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

The action of many agonists results in rapid production of sn-1,2-diacylglycerol (DAG). Using platelets as a model system, we previously identified a delayed phase of DAG accumulation that is temporally associated with secondary aggregation and secretion. In the present study, we examined the quantitative relationship between this delayed DAG accumulation and platelet aggregation and secretion. To quantitate the low levels of DAG in platelets, we used the sensitive DAG kinase assay and simultaneously compared DAG levels with aggregation and ATP secretion. In platelets stimulated by gamma-thrombin or collagen, there was a dose response between concentration of agonist and DAG accumulation. Significantly, a dose response was observed between DAG accumulation and extent of aggregation and secretion in platelets stimulated by either agonist. A concentration of either gamma-thrombin or collagen that caused secondary aggregation and secretion was associated with DAG accumulation above 0.2 pmol of DAG/nmol of phospholipid. Subthreshold concentrations of gamma-thrombin or collagen resulted in DAG levels less than 0.2 pmol/nmol of phospholipid. Thus, these data suggest that a to occur. Moreover, secretion was blocked when DAG production was blocked with aspirin or when protein kinase C was inhibited with sphingosine. We conclude that endogenously formed DAG plays a critical role in regulating secondary aggregation and secretion and, therefore, represents an important target for future antiplatelet agents.
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PMID:Quantitative analysis of diacylglycerol second messengers in human platelets: correlation with aggregation and secretion. 153 14

Phosphorylation of beta-connectin (titin 2), an elastic protein of chicken breast muscle, occurred in the presence of [gamma-32P] ATP, 0.2 mM CaCl2 and 25 mM phosphate buffer, pH 7.0. Addition of 3 mM MgCl2 did not affect the phosphorylation. However, Ca2+ ions were required for the phosphorylation and EGTA inhibited it even if MgCl2 were present. Myosin light chain kinase (gizzard MLCK), cAMP dependent protein kinase (A kinase), and protein kinase C (C kinase) did not phosphorylate beta-connectin in vitro under optimal conditions. Thus it appears that beta-connectin, possibly containing a domain homologous with MLCK, has an autophosphorylating action.
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PMID:Autophosphorylation of beta-connectin (titin 2) in vitro. 154 1

In order to understand the mechanisms underlying the T lymphocyte dysfunction associated to allogeneic bone marrow transplantation (BMT), we assessed two different protein kinase C (PKC) dependent events in T cells from BMT recipients: the PKC-dependent membrane expression and function of the CD69 early activation antigen; and the rapid phorbol ester-induced phosphorylation of PKC protein substrates in lysates from T cells permeabilized with digitonin, in the presence of (gamma-32P)ATP. Most BMT recipient T cells detectably expressed the CD69 surface antigen after 24 h of stimulation with either phorbol 12-myristate 13-acetate (PMA) or anti-CD3 MoAb and PMA, thus indicating that PKC activity is sufficient to induce de novo gene expression. Nevertheless, it is noteworthy that the fluorescent staining intensity with anti-CD69 MoAbs was significantly lower in BMT recipient T cells than in normal T lymphocytes, although no clear-cut correlation was found between the expression of CD69 and the proliferative capacity. However, the pattern of PMA-induced phosphoproteins analysed as early as 1 min after PKC activation in T cells from BMT recipients displaying a low response to mitogenic stimuli, was undistinguishable from that detected in T cells from healthy subjects. In all cases a major 110-kD phosphoprotein was observed, which was inducible with PMA, phorbol 12,13-dibutyrate (PDBu), 1-oleoyl-2-acetylglycerol (OAG) and a phorbol-ester-related activator of PKC (mezerein); moreover, its phosphorylation was blocked by pretreating cells with the PKC inhibitor H-7. Altogether our results suggest that the depressed mitogenic responses, which were also observed in the present study when T cells were stimulated via CD69, cannot be simply attributed to a defective PKC activity.
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PMID:Analysis of different protein kinase C-dependent events in T cells from allogeneic bone marrow transplantation recipients. 154 33

A hypothetical mode of inhibition of protein kinase C (PKC) by the natural product staurosporine has been used as a basis for the design of substituted bisindolylmaleimides with improved potency over the parent compound. Structure-activity relationships were consistent with the interaction of a cationic group in the inhibitor with a carboxylate group in the enzyme, and the most potent compound had a Ki of 3 nM. The inhibitors were competitive with ATP but inhibited cAMP-dependent protein kinase (PKA) only at much higher concentrations despite the extensive sequence homology between the ATP-binding regions of PKA and PKC. Three compounds were evaluated further and found to inhibit a human allogeneic mixed lymphocyte reaction pointing to the potential utility of PKC inhibitors in immunosuppressive therapy. One of these compounds was orally absorbed in the rat and represents an attractive lead in the development of PKC inhibitors as drugs.
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PMID:Inhibitors of protein kinase C. 2. Substituted bisindolylmaleimides with improved potency and selectivity. 155 13

Protein kinase C was purified 6,900-fold from rabbit pancreas with a total yield of 15% by a procedure involving ammonium sulfate fractionation, diethyl aminoethyl ion exchange chromatography, hydroxylapatite chromatography, and finally protamine-agarose affinity chromatography. After these purification steps the protein kinase C preparation contained two major protein bands as judged by silver staining after SDS-polyacrylamide gel electrophoresis: 80 and 69-kDa bands. Monoclonal antibodies directed against bovine brain protein kinase C (alpha- and beta-subtype) recognized only the 80-kDa band. On the other hand, both the 80 and 69-kDa proteins were recognized by a polyclonal monospecific antibody directed against rat brain protein kinase C. Analysis of rabbit pancreas protein kinase C subtypes by means of hydroxylapatite chromatography showed the presence of the III (alpha) subtype as the major subtype. The enzyme depended absolutely on the presence of both phosphatidylserine and Ca2+ for its activity, with apparent Ka values of 3.1 micrograms/ml and 247 microM for phosphatidylserine and Ca2+, respectively. When dioctanoylglycerol or the phorbol ester 12-O-tetradecanoyl-phorbol 13 acetate (TPA) was present, the Ka value for Ca2+ decreased to 10 and 18 microM, respectively. In the presence of the phorbol ester, pancreatic protein kinase C could be activated without added Ca2+. The enzyme also required Mg2+ for its activity. The Ka value was 3.6 mM and maximal activity was reached at 10 mM Mg2+. Pancreatic protein kinase C activity showed a broad pH dependence, with optimal activity at pH 6.75. The Km value for ATP and for histone-H1 was 8.5 microM and 20.4 micrograms/ml, respectively. The present study shows that the kinetic properties of protein kinase C purified from rabbit pancreas closely resemble those found in other tissues.
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PMID:Purification and characterization of rabbit pancreas protein kinase C. 155 44

The role of protein kinase C (PKC) in the regulation of phosphatidylcholine-hydrolyzing phospholipase D (PLD) was investigated. In membranes from Chinese hamster lung fibroblasts that had been incubated with [14C]choline to label endogenous phosphatidylcholine, phorbol 12-myristate 13-acetate (PMA) failed to stimulate production of [14C]choline. However, stimulation was observed if fibroblast cytosolic fraction or PKC partially purified from this fraction was added. When incubated with membranes in the presence of PMA, pure PKC from rat brain stimulated [14C]choline production in a concentration-dependent manner, with a maximal 2-3-fold effect. PMA similarly stimulated [14C]phosphatidylpropanol formation from propanol using membranes from [14C]myristic acid-prelabeled cells, confirming the activation of PLD. None of the effects described required exogenous ATP. To probe the role of phosphorylation in the PKC effect, we included high concentrations of apyrase in the assay. This ATPase had no effect on the ability of PKC to activate PLD, but under exactly the same conditions, it eliminated autophosphorylation of PKC. The results provide conclusive evidence for the involvement of PKC in the activation of PLD and suggest that ATP-dependent phosphorylation is not required.
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PMID:Activation of phospholipase D by protein kinase C. Evidence for a phosphorylation-independent mechanism. 155 64

In this study we used the bovine thoracic aorta endothelial cell line AG 4762 and primary bovine aortic endothelial cells to investigate the formation of phosphatidic acid (PA) in response to activation of P2-purinergic receptors. 2-Methylthio ATP (2MeSATP) stimulated the formation of [32P]-PA in bovine aortic endothelial cells labelled with 32P(i) for 2.5 hr. A comparison of the response to other ATP analogues suggests that this was mediated via a P2Y-purinergic receptor. Using various agonists at 30 microM there was a correlation between the formation of [32P]PA and of total inositol phosphates in the presence of lithium. The 2MeSATP-stimulated accumulation of [32P]PA showed an initial high rate, followed by a more sustained slower rate. The initial response was independent of extracellular calcium while the later response was dependent on calcium influx. The protein kinase C stimulator phorbol myristate acetate (PMA) produced only a very small enhancement of [32P]PA accumulation compared to 2MeSATP. The 2MeSATP stimulation of both inositol phosphates and [32P]PA was almost eliminated by the presence of PMA. Using cells prelabelled with [3H]methylcholine 2MeSATP produced only a small non-significant enhancement of [3H]choline formation; PMA by contrast formed a much larger amount of [3H]choline. There was no evidence of a change in [3H]phosphocholine. The dissociation between phospholipase D (PLD) activation and [32P]PA accumulation and the correlation between stimulation of [32P]PA accumulation and phospholipase C (PLC) activation all suggest that, using this protocol for labelling cells, the principle route of the stimulation of formation of [32P]PA is via the activation of PLC followed by metabolism of diacylglycerol (DAG) by DAG kinase. These results show that activation of P2Y-purinergic receptors on aortic endothelial cells leads to the formation of phosphatidic acid and that both PLD and PLC pathways are likely to contribute to this response.
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PMID:Stimulation of phosphatidic acid synthesis in bovine aortic endothelial cells in response to activation of P2-purinergic receptors. 156 76

The P2T purinergic receptor for ADP has previously been found only in platelets. We investigated the effect of ADP on the concentration of intracellular free calcium ([Ca++]i) in fura-2-loaded K562 leukemia cells, a cell line with the potential for megakaryocytic differentiation. ADP causes a rapid and transient increase in [Ca++]i, which peaks within 5 to 10 sec. The EC50 for this response is 0.4 microM. A major portion of the increased calcium is due to mobilization of intracellular stores because the response to ADP is only partially reduced in the absence of extracellular calcium. Exposure to ADP desensitizes K562 cells to additional administrations of this nucleotide. Pretreatment of K562 cells with the protein kinase C activator phorbol 12-myristate 13-acetate completely blocks the response to ADP. This effect of phorbol 12-myristate 13-acetate is prevented by the protein kinase C inhibitor staurosporine, but staurosporine does not affect the progression of desensitization after repeated ADP exposures. ATP does not increase [Ca++]i in K562 cells, but antagonizes the response to ADP. We propose that the P2T receptor for ADP in K562 cells is an early marker for megakaryocytic differentiation. Furthermore, this immortalized nucleated cell line may be a useful model to decipher the signal transduction pathways involved in the ADP response.
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PMID:K562 leukemia cells express P2T (adenosine diphosphate) purinergic receptors. 157 75

Both 86Rb+ efflux experiments and electrophysiological studies have shown that arachidonic acid and other nonesterified fatty acids activate ATP-sensitive K+ channels in insulinoma cells (HIT-T15). Activation was observed with arachidonic, oleic, linoleic, and docosahexaenoic acid but not with myristic, stearic, and elaidic acids. Fatty acid activation of ATP-sensitive K+ channels was blocked by antidiabetic sulfonylureas such as glibenclamide. The activating effect of arachidonic acid was unaltered by indomethacin and by nordihydroguaiaretic acid, indicating that it is not due to metabolites of arachidonic acid via cyclooxygenase or lipoxygenase pathways. Moreover, the nonmetabolizable analogue of arachidonic acid, eicosatetraynoic acid, was an equally potent activator. Activation of ATP-sensitive K+ channels by fatty acids was potentiated by diacylglycerol and was inhibited by calphostin C, an inhibitor of protein kinase C. These findings indicate that fatty acid activation of ATP-sensitive K+ channels is most likely due to the participation of arachidonic acid (and other fatty acid)-activated protein kinase C isoenzymes. Activation of ATP-sensitive K+ channels by nonesterified fatty acids is not involved in the control of insulin secretion since arachidonic acid stimulates insulin secretion from insulinoma cells instead of inhibiting it.
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PMID:ATP-sensitive K+ channels in insulinoma cells are activated by nonesterified fatty acids. 158 15

To determine the role of protein tyrosine phosphorylation in the activation of phospholipase D (PLD), electropermeabilized HL-60 cells labeled in [3H]alkyl-phosphatidylcholine were treated with vanadate derivatives. Micromolar concentrations of vanadyl hydroperoxide (V(4+)-OOH) induced accumulation of tyrosine-phosphorylated proteins. Concomitantly, V(4+)-OOH or a combination of vanadate and NADPH elicited a concentration- and time-dependent accumulation of phosphatidic acid (PtdOH). In the presence of ethanol a sustained formation of phosphatidylethanol was observed, indicating that a type D phospholipase was activated. A good correlation was found to exist between the accumulation of tyrosine-phosphorylated proteins and activation of PLD. The V(4+)-OOH concentration dependence of the two responses was nearly identical, and the time course of activation was similar, with tyrosine phosphorylation preceding PLD activation by approximately 1 min. The ability of V(4+)-OOH to induce both responses was found to be strictly dependent on the presence of ATP and/or Mg2+, suggesting that PLD activation involves phosphotransferase reactions. Accordingly, ST638, a tyrosine kinase inhibitor, reduced concomitantly tyrosine phosphorylation and PLD activation elicited by V(4+)-OOH. The mechanism of action of V(4+)-OOH was investigated. The diacylglycerol kinase inhibitors, dioctanoylethylene glycol and R59022 potentiated PLD stimulation by exogenous diacylglycerol but not by V(4+)-OOH. Moreover, stimulation by V(4+)-OOH and by phorbol esters was synergystic. Therefore, diacylglycerol-induced activation of protein kinase C is unlikely to mediate the effects of V(4+)-OOH. The response of PLD to V(4+)-OOH was larger than that to guanosine 5'-(gamma-thio)triphosphate. Moreover, the effects of GTP gamma S and V(4+)-OOH were additive. Hence, activation of G proteins cannot account for the stimulation of PLD by V(4+)-OOH. V(4+)-OOH also triggers a burst of O2 consumption by the NADPH oxidase. Inhibition of PtdOH accumulation by addition of ethanol or by ST638 abolished this respiratory burst. Together, the results establish a strong correlation between tyrosine phosphorylation, PLD activation, and stimulation of the NADPH oxidase in HL-60 cells, suggesting a causal relationship.
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PMID:Peroxides of vanadate induce activation of phospholipase D in HL-60 cells. Role of tyrosine phosphorylation. 160 60


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