone was able to stimulate PLD activity, but this response was inhibited 50% by Ro-31-8220. Thapsigargin was unable to stimulate PLD activity and had no modulatory effect upon bombesin-stimulated PLD activity at any agonist concentration. The results are discussed in terms of the role of PLD in DG generation and the regulation of PLD activity both by bombesin and by PMA.
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PMID:The regulation of phospholipase D activity and its role in sn-1,2-diradylglycerol formation in bombesin- and phorbol 12-myristate 13-acetate-stimulated Swiss 3T3 cells. 174 19

Oxidized LDL (oxLDL) has been identified as a potent stimulus of leukocyte adhesion to endothelium, a hallmark of early atherogenesis. A cytofluorometric study was performed to further characterize the mechanisms by which oxLDL stimulates the rapid adhesion of leukocytes to endothelium in vitro and in vivo. Incubation (30 minutes at 37 C) of whole blood (diluted with buffered saline to 1 x 10(6) leukocytes/ml) with oxLDL (0.85 mg LDL cholesterol/ml; oxidized by 7.5 mumol/L Cu2+ for 18 hours) but not native LDL stimulated the upregulation of CD11b/CD18 adhesion receptors on neutrophils (anti-leu-15 binding: 178 +/- 16% of baseline, P < 0.01, means +/- SD of n = 10 experiments) and on monocytes (169 +/- 34% of baseline, P < 0.01). This phenomenon was almost entirely inhibited by n-butanol or the vasoactive drug pentoxifylline (PTX), which also significantly reduced oxLDL-induced leukocyte adhesion to venular and arteriolar endothelium, as assessed by intravital microscopy on the dorsal skinfold chamber in hamsters (venules: 49 +/- 19 versus 120 +/- 34 cells/mm2, P < 0.05; arterioles: 9 +/- 4 versus 52 +/- 7 cells/mm2, P < 0.01) 30 minutes after intravenous injection of oxLDL (4 mg/kg body weight; means +/- SD of n = 7 hamsters per group). Butanol and PTX also significantly reduced the upregulation of CD11b/CD18 by f-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) but not by phorbol myristate acetate (PMA). Whereas fMLP and PAF stimulate leukocytes via binding to specific cell surface receptors and triggering complex signal transduction pathways, PMA bypasses these pathways and directly activates intracellular protein kinase C. By analogy, we propose that oxLDL upregulates CD11b/CD18 through its previously documented ability to stimulate the generation of second messengers. The effect of n-butanol and PTX on receptor presentation cannot be explained by changes in plasma membrane fluidity, as both agents failed to reverse the decrease in plasma membrane fluidity of neutrophils after stimulation with oxLDL, as assessed by fluorescence anisotropy measurement of the membrane marker diphenylhexatriene. Incubation of isolated neutrophils but not of whole blood with oxLDL resulted in a significant loss of L-selectin from the neutrophil surface (anti-TQ-1 binding: 40 +/- 13% of baseline, P < 0.01). A significant loss of this adhesion receptor on neutrophils and monocytes was also observed after stimulation of isolated neutrophils and whole blood with fMLP, PAF, and PMA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:In vitro effects of oxidized low density lipoprotein on CD11b/CD18 and L-selectin presentation on neutrophils and monocytes with relevance for the in vivo situation. 753 48

The early signalling events that may ultimately contribute to the assembly and subsequent activation of the NADPH oxidase in guinea-pig peritoneal eosinophils were investigated in response to leukotriene B4 (LTB4). LTB4 promoted a rapid, transient and receptor-mediated increase in the rate of H2O2 generation that was potentiated by R 59 022, a diradylglycerol (DRG) kinase inhibitor, implicating protein kinase C (PKC) in the genesis of this response. This conclusion was supported by the finding that the PKC inhibitor, Ro 31-8220, attenuated (by about 30%) the peak rate of LTB4-induced H2O2 generation under conditions where the same response evoked by 4 beta-phorbol 12,13-dibutyrate (PDBu) was inhibited by more than 90%. Paradoxically, Ro 31-8220 doubled the amount of H2O2 produced by LTB4 which may relate to the ability of PKC to inhibit cell signalling through phospholipase C (PLC). Indeed, Ro 31-8220 significantly enhanced LTB4-induced Ins(1,4,5)P3 accumulation and the duration of the Ca2+ transient in eosinophils. Experiments designed to assess the relative importance of DRG-mobilizing phospholipases in LTB4-induced oxidase activation indicated that phospholipase D (PLD) did not play a major role. Thus, although H2O2 generation was abolished by butan-1-ol, this was apparently unrelated to the inhibition of PLD, as LTB4 failed to stimulate the formation of Ptd[3H]BuOH in [3H]butan-1-ol-treated eosinophils. Rather, the inhibition was probably due to the ability of butan-1-ol to increase the eosinophil cyclic AMP content. In contrast, Ca(2+)- and PLC-driven mechanisms were implicated in H2O2 generation, as LTB4 elevated the Ins(1,4,5)P3 content and intracellular free Ca2+ concentration in intact cells, and cochelation of extracellular and intracellular Ca2+ significantly attenuated LTB4-induced H2O2 generation. Pretreatment of eosinophils with wortmannin did not affect LTB4-induced H2O2 production at concentrations at which it abolished the respiratory burst evoked by formylmethionyl-leucylphenylalanine in human neutrophils. Collectively, these data suggest that LTB4 activates the NADPH oxidase in eosinophils by PLD- and PtdIns 3-kinase-independent mechanisms that involve Ca2+, PLC and PKC. Furthermore, the activation of additional pathways that do not require Ca2+ is also suggested by the finding that LTB4 evoked a significant respiratory burst in Ca(2+)-depleted cells.
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PMID:Early signalling events implicated in leukotriene B4-induced activation of the NADPH oxidase in eosinophils: role of Ca2+, protein kinase C and phospholipases C and D. 757 12

Stimulation of protein kinase C (PKC) activity is achieved in vivo by diacylglycerol but can also be obtained with tumor-promoting phorbol esters. Evidence is presented indicating that these two classes of activator may interact at different regions of the enzyme. The activity of a calcium-dependent PKC isoform (PKC-I) preparation was determined using 1,2-dioleoylglycerol (DOG) together with the phorbol ester 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA). The resulting PKC activity was in excess of that attained with either activator alone, each being at a maximum concentration for activation. A similar result was obtained with purified PKC-alpha and -epsilon isoforms, indicating that the additive effect was not due to sites being on distinct enzyme molecules. Support for two dissimilar activator sites came from the observation that the inactive phorbol ester 4 alpha-TPA competed for TPA but not for DOG in PKC activation. Other differences were observed between TPA- and DOG-activated PKC. It was found that 1-butanol inhibited DOG-activated PKC-I, while being without effect on stimulation by TPA. Also, the inclusion of phosphatidylethanolamine in the lipid vesicles led to a potentiation of PKC-I activity which was greater when activation was achieved by DOG compared to TPA. Further, the calcium- and DOG-dependent active conformational change of PKC was fully reversible upon calcium chelation, while that stimulated by TPA was only partially reversible. These experiments taken together suggest that diacylglycerols and phorbol esters bind with different affinities and at different sites on PKC, and induce distinct activated conformational forms of the enzyme.
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PMID:Evidence for discrete diacylglycerol and phorbol ester activator sites on protein kinase C. Differences in effects of 1-alkanol inhibition, activation by phosphatidylethanolamine and calcium chelation. 800 23

In the absence of inositol-lipid hydrolysis, mitogenic concentrations of basic fibroblast growth factor (bFGF) stimulated phosphatidylbutanol formation in the presence of butan-1-ol in [3H]myristate-labeled human umbilical vascular endothelial (HUVE) cells, indicating that the fibroblast growth factor receptor was able to couple to the activation of phospholipase D (PLD). The ability of bFGF and 12-O-tetradecanoylphorbol-13-acetate (TPA) to stimulate PLD activity was completely abolished in cells pretreated with 400 nM TPA for 48 h to downregulate protein kinase C (PKC). bFGF-stimulated PLD activity was inhibited by genistein (5 microM; P < 0.02) and the PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 5 microM; P < 0.001) as well as by the removal of calcium from extracellular environment. bFGF induced DNA synthesis in a dose-dependent manner, and pretreatment of cells with H-7 inhibited the mitogenic activity of bFGF. These results indicate that activation of PKC is responsible for bFGF-induced PLD activation and the mitogenic activity of bFGF in HUVE cells. A coupled PLD/3-sn-phosphatidate phosphohydrolase pathway may play a role in the regulation of endothelial cell proliferation.
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PMID:Basic FGF activates phospholipase D in endothelial cells in the absence of inositol-lipid hydrolysis. 830 17

Conditions were established for the primary culture of guinea-pig tracheal smooth muscle cells, the identity of which was confirmed by the presence of smooth muscle alpha-actin by western blotting. Cells were preincubated with [3H]palmitate which was incorporated, almost exclusively, into phosphatidylcholine. When these cells were stimulated by either bradykinin or phorbol 12-myristate 13-acetate (PMA), in the presence of butan-1-ol, the non-metabolizable product [3H]phosphatidylbutanol ([3H]PtdBut) accumulated by virtue of the phosphatidyltransferase activity of phospholipase D. The activation of phospholipase D by bradykinin was inhibited by 86 +/- 11% (N = 3 experiments) in the presence of the protein kinase C inhibitor, staurosporine (1 microM) and by 88 +/- 11% (N = 3 experiments) in cells that had been chronically treated with PMA to down-regulate their protein kinase C. PMA-stimulated phospholipase D was similarly affected (92 +/- 2% inhibited by staurosporine, 87 +/- 6% inhibited by protein kinase C down-regulation). Removal of extracellular Ca2+ markedly reduced the bradykinin-stimulated phospholipase D response (by 73 +/- 10%, N = 3 experiments) but had only a limited effect upon PMA-stimulated phospholipase D activity (by 23 +/- 6%, N = 3 experiments). [AIF4](-)-stimulation of the cells also resulted in the activation of phospholipase D, indicating the involvement of a G-protein. However, this was not Gi since pertussis-toxin pretreatment of the cells failed to abolish either bradykinin-stimulated inositol (1,4,5)trisphosphate formation or [3H]PtdBut accumulation. Western blotting revealed the presence of Gq/G11 which couples to the inositol lipid-directed phospholipase C. Indomethacin (10 microM) was without effect upon bradykinin-stimulated phospholipase D activity, suggesting that the bradykinin effects were not mediated indirectly by cyclooxygenase products. The role of phospholipase D activation in tracheal smooth muscle may be to, indirectly, produce diacylglycerol for the activation of protein kinase C which has been implicated in sustained contraction. However, the immediate product of phospholipase D, phosphatidate, has been proposed to have a number of second messenger roles and may itself, by an undefined mechanism, be involved in the sustained contraction of airway smooth muscle.
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PMID:Bradykinin stimulates phospholipase D in primary cultures of guinea-pig tracheal smooth muscle. 844 59

Hepatocyte growth factor (HGF) activated phospholipase D (PLD) in primary-cultured rat hepatocytes, as assessed by the formation of phosphatidylbutanol (PBut), a specific and stable product of PLD activity in the presence of 0.3% butanol. PLD hydrolyzes phosphatidylcholine to choline and phosphatidic acid (PA), which is further metabolized to diacylglycerol (DG) by PA phosphohydrolase (PAP). In HGF-stimulated hepatocytes, butanol prevented the formation of PA and DG. A PAP inhibitor, propranolol, inhibited DG production with a reciprocal increase of PA, implying that PLD played a role in the formation of not only PA but DG. Inhibitors for protein kinase C (PKC), Ro31-8425, H-7, and calphostin C, reduced HGF-induced PLD activation. A protein tyrosine kinase (PTK) inhibitor, genistein but not its inactive analogue daidzein, inhibited PLD activation by HGF. Moreover, depletion of extracellular Ca2+ by omission of Ca2+ or by chelating residual Ca2+ with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) abolished HGF-induced PLD activation. HGF, phorbol myristate acetate (PMA) and a DG analog, oleylacetylglycerol (OAG), activated the expression of c-jun and c-fos messenger RNAs (mRNAs). Ro31-8425, calphostin C, and genistein, which prevented HGF-induced PLD activation, inhibited induction of these immediate early genes. Butanol and propranolol at concentrations which effectively inhibited the formation of DG, suppressed HGF-induced expression of c-jun and c-fos mRNAs. However, HGF-induced mitogen-activated protein kinase (MAPK) activation was not affected by both butanol and propranolol. These results suggest that PTK, PKC, and Ca2+ regulate HGF-induced PLD activation, and that DG produced by PLD pathway may play a role in the induction of immediate early genes, which is activated in MAPK-independent manner, in rat hepatocytes.
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PMID:Phospholipase D activation in hepatocyte growth factor-stimulated rat hepatocytes mediates the expressions of c-jun and c-fos: involvement of protein tyrosine kinase, protein kinase C, and Ca2+. 890 10


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