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)

Cytosolic phospholipase A2 (cPLA2) selectively catalyses the release of arachidonic acid from the sn-2 position of glycero-phospholipids to produce prostaglandins and leukotrienes. In this study, vitamin E enrichment of rat heart myoblastic H9c2 cells caused an increase in the release of arachidonate during ionophore (A23187) stimulation. PLA2 activity in the cytosolic fraction was also enhanced but enzyme activity in the particulate fraction was not affected by this treatment. Immunoblotting analysis with a polyclonal anti-cPLA2 antibody showed an increased level of the enzyme in vitamin E-treated cells. Direct incorporation of vitamin E into lipid vesicles in the assay mixture resulted in modulation of enzyme activity in a biphasic manner. Pretreatment of cells with phorbol 12-myristate 13-acetate, a known activator of protein kinase C, synergistically potentiated the ionophore-induced arachidonate release in both the control and vitamin E-treated cells. However, vitamin E treatment by itself did not affect the protein kinase C activity, indicating that the vitamin E-induced activation of cPLA2 was independent of the protein kinase C cascade. Collectively, these results suggest that vitamin E potentiates arachidonate release through the direct and/or indirect modulation of cPLA2 activity.
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PMID:Vitamin E potentiates arachidonate release and phospholipase A2 activity in rat heart myoblastic cells. 891 71

Low concentrations of angiotensin II (Ang II) increase, whereas high concentrations inhibit the apical Na/H antiporter activity in the proximal tubule, but the respective roles of the different signaling pathways in mediating these effects remains unsettled. We studied the effects of both low and high doses of Ang II in the presence of selective signaling pathway inhibitors, on the apical Na/H antiport activity of rat proximal tubule. Experiments were carried out in intact cells of freshly prepared tubule fragments obtained from the outer third of cortex, that is, devoid of basolateral Na/H antiport activity in the absence of bicarbonate transport and H(+)-ATPase activity. In tubules acid-loaded by an NH4Cl prepulse, Na/H antiport activity was assessed by the initial rate of intracellular pH recovery (dpHi/dt), measured with BCECF. When tubules were preincubated with low dose Ang II (10(-11) M for 3 min), dpHi/dt increased by 25 +/- 8%, whereas incubation with high dose Ang II (10(-7) M for 3 min) decreased dpHi/dt by 30 +/- 4%, compared to control (P < 0.01 in both cases). Both effects were abolished in the presence of 2.10(-3) M amiloride. Low dose Ang II-induced increase in dpHi/dt was not affected by preincubation with a specific PKA inhibitor, Rp-CPT-cAMP 10(-4) M, and was completely abolished by preincubation with PKC inhibitors, staurosporine 10(-7) M, sphingosine 5.10(-6) M, or calphostin 10(-6) M. In addition, pretreatment of rats with pertussis toxin led to a partial inhibition of the effect of low dose Ang II. The high dose-Ang II-induced decrease in dpHi/dt was not affected by pretreatment with a calcium-calmodulin kinase inhibitor W-7 10(-4) M. Conversely, pretreatment with the cytochrome P-450 inhibitor econazole 10(-5) M reversed the inhibitory effect of high dose Ang II to a stimulatory effect (24 +/- 8%, P < 0.01), quantitatively similar to the effect of low dose Ang II. In addition, arachidonate was found to exert an econazole-sensitive dose-dependent inhibitory effect on dpHi/dt, and 5,6-EET 10(-6) M, a cytochrome P-450 derived-arachidonic acid metabolite, induced a 38 +/- 9% inhibition, similar to that observed with high dose Ang II alone. There was no additive effect of 5,6-EET and high dose Ang II. Finally, pretreatment with two PLA2 inhibitors (BromoPhenacylBromide, 6.10(-6) M, and oleyloxyethyl phosphorylcholine, 5.10(-6) M) reversed the inhibitory effect of high dose Ang II to a stimulatory effect (32 +/- 11% and 25 +/- 11%, respectively, P < 0.05 for both inhibitors). We conclude that, in intact rat proximal cells, low dose Ang II stimulates the apical Na/H antiport through a pertussis toxin-sensitive G protein-dependent PKC pathway, whereas high dose Ang II inhibits the Na/H antiport activity through the PLA2- and cytochrome P-450-dependent metabolites of arachidonate.
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PMID:Signaling pathways in the biphasic effect of angiotensin II on apical Na/H antiport activity in proximal tubule. 891 15

Numerous studies have identified phospholipase metabolites as membrane fusogens, and phospholipase D (PLD) (J.R. Coorssen and R.J. Haslam. FEBS Lett. 316: 170-174, 1993), C (PLC), and A2 (PLA2) activities correlate with secretion. Do these enzymes have essential or modulatory roles? This study confirms that secretion does not require Ca2+ or PLC (Coorssen et al. Cell Regul. 1: 1027-1041, 1990). Arachidonic acid (AA), phosphatidic acid (PA) and analogues, exogenous metabolites of PLA2 and PLD, were tested in electropermeabilized human platelets. AA potentiated guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-induced secretion, and eicosanoids were not essential. Endogenous [3H]AA formation correlated with GTP gamma S-induced secretion, and phorbol 12-myristate 13-acetate (PMA) promoted these effects. Inhibitors were used to probe phospholipase influences on secretion. Only PLD inhibitors blocked secretion. However, PMA blocked inhibition of protein kinase C (PKC) and secretion by quercetin, suggesting that PA formed by PLD supports PKC activation and GTP gamma S-induced secretion. Thus PA analogues had no effect alone but enhanced GTP gamma S-induced PKC activity and secretion. Slower PLD activation compared with secretion also indicates a nonessential role. This is the first report of a Ca(2+)-independent PLA2 activity in human platelets, use of quercetin as a PLD inhibitor, and dissociation of PLA2, PLC, and PLD activities from secretion. No major phospholipase activities are essential to the final steps in exocytosis, but modulatory roles are indicated.
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PMID:Phospholipase activation and secretion: evidence that PLA2, PLC, and PLD are not essential to exocytosis. 892 43

The present study investigated the role of kinases and G-proteins in arachidonic acid (AA) mobilization by resident mouse peritoneal macrophages in response to phagocytosis of opsonized zymosan. Stimulation of resident murine peritoneal macrophages with opsonized zymosan caused an increase in [3H] arachidonic acid release. This increase was dose-dependent and was not accompanied by de novo synthesis of proteins. Neither staurosporine, a protein kinase C inhibitor, nor genistein, a tyrosine kinase inhibitor, had any effect on [3H] AA mobilization, although trifluoperazine significantly inhibited AA release. The involvement of G proteins and phospholipase C (PLC) in the regulation of arachidonic acid release induced by opsonized zymosan was also examined in mouse peritoneal macrophages. Prior treatment of cells with pertussis toxin induced a partial decrease in AA mobilization. However, neomycin or aspirin, at doses that inhibit inositol phosphate formation (PLC activity), did not [3H] AA mobilization by PLA2. We proposed that the AA release by peritoneal macrophages in response to opsonized zymosan phagocytosis could be due to the participation of enzymes other than PLC and PKC, or proteins other than G proteins.
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PMID:Role of kinases and G-proteins on arachidonate release induced by zymosan in mouse peritoneal macrophages. 902 57

Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)-mediated AA release by P2U-purinergic receptors in MDCK-D1 cells. Treatment of cells with the P2U receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anti-cPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P2U receptors in MDCK-D1 cells. P2U receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13-acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P2U receptors. Differential effects of GF109203X on cPLA2-mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKCalpha, PKCalpha may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKCalpha expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P2U receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKCalpha regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby alpha1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.
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PMID:Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and -independent pathways. 904 86

Annexin V belongs to a family of proteins that interact with phospholipids in a Ca2+-dependent manner. This protein has been demonstrated to have anti-phospholipase A2 activity. However, this effect has never yet been reported with the 85-kDa cytosolic PLA2 (cPLA2). We studied, in a model of differentiated and streptolysin O-permeabilized HL-60 cells, the effect of annexin V on cPLA2 activity after stimulation by calcium, GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), formyl-Met-Leu-Phe, or phorbol 12-myristate 13-acetate. Both recombinant and human placental purified annexin V inhibit cPLA2 activity whatever the stimulus used. The decrease of arachidonic acid release is of 40 and 50%, respectively, at [Ca2+] of 3 and 10 microM. The mechanism of inhibition was also analyzed. cPLA2 requires calcium and protein kinase C (PKC) or mitogen-activated protein kinase phosphorylation for its activation. As annexin V was shown to be an endogenous inhibitor of PKC, PKC-stimulated cPLA2 activity was analyzed. Using GF109203x, a specific PKC inhibitor, we demonstrated that this pathway is of minor importance in our model. cPLA2 inhibition by annexin V is not linked to PKC inhibition. To test the hypothesis of phospholipid depletion, mutants of annexin V were constructed using mutagenesis directed to Ca2+ site. We demonstrate that the Ca2+ site located in domain I is necessary for the inhibitory effect of annexin V on cPLA2 activity. The site in domain IV is also involved but with less efficiency. In contrast, mutations in site II and III do not modify this effect. Moreover, annexin V mutated on all sites does not inhibit cPLA2. Thus, we propose a predominant role of module (I/IV) in the biological action of annexin V, which, in physiological conditions, may control cPLA2 activity by depletion of the phospholipid substrate.
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PMID:Inhibition of cytosolic phospholipase A2 by annexin V in differentiated permeabilized HL-60 cells. Evidence of crucial importance of domain I type II Ca2+-binding site in the mechanism of inhibition. 909 90

The effects of phorbol myristate acetate, an activator of protein kinase C, on the release of [3H]arachidonic acid and prostaglandin synthesis were studied in an osteoblast cell line (MC3T3-E1). Phorbol myristate acetate (20 uM) liberated 16 and 55% of the [3H]arachidonate in prelabeled phosphatidylinositol and phosphatidylethanolamine, respectively; and evoked a 19-fold stimulation in the synthesis of prostaglandin E2. Phorbol myristate acetate doubled the cellular mass of 1,2-diacylglycerol and stimulated the liberation of [3H]arachidonate from the diacylglycerol pool in prelabeled cells. The diacylglycerol lipase inhibitor RHC 80267 blocked 75-80% of the phorbol ester-promoted (total) cellular liberation of [3H]arachidonic acid and production of prostaglandin E2. In comparison, the release of [3H]arachidonate from phosphatidylethanolamine (but not phosphatidylinositol) was only partially antagonized (to the same degree) by the PLA2 inhibitor p-bromophenacylbromide and the protein kinase C inhibitor Et-18-OMe, PMA-induced formation of diacylglycerol or synthesis of PGE2 was not affected by the prior inhibition of protein kinase C. Therefore, we have shown a novel pathway for the liberation of arachidonic acid in osteoblasts involving the nonspecific hydrolysis of phosphatidylinositol and phosphatidylethanolamine by phospholipase C followed by the deesterification of diacylglycerol. This pathway can be activated by a phorbol ester through a protein kinase C-independent mechanism.
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PMID:Protein kinase C-independent activation of a novel nonspecific phospholipase C pathway by phorbol myristate acetate releases arachidonic acid for prostaglandin synthesis in MC3T3-E1 osteoblasts. 913 31

Cytosolic phospholipase A2 (cPLA2) is a signal-responsive enzyme that is highly selective to the nature of phospholipid substrates. A mechanism for cPLA2 activity regulation through a signal transduction pathway has been proposed and this signaling appears to be influenced by oxidants. Oxidant-mediated signaling of PLA2 may serve as an alternative mechanism for enzyme regulation; however, the manner of regulation has yet to be delineated. In this report we demonstrate that there is a direct effect of membrane oxidation on cPLA2 phosphorylation and activity. A simple in vitro system consisting of purified cPLA2 and phospholipid vesicles was used to facilitate protein kinase C (PKC) activity and provide substrates for cPLA2. Using these vesicles we found that the activity of cPLA2 was enhanced twofold when the vesicles contained as little as 5 mol% phosphatidylcholine hydroperoxides (PLPCOOH). The order of hydrolytic preference for fatty acyl species was 20:4 > 18:2 > 18:1 > 16:0, and the presence of PLPCOOH stimulated hydrolysis largely of phosphatidylcholine containing 20:4. The Ca2+ concentrations required for stimulated hydrolytic activity were also twofold lower for oxidized compared to unoxidized vesicles. Using phospholipid micelles as substrates, PKC-mediated phosphorylation of cPLA2 increased hydrolytic activity 71% compared to preparations lacking PKC. Using phospholipid vesicles as substrates, PKC-mediated phosphorylation resulted in an 85% increase in cPLA2 activity compared to preparations without PKC. PKC-mediated phosphorylation of cPLA2, therefore, stimulates catalytic activity toward membrane phospholipids and the extent of activation is enhanced directly by peroxidation of membrane phospholipids and involves a peroxide-induced stimulation of cPLA2 phosphorylation.
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PMID:Phospholipid peroxidation induces cytosolic phospholipase A2 activity: membrane effects versus enzyme phosphorylation. 921 Jun 45

Signal transduction in gastric and intestinal smooth muscle is mediated by receptors coupled via distinct G proteins to various effector enzymes, including PI-specific PLC-beta 1 and PLC-beta 3, and phosphatidylcholine (PC)-specific PLC, PLD and PLA2. Activation of these enzymes is different in circular and longitudinal muscle cells, generating Ca(2+)-mobilizing (IP3, AA, cADPR) and other (DAG) messengers responsible for the initial and sustained phases of contraction, respectively. IP3-dependent Ca2+ release occurs only in circular muscle. Ca2+ mobilization in longitudinal muscle involves a cascade initiated by agonist-induced transient activation of PLA2 and formation of AA, AA-dependent depolarization of the plasma membrane and opening of voltage-sensitive Ca2+ channels. The influx of Ca2+ induces Ca2+ release by activating sarcoplasmic ryanodine receptor/Ca2+ channel and stimulates cADPR formation which enhances Ca(2+)-induced Ca2+ release. The initial [Ca2+]i transient in both muscle cell types results in Ca2+/calmodulin-dependent activation of MLC kinase, phosphorylation of MLC20 and interaction of actin and myosin. The sustained phase is mediated by a Ca(2+)-independent isoform of PKC, PKC-epsilon DAG for this process is generated by PLC- and PLD-mediated hydrolysis of PC. Relaxation is mediated by cAMP-and/or cGMP-dependent protein kinase which inhibit the initial [Ca2+]i transient and reduce the sensitivity of MLC kinase to [Ca2+]i. Relaxation induced by the main neurotransmitters, VIP and PACAP, involves two cascades, one of which reflects activation of adenylyl cyclase. A distinct cascade involves G-protein-dependent stimulation of Ca2+ influx leading to Ca2+/calmodulin-dependent activation of a constitutive eNOS in muscle cells; the generation of NO activates soluble guanylyl cyclase. The resultant activation of PKA and PKG is jointly responsible for muscle relaxation.
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PMID:Signal transduction in gastrointestinal smooth muscle. 921 27

Hexadecylphosphocholine (HePC) is the main representative of a new group of antineoplastic agents, the alkylphosphocholines, which were originally derived from cytotoxic etherlysophospholipids. HePC shows antiproliferative action against a whole variety of tumor cells and tumors in vitro and in vivo. Furthermore, it also induces differentiation in some hematologic cell lines and prevents invasive growth of neoplastic cells in vitro. To date, the precise molecular mechanisms mediating the biological effects of HePC have not been identified yet. As etherlysophospholipids seem to inhibit some pathways of lipid-dependent intracellular signalling, similar effects may be relevant for HePC. We therefore investigated the influence of HePC on phospholipase A2 (PLA2-EC 3.1.1) in the human leukemia cell line U 937. HePC seems to inhibit enzyme activity independently of protein kinase C (PKC) in differentiated U 937 cells stimulated by tumor necrosis factor alpha (TNFalpha). Inhibition of purified secretory PLA2 from snake venom (EC 3.1.1.4) in vitro shows characteristics of a non-competitive mode. In contrast, HePC leads to an enhancement of PLA2 activity in immature cells which cannot be explained by changes in membrane composition. Our data suggest that PLA, inhibition is most probably not the mechanism by which HePC mediates its antiproliferative effects.
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PMID:Differential regulation of phospholipase A2 in human leukemia cells by the etherphospholipid analogue hexadecylphosphocholine. 926 26


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