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 rat basophilic leukemic (RBL-2H3) cell line was stably transfected with the endogenously expressed Ca2+-dependent protein kinase C-alpha (PKC-alpha) and -betaI and the Ca2+-independent delta and epsilon isoforms to study their functional roles. In addition, the Ca2+-independent PKC-eta was expressed. All transfected PKC isoforms translocated to the membrane-containing fraction in response to aggregation of the IgE-sensitized high affinity receptor for IgE (Fc epsilonRI) with the Ag dinitrophenyl(25)-BSA. All PKC transfectants, except PKC-eta, showed increased proliferative responses, and aggregation of Fc epsilonRI further enhanced the rate of proliferation. The PKC transfectants also showed increased phosphoinositide hydrolysis in response to Ag aggregation of receptors. No marked differences in the Ca2+ responses of the transfectants to Ag or thapsigargin were observed. Overexpression of PKC-alpha or -epsilon specifically inhibited receptor-dependent cytosolic phospholipase A2 (cPLA2) activity, whereas this activity was enhanced in the PKC-betaI transfectant. Analysis of the secretory response revealed that overexpression of PKC-betaI and -eta significantly enhanced secretion. A broad spectrum of cytokine mRNAs was detected in all transfectants, and overexpression of PKC-betaI significantly enhanced the receptor-dependent production of IL-2 and IL-6 mRNA. These studies identify PKC-alpha and -epsilon as negative regulators of cPLA2 activity and demonstrate the importance of PKC-beta as a positive modulator of secretion, cPLA2 activity, and cytokine production in this mast cell line.
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PMID:Functional effects of overexpression of protein kinase C-alpha, -beta, -delta, -epsilon, and -eta in the mast cell line RBL-2H3. 930 Jun 81

The role of cytosolic phospholipase A2 (cPLA2) and its mode of activation by opsonized zymosan (OZ) was studied in human neutrophils in comparison with activation by PMA. The activation of cPLA2 by 1 mg/ml OZ or 50 ng/ml PMA is evidenced by its translocation to the membrane fractions on stimulation. This translocation is consistent with dithiothreitol (DTT)-resistant phospholipase A2 (PLA2) activity detected in the membranes of activated cells. Neutrophils stimulated by either OZ or PMA exhibited an immediate stimulation of extracellular-signal-regulated kinases (ERKs). The inhibition of ERKs, DTT-resistant PLA2 and NADPH oxidase activities by the MAP kinase kinase inhibitor PD-98059 indicates that ERKs mediate the activation of cPLA2 and NADPH oxidase stimulated by either OZ or PMA. The protein kinase C (PKC) inhibitor GF-109203X inhibited epidermal growth factor receptor peptide kinase activity, the release of [3H]arachidonic acid, DTT-resistant PLA2 activity and superoxide generation induced by PMA, but did not inhibit any of these activities induced by OZ. PKC activity was similarly inhibited by GF-109203X in membrane fractions separated from neutrophils stimulated by either PMA or OZ. In the presence of the tyrosine kinase inhibit orgenistein, ERKs, PLA2 and NADPH oxidase activities were inhibited in cells stimulated by OZ, whereas they were hardly affected in cells stimulated by PMA. The results suggest that the activation of cPLA2 by PMA or OZ is mediated by ERKs. Whereas PMA stimulates ERKs activity through a PKC-dependent pathway, signal transduction stimulated by OZ involves tyrosine kinase activity leading to activation of ERKs via a PKC-independent pathway.
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PMID:Cytosolic phospholipase A2 and its mode of activation in human neutrophils by opsonized zymosan. Correlation between 42/44 kDa mitogen-activated protein kinase, cytosolic phospholipase A2 and NADPH oxidase. 930 39

The C2 domain is a Ca2+-dependent, membrane-targeting motif originally discovered in protein kinase C and recently identified in numerous eukaryotic signal-transducing proteins, including cytosolic phospholipase A2 (cPLA2) of the vertebrate inflammation pathway. Intracellular Ca2+ signals recruit the C2 domain of cPLA2 to cellular membranes where the enzymatic domain hydrolyzes specific lipids to release arachidonic acid, thereby initiating the inflammatory response. Equilibrium binding and stopped-flow kinetic experiments reveal that the C2 domain of human cPLA2 binds two Ca2+ ions with positive cooperativity, yielding a conformational change and membrane docking. When Ca2+ is removed, the two Ca2+ ions dissociate rapidly and virtually simultaneously from the isolated domain in solution. In contrast, the Ca2+-binding sites become occluded in the membrane-bound complex such that Ca2+ binding and dissociation are slowed. Dissociation of the two Ca2+ ions from the membrane-bound domain is an ordered sequential process, and release of the domain from the membrane is simultaneous with dissociation of the second ion. Thus, the Ca2+-signaling cycle of the C2 domain passes through an active, membrane-bound state possessing two occluded Ca2+ ions, one of which is essential for maintenance of the protein-membrane complex.
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PMID:Ca2+-signaling cycle of a membrane-docking C2 domain. 934 10

Incubation of rat glomerular mesangial cells with potent proinflammatory cytokines like interleukin 1beta, (IL- 1beta) triggers the expression of a non-pancreatic secretory phospholipase A2 (sPLA2) and increases the formation of prostaglandin E2. We show here that sPLA2 acts in an autocrine fashion on mesangial cells and induces a rapid activation of protein kinase C (PKC) isoenzymes delta and epsilon and of p42 mitogen-activated protein kinase (MAPK), two putative activators of cytosolic phospholipase A2 (cPLA2). sPLA2 also activates Raf-1 kinase in mesangial cells which integrates the signals coming from PKC for further processing along the MAPK cascade. Subsequently a phosphorylation and activation of cPLA2 is observed, thus arguing for a cross-talk between the two classes of PLA2. Pretreatment of cells with either the highly specific PKC inhibitor Ro-318220 or the highly specific MAPK kinase (MEK) inhibitor PD 98059 completely blocked the sPLA2-induced cPLA2 activation, indicating that both kinases are essential for the cross-talk between the two types of PLA2. The effect of sPLA2 is mimicked by lysophosphatidylcholine (LPC), a reaction product of sPLA2 activity. LPC stimulates PKC-epsilon, Raf-1 kinase and MAPK activation as well as cPLA2 activation with a subsequent increase in arachidonic acid release from mesangial cells. These data suggest that sPLA2 by cleaving membrane phospholipids and generating LPC and other lysophospholipids activates cPLA2 via the PKC/Raf-1/MAPK signalling pathway. Hence a network of interactions between different PLA2s is operative in mesangial cells and may contribute to the progression of glomerular inflammatory processes.
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PMID:Cross-talk between secretory phospholipase A2 and cytosolic phospholipase A2 in rat renal mesangial cells. 936 43

Bradykinin stimulates cAMP synthesis in cultured airway smooth muscle (ASM) cells. This occurs via a pathway that involves: (1) the protein kinase C (PKC)-dependent activation of mitogen-activated protein kinase (MAPK); (2) the MAPK-dependent phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) and (3) the utilization of cPLA2-derived arachidonate by the cyclo-oxygenase pathway to produce prostaglandin E2 (PGE2). PGE2 is released and binds to cell surface receptors to stimulate intracellular cAMP synthesis. The signalling pathway was confirmed by the use of PD098059 [the inhibitor of MAPK kinase-1 (MEK-1) activation], AACOCF3 (an inhibitor of cPLA2) and indomethacin (an inhibitor of cyclo-oxygenase), which all reduced bradykinin-stimulated cAMP synthesis. Bradykinin also elicits the inhibition of approx. 60% of the total cAMP phosphodiesterase activity in the cell [Stevens, Pyne, Grady and Pyne (1994) Biochem. J. 297, 233-239]. This is likely to decrease the rate of cAMP degradation markedly and therefore to potentiate PGE2-stimulated cAMP synthesis. Acute treatment of ASM cells with PMA (a direct activator of PKC) also stimulated the MAPK-dependent phosphorylation of cPLA2. However, in contrast with bradykinin, PMA did not stimulate arachidonate release, suggesting that additional signals (e.g. Ca2+ ions) are required for phosphorylation by MAPK to activate cPLA2. PMA was also without effect on PGE2 release and cAMP synthesis. Evidence that PKC can also directly regulate adenylate cyclase was obtained by using cells pretreated with cholera toxin. Under these conditions, PMA stimulated cAMP synthesis independently of arachidonate metabolites. Furthermore the combined treatment of cells with PMA (to activate PKC) and PGE2 (to activate Gs) stimulated synergistic cAMP synthesis. This might be due to the presence of the type 2 adenylate cyclase, which is synergistically activated by Gs and PKC.
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PMID:Bradykinin stimulates cAMP synthesis via mitogen-activated protein kinase-dependent regulation of cytosolic phospholipase A2 and prostaglandin E2 release in airway smooth muscle. 937 32

Tracheal epithelial cells and skin fibroblasts from different cystic fibrosis (CF) patients bearing the deltaF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) released more arachidonic acid in response to bradykinin than do other CF and normal cells. Immortalized tracheal epithelial cell lines were used as models to study the mechanisms of this dysregulation. An 85 kD cytosolic phospholipase A2 (cPLA2) was found in these cells and bradykinin increased its binding to membranes of deltaF508 cells (CFT-2) but not to those of a double heterozygous CF cells (CFT-1), or of control cells (NT-1). The expression of G alpha(q)/11 protein was also increased in deltaF508 cells, with increased stimulation of phosphatidylinositol diphosphate-specific phospholipase C (PLC) by bradykinin, and an early, transient activation of mitogen-activated protein (MAP) kinase. As the binding of cPLA2 to membranes is Ca2+-dependent, the increased coupling to PLC could cause the hypersensitivity to bradykinin. Comparison of the effects of bradykinin to those observed with thapsigargin, an inhibitor of calcium reuptake, suggests that the increase of intracellular calcium is not the only mechanism involved in arachidonic acid release by bradykinin in deltaF508 cells. The lack of effect of calcium ionophore A23187 or TPA on arachidonic acid release from any of the cell lines suggested that activation needs a PKC-independent cPLA2 phosphorylation step, perhaps via MAP kinase activation. The binding of cPLA2 to membranes after bradykinin stimulation still occurred in CFT2 cells (deltaF508) homogenized in EDTA, suggesting that a membrane component plus increased intracellular calcium influenced cPLA2 anchoring to membranes. The defective processing of deltaF508 CFTR seems to increase cPLA2 stimulation by bradykinin, since the bradykinin-stimulated release of arachidonic acid is reversed by growing cells at 28 degrees C for 48 h. The deltaF508 mutation of CFTR appears to increase the stimulation of cPLA2 by Gq-mediated receptors in a PKC-independent and MAP kinase-dependent manner. Hence normal CFTR, or normally processed deltaF508 CFTR, inhibit cPLA2 stimulation. The greater reactivity of deltaF508 CFTR cells to inflammatory mediators might be part of the increased sensitivity of CF patients to lung inflammation.
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PMID:Differential stimulation of cytosolic phospholipase A2 by bradykinin in human cystic fibrosis cell lines. 937 23

A cDNA clone, called CLB1, was isolated from a cDNA library from tomato (Lycopersicon esculentum) and characterized. The CLB1 cDNA contains an open reading frame of 1518 bp, and encodes a putative protein of 506 amino acids with a predicted molecular mass of 54,633 Da. The deduced CLB1 amino acid sequence contains a domain that exhibits from 26% to 37% identity with the Ca2+-dependent lipid-binding domains of cytosolic phospholipase A2, protein kinase C, Rabphilin-3A, and Synaptotagmin 1 of animals. Southern blot analysis indicates that the CLB1 gene belongs to a small gene family in the tomato genome. The CLB1 mRNA is preferentially expressed in fruit tissues.
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PMID:A novel gene of tomato preferentially expressed in fruit encodes a protein with a Ca2+-dependent lipid-binding domain. 942 16

The Ca(2+)-dependent lipid binding domain of the 85-kDa cytosolic phospholipase A2 (cPLA2) is a homolog of C2 domains present in protein kinase C, synaptotagmin, and numerous other proteins involved in signal transduction. NH2-terminal fragments of cPLA2 spanning the C2 domain were expressed as inclusion bodies in Escherichia coli, extracted with solvent to remove phospholipids, and refolded to yield a domain capable of binding phospholipid vesicles in a Ca(2+)-dependent manner. Unlike other C2 domains characterized to date, the cPLA2 C2 domain bound preferentially to vesicles comprised of phosphatidylcholine in response to physiological concentrations of Ca2+. Binding of the cPLA2 C2 domain to vesicles in the presence of excess Ca2+ chelator was induced by high concentrations of salts that promote hydrophobic interactions. Despite the selective hydrolysis of arachidonyl-containing phospholipid vesicles by cPLA2, the cPLA2 C2 domain did not discriminate among phospholipid vesicles containing saturated or unsaturated sn-2 fatty acyl chains. Moreover, the cPLA2 C2 domain bound to phospholipid vesicles containing sn-1 and -2 ether linkages and sphingomyelin at Ca2+ concentrations that caused binding to vesicles containing ester linkages, demonstrating that the carbonyl oxygens of the sn-1 and -2 ester linkage are not critical for binding. These results suggest that the cPLA2 C2 domain interacts primarily with the headgroup of the phospholipid. The cPLA2 C2 domain displayed selectivity among group IIA cations, preferring Ca2+ approximately 50-fold over Sr2+ and nearly 10,000-fold over Ba2+ for vesicle binding. No binding to vesicles was observed in the presence of greater than 10 mM Mg2+. Such strong selectivity for Ca2+ over Mg2+ reinforces the view that C2 domains link second messenger Ca2+ to signal transduction events at the membrane.
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PMID:Independent folding and ligand specificity of the C2 calcium-dependent lipid binding domain of cytosolic phospholipase A2. 943 Jun 70

The Cl- secretory response of colonic cells to Ca(2+)-mediated agonists is transient despite a sustained elevation of intracellular Ca2+. We evaluated the effects of second messengers proposed to limit Ca(2+)-mediated Cl- secretion on the basolateral membrane, Ca(2+)-dependent K+ channel (Kca) in colonic secretory cells, T84. Neither protein kinase C (PKC) nor inositol tetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affected Kca in excised inside-out patches. In contrast, arachidonic acid (AA; 3 microM) potently inhibited Kca, reducing NP0, the product of number of channels and channel open probability, by 95%. The apparent inhibition constant for this AA effect was 425 nM. AA inhibited Kca in the presence of both indomethacin and nordihydroguaiaretic acid, blockers of the cyclooxygenase and lipoxygenase pathways. In the presence of albumin, the effect of AA on Kca was reversed. A similar effect of AA was observed on Kca during outside-out recording. We determined also the effect of the cis-unsaturated fatty acid linoleate, the trans-unsaturated fatty acid elaidate, and the saturated fatty acid myristate. At 3 microM, all of these fatty acids inhibited Kca, reducing NP0 by 72-86%. Finally, the effect of the cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3) on the carbachol-induced short-circuit current (Isc) response was determined. In the presence of AACOCF3, the peak carbachol-induced Isc response was increased approximately 2.5-fold. Our results suggest that AA generation induced by Ca(2+)-mediated agonists may contribute to the dissociation observed between the rise in intracellular Ca2+ evoked by these agonists and the associated Cl- secretory response.
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PMID:Modulation of K+ channels by arachidonic acid in T84 cells. I. Inhibition of the Ca(2+)-dependent K+ channel. 945 22

Hyperglycemia is the major causal factor in the development of diabetic vascular complications and can mediate their adverse effects through multiple pathways. One of those mechanisms is the activation of protein kinase C (PKC) by hyperglycemia-induced increases in diacylglycerol (DAG) level, partly due to de novo synthesis. The activation of PKC regulates various vascular functions by modulating enzymatic activities such as cytosolic phospholipase A2 and Na+,K+-ATPase, and gene expressions including extracellular matrix components and contractile proteins. Some of the resulting vascular abnormalities include changes in retinal and renal blood flow, contractility, permeability, proliferation, and basement membrane. Among the various isoforms of PKC predominantly the beta isoforms are activated in cultured vascular cells exposed to high glucose and vascular tissues isolated from animal models of diabetes mellitus. Administration of vitamin E, which decreases DAG level possibly through the activation of DAG kinase, prevents hemodynamic changes in retina and renal glomeruli of diabetic rats. In addition, the inhibition of PKC beta isoforms by a specific inhibitor (LY333531) can normalize the changes in gene expression of cytokines, caldesmon, and hemodynamics. These results provide supportive evidence that the activation of PKC, especially the beta isoforms, is involved in the development of diabetic vascular complications, and that PKCbeta inhibitors can be used in the treatment of diabetic vascular complications.
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PMID:Protein kinase C activation and its role in the development of vascular complications in diabetes mellitus. 946 65


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