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)

Morphological and biochemical evidence indicates that in several cell types, lysozyme is found in both lysosomes and the medium. Here we report that in calcitriol-treated human promonocytes U937, in which approx. two-thirds of the synthesized lysozyme is secreted, most of the intracellular lysozyme co-localizes with cathepsin D in lysosomal organelles. In the presence of NH4Cl the lysosomal targeting of procathepsin D, but not that of lysozyme, is inhibited. In the presence of 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA; 'TPA'), the lysosomal packaging of lysozyme is almost completely inhibited, while that of procathepsin D is only partially so. However, the inhibition of the lysosomal targeting of procathepsin D by NH4Cl and 4 beta-PMA is additive. The targeting of lysozyme is partially inhibited in the presence of R-59022, an inhibitor of diacylglycerol kinase, whereas it is not affected by 4 alpha-phorbol 12-myristate 13-acetate, an isomer of 4 beta-PMA that does not activate protein kinase C. It is concluded that in U937 cells both carbohydrate-dependent and -independent recognition contributes to the lysosomal targeting of soluble proteins. We suggest that the carbohydrate-independent traffic of proteins to lysosomal compartments is controlled by a signalling pathway involving protein kinase C.
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PMID:Distinctive inhibition of the lysosomal targeting of lysozyme and cathepsin D by drugs affecting pH gradients and protein kinase C. 809 11

Caco-2 cells are an enterocyte-like cell line derived from a human colonic adenocarcinoma. Paracellular permeability was assessed in monolayers of these cells by transmonolayer resistance and by the permeation of [3H]mannitol across the monolayer. Paracellular permeability was increased by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (50 nM), carbachol (500 microM), and the combination of carbachol (50 microM) and monolein (100 microM), an inhibitor of diacylglycerol kinase, as manifested by a decrease in transmonolayer resistance and an increase in mannitol permeation. The effects of all of these stimuli on transmonolayer resistance were inhibited by staurosporine (3 nM), an inhibitor of PKC. The effects of carbachol plus monolein were also inhibited by atropine (0.1 microM), a muscarinic antagonist. Treatment of the monolayers with each of the stimuli was associated with translocation of PKC activity from cytosol to a membrane-associated state. Stimulation of Caco-2 cell monolayers with phorbol myristate acetate or with the combination of carbachol and monolein was also associated with phosphorylation of the MARCKS protein, an endogenous substrate of PKC. These data support the hypothesis that intestinal paracellular permeability is regulated by the activity of enterocyte PKC and demonstrate that the increase in paracellular permeability induced by binding of carbachol to the muscarinic receptor is mediated by activation of PKC.
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PMID:Regulation of paracellular permeability in Caco-2 cell monolayers by protein kinase C. 823 25

Migration activated by fMet-Leu-Phe is inhibited by GTP[S] and is little affected by protein kinase C inhibitors. We investigated the effects of GTP[S] and the protein kinase C inhibitor AMG-C16 on dioctanoyl-sn-glycerol (DiC8)-activated migration of rabbit neutrophils and compared them with the effects on fMet-Leu-Phe-activated migration and random migration. GTP[S] did not inhibit DiC8-activated migration or random migration but inhibited fMet-Leu-Phe-activated migration. AMG-C16 gave a strong inhibition of DiC8-activated migration but had only a small effect on fMet-Leu-Phe-activated migration and random migration. When fMet-Leu-Phe and DiC8 were added together in suboptimal concentrations an additive effect was found. Pretreatment with the diacylglycerol kinase inhibitor R59022 enhanced random migration. The enhancement was completely inhibited by AMG-C16 and was unaffected by GTP[S]. These findings suggest that DiC8-activated migration and fMet-Leu-Phe-activated migration are controlled by different pathways.
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PMID:Activation of neutrophil migration by dioctanoyl-sn-glycerol and fMet-Leu-Phe is controlled by different pathways. 831 8

Exposure of human synovial fibroblasts prelabelled with [3H]arachidonic acid to bradykinin causes a rapid and sustained increase in arachidonic acid release, a transient increase in cytosolic calcium and an increase in radiolabelled diacylglycerol. Activation of arachidonic acid release by bradykinin was potentiated by interleukin-1 added either simultaneously with bradykinin or to cultures 24 h before addition of bradykinin. In contrast, interleukin-1 did not modify bradykinin-induced increases in cytosolic calcium or diacylglycerol. The stimulation of arachidonic acid release in response to bradykinin, in the absence or presence of interleukin-1, was not affected by RHC-80267, an inhibitor of diacylglycerol kinase, suggesting that deacylation of diacylglycerol was not an important pathway of arachidonic acid production in cultures exposed to bradykinin. This conclusion is supported by the observation that increased release of arachidonic acid was not accompanied by increased release of [14C]stearic acid in cultures labelled with both isotopes. Bradykinin-stimulated release of arachidonic acid was prevented by down-regulating protein kinase C by pretreatment with phorbol 12-myristate 13-acetate and was unaffected by inhibitors of protein synthesis actinomycin D or cycloheximide. On the other hand, interleukin-1 amplification of bradykinin-stimulated release of arachidonic acid was blocked by actinomycin D and cycloheximide. The results from this study point to activation of phospholipase A2 as the source of arachidonic acid in response to bradykinin. Our data further indicate that interleukin-1 selectively potentiates bradykinin activation of a phospholipase A2 by a mechanism requiring protein synthesis, but has no effect on bradykinin activation of phospholipase C.
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PMID:Interleukin-1 selectively potentiates bradykinin-stimulated arachidonic acid release from human synovial fibroblasts. 837 25

R59022, a diacylglycerol kinase inhibitor, enhances the prostaglandin F2 alpha (PGF2 alpha)-induced diacylglycerol (DAG) synthesis in Swiss 3T3 cells. It also potentiates the PGF2 alpha-mediated protein kinase C (PKC)-dependent 80 kDa protein (80K) phosphorylation and initiation of DNA replication. R59022 enhances the PGF2 alpha mitogenic response by increasing the rate of entry into the S phase. Insulin does not cause 80K phosphorylation, and does not enhance its induction but it potentiates the PGF2 alpha mitogenic response. These results suggest that mitogenically triggered fluctuations in DAG content and PKC activity play a pivotal role in controlling the PGF2 alpha-induced DNA synthesis while insulin acts via a different mechanism.
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PMID:Early cell cycle diacylglycerol (DAG) content and protein kinase C (PKC) activity enhancement potentiates prostaglandin F2 alpha (PGF2 alpha) induced mitogenesis in Swiss 3T3 cells. 838 Jul 77

The primary phase of ADP-induced aggregation of human platelets does not involve appreciable formation of thromboxane A2 or release of granule contents; lack of formation of inositol trisphosphate has also been noted. Because these responses of platelets to ADP differ so markedly from their responses to other aggregating agents, the roles in ADP-induced aggregation of diacylglycerol, protein kinase C, increases in cytosolic [Ca2+], phosphorylation of pleckstrin (47 kDa) and phosphatases 1 and 2a were investigated. Washed human platelets, prelabelled with [14C]5-hydroxytryptamine and suspended in Tyrode solution (2 mM Ca2+, 1 mM Mg2+), were used for comparisons between the aggregation induced by 2-4 microM ADP, in the presence of fibrinogen, and that induced by 0.05 units/ml thrombin. The diacylglycerol kinase inhibitor 6-(2-[(4-fluorophenyl)phenyl-methylene]-1-piperidinylethyl)-7-meth yl-5H-thiazolo[3,2-a]-pyrimidin-5-one (R59022; 25 microM) had no, or only a slight, enhancing effect on ADP-induced aggregation, but potentiated thrombin-induced responses to a much greater extent. 1,2-Dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol (25 microM) added with or 30-90 s before ADP greatly potentiated aggregation without formation of thromboxane; staurosporine, an inhibitor of protein kinase C, reduced this potentiation. Staurosporine (25 nM) did not inhibit ADP-induced aggregation, although it strongly inhibited thrombin-induced aggregation and release of [14C]5-hydroxytryptamine. All these observations indicate little or no dependence of primary ADP-induced aggregation on the formation of diacylglycerol or on the activation of protein kinase C. At 2-4 microM, ADP did not significantly increase the phosphorylation of pleckstrin (studied with platelets prelabelled with [32P]orthophosphate), but 1,2-dihexanoyl-sn-glycerol- induced phosphorylation of pleckstrin was increased by ADP. Surprisingly, the diacylglycerols strongly inhibited the ADP-induced rise in cytosolic [Ca2+] concurrently with potentiation of ADP-induced aggregation; thus the extent of primary aggregation is independent of the level to which cytosolic [Ca2+] rises. Incubation of platelets with 1,2-dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol for several minutes reversed their potentiating effects on aggregation, and inhibition was observed. Incubation of platelets with okadaic acid, an inhibitor of phosphatases 1 and 2a, inhibited ADP- and thrombin-induced aggregation; although the reason for this effect is unknown, it is unlikely to involve inhibition of phospholipase C, since formation of diacylglycerol appears to have little involvement in the primary phase of ADP-induced aggregation.
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PMID:Activation of phospholipase C and protein kinase C has little involvement in ADP-induced primary aggregation of human platelets: effects of diacylglycerols, the diacylglycerols, the diacylglycerol kinase inhibitor R59022, staurosporine and okadaic acid. 838 48

The diacylglycerol kinase inhibitor R59022 induced chemotaxis in neutrophils. The response to R59022 was primarily chemotactic and only very little chemokinetic. Pretreatment with the protein kinase C inhibitors staurosporine and AMG-C16 inhibited chemotaxis induced by R59022 indicating the involvement of protein kinase C. In contrast, chemotaxis induced by fMet-Leu-Phe was only slightly inhibited by staurosporine and AMG16. The effects of R59022 were comparable to the effects of the protein kinase C activators DiC8 and PMA and suggest an involvement of protein kinase C. Pretreatment with pertussis toxin inhibited R59022-induced migration, fMet-Leu-Phe-induced migration, and random migration. GTP gamma S, which stimulates migration of electropermeabilized neutrophils by itself, causes an additive increase of migration in electropermeabilized neutrophils stimulated with a suboptimal concentration R59022, but causes a synergistic increase of migration in cells stimulated with a suboptimal concentration fMet-Leu-Phe. The effects of GTP gamma S on migration are completely inhibited by AMG-C16. This suggests that the GTP-binding protein involved in R59022-activated migration is the G protein that is associated with random migration.
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PMID:Neutrophil chemotaxis induced by the diacylglycerol kinase inhibitor R59022. 839 81

p74raf-1, a serine/threonine kinase, is structurally related to the protein kinase C (PKC) family and contains a cysteine motif in its N-terminal domain, which is essential for its regulation. It has been shown that p74raf-1 functions upstream of mitogen-activated protein (MAP) kinase kinase. We have constructed a p74raf-1 mutant (N delta raf) that only contains the N-terminal regulatory domain. When transiently expressed in COS-M6 cells, N delta raf efficiently blocked the activation of the MAP extracellular signal regulated kinase (ERK2), induced by either epidermal growth factor, phorbol ester, serum, or oncogenic p21ras. Similar constructs with the cysteine motifs from either PKC-alpha or diacylglycerol kinase did not inhibit activation of ERK2. Overexpression of full-length p74raf-1 rescued the inhibition of ERK2 by N delta raf in a stimulus dependent manner, indicating that N delta raf acts as a competitive inhibitor of wild-type p74raf-1. In contrast, overexpression of either PKC-alpha, -epsilon, or -zeta in N delta raf-containing cells could not rescue the inhibition of ERK2. We conclude that p74raf-1 is an essential mediator of epidermal growth factor- and phorbol ester-induced ERK2 activation and that the MAP kinase kinase activity of p74raf-1 cannot be substituted with either PKC-alpha, -epsilon or -zeta.
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PMID:A dominant-negative mutant of raf blocks mitogen-activated protein kinase activation by growth factors and oncogenic p21ras. 839 1

Cholinergic stimulation of the HT29-18N2 goblet cell line increased mucin secretion as assessed: (1) with a mucin-specific immunoassay, (2) using whole-mount immunocytochemistry, or (3) by morphometric quantification of intracellular mucous granule stores. Cholinergic stimulation did not, however, result in the apical plasmalemmal membrane cavitation that is characteristic of recent compound exocytotic activity. The response was not dependent on protein kinase C activation since it was not inhibited by the kinase C antagonist H7 or potentiated by the diacylglycerol kinase antagonist R59022. Calcium ionophore A23187 also accelerated mucin secretion by a noncompound exocytotic pathway. Activation of protein kinase C by phorbol 12-myristate 13-acetate, on the other hand, increased mucin secretion by a compound exocytotic pathway. The results provide insight into the signal transduction pathways underlying secretory responses of goblet cells observed in situ.
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PMID:Signal transduction pathways mediating mucin secretion from intestinal goblet cells. 850 99

We have investigated the presence of Na-K-Cl cotransport in alveolar type II cells using uptake of 86Rb. Several data support the presence of a Na-K-Cl cotransport in these cells. First, a large fraction of ouabain-resistant 86Rb uptake was inhibited by bumetanide and furosemide. Second, bumetanide-sensitive 86Rb uptake required the presence of Na+ and Cl- in the incubation medium; dependency on extracellular Na+ and K+ was hyperbolic, with a Km of 14.6 mM and 8.3 mM, respectively, while dependency on extracellular Cl- was sigmoidal, which suggests a 1:1:2 stoichiometry. Third, a fraction of amiloride-insensitive 22Na influx was deeply inhibited by bumetanide. 22Na influx was dependent on the presence of extracellular K+ and Cl-. Since Na-K-Cl activity dramatically decreased with time in culture, further characterization of the cotransport on polarized cells could not be performed. The phorbol ester PMA inhibited Na-K-Cl cotransport in a time- and concentration-dependent manner. This inhibition was mimicked by oleoylacetylglycerol, dioctanoylglycerol, and the diacylglycerol kinase inhibitor R59022, and was reversed by an antagonist of PKC, staurosporine. Since the Na-K-Cl cotransport has been reported to be involved in cell volume regulation, we investigated its modulation by changes in extracellular osmolarity. Na-K-Cl activity was increased after a two-step procedure: swelling in hypotonic medium followed by shrinking in hypertonic medium. Under these conditions, cotransport activity increased whenever PKC activity was up- or downregulated, which suggests that the cell volume-induced modulation of the cotransport is independent from the PKC activity. Though we were not able to determine the polarity of the cotransport, it may also be involved in the absorptive function of alveolar type II cells, and would provide an alternate pathway for sodium entry.
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PMID:Evidence for Na-K-Cl cotransport in alveolar epithelial cells: effect of phorbol ester and osmotic stress. 855 95


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