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 role of protein kinase C (PKC) in regulating the contractile state of smooth muscle was investigated using the constitutively active catalytic fragment of PKC (PKM) with skinned (demembranated) chicken gizzard fibres. PKM attenuated a submaximal contraction in gizzard smooth muscle skinned fibres, but not in rabbit cardiac skinned fibres. PKM-mediated relaxation of submaximal contractions of smooth muscle was accompanied by a reduction in the rate of ATP hydrolysis in the fibre and by phosphorylation of the 20 kDa light chain of gizzard myosin at the PKC sites (serine-1, serine-2 and threonine-9). In addition, several other endogenous proteins were phosphorylated by PKM. However, the inhibitory effects on tension and ATPase are consistent with the biochemical effects of PKC-catalysed phosphorylation of myosin, i.e. reduction of the actin-activated MgATPase activity of myosin prephosphorylated at serine-19 by myosin light chain kinase. Pretreatment of skinned fibres with PKM and ATP gamma S in the absence of Ca2+ had no inhibitory effect on the subsequent submaximal Ca(2+)-activation of force. Consistent with this observation, PKC was not able to utilize ATP gamma S as a substrate, confirming that the observed effects were the result of PKM-catalysed protein phosphorylation. We suggest that PKC may have two distinct effects on smooth muscle contraction: translocation of PKC to the sarcolemma on stimulation results in phosphorylation of a protein(s) other than myosin and a slow, sustained contraction; in some circumstances PKC may undergo proteolysis to PKM resulting in myosin phosphorylation at PKC-specific sites, a reduction in ATPase activity and relaxation of the muscle.
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PMID:Effects of the constitutively active proteolytic fragment of protein kinase C on the contractile properties of demembranated smooth muscle fibres. 153 85

The rapid redistribution of cytosolic protein kinase C (PKC) to membranes and its subsequent proteolytic activation to PKM have been implicated in the DMSO/HMBA-induced differentiation of murine erythroleukemia (MEL) cells. However, DMSO was found not to induce detectable changes in PKC distribution in a MEL cell subline (MEL1) which differentiated normally in response to the agent. Nevertheless, the differentiation of MEL1 cells appeared dependent on an early PKC-related event because hemoglobinization was partially blocked by the PKC inhibitor H-7 added to cells within the first 2 h after DMSO induction. Indeed, a rapid (15-60 min) increase in membrane PKC activity was detected in DMSO-treated MEL1 cells using a novel method which quantitates the amount of 'active' PKC in intact membranes. This transient PKC increase resulted from the activation of 'inactive' enzyme already associated with membranes, and not from the translocation of cytosolic PKC. Conventional PKC assays cannot distinguish between active and inactive membrane PKC pools. DMSO also activated inactive membrane PKC in HL-60 cells, but not in S49T-lymphoma and WEHI-231 B-lymphoma cells which do not differentiate in response to DMSO. The results suggest that a rapid and transient increase in membrane PKC activity may be an important early step in DMSO-induced differentiation of erythroleukemia cells.
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PMID:The activation of inactive membrane-associated protein kinase C is associated with DMSO-induced erythroleukemia cell differentiation. 164 17

Recent evidence from our laboratory has demonstrated that NK/LAK cell activation of human lymphocytes is protein kinase C (PKC)-dependent. Here, we have investigated the translocation of PKC in human NK cells exposed to sensitive targets or to PMA, a phorbol ester. In NK cells exposed to K562 for 6 hr, we observed a weak translocation of PKC whereas in NK cells exposed to PMA more than 90% of cytosolic PKC was translocated to the membrane in less than 5 min. Stimulation of NK cells with an NK-resistant target, however, did not translocate PKC even after 6 hr. Translocation of PKC to the membrane was followed by the appearance of PKM, the cytosolic calcium/phospholipid (Ca2+/PL)-independent form of PKC. The conversion of PKC to PKM was mediated by calpain, an intracellular calcium-dependent thiol proteinase. When we used two inhibitors of calpain, calpain inhibitor I (CI-I) and calpain inhibitor II (CI-II), both caused a dose-related enhancement of NK-CMC when the inhibitors were present throughout the 3-hr chromium release assay. This enhancement could be circumvented by PMA or by the PKC inhibitor H-7. CI-I and CI-II added together caused a greater increase in NK-CMC than when each was added alone. CI-I and CI-II also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), substantiating further our previous contention that the activation of both NK-CMC and ADCC may involve a common lytic pathway. Activation of NK cells with IL-2 for 18 hr at 37 degrees C was inhibited in the presence of CI-I. To investigate a possible feedback inhibition mechanism due to the buildup of PKC, we examined phosphatidylinositol (PI) metabolism in NK cells activated by IL-2 in either the presence or the absence of CI-I. We observed a significant decrease in PI turnover when NK cells, activated in the presence of IL-2 and CI-I, were stimulated with K562 as compared to NK cells activated by IL-2 alone, then stimulated with K562.
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PMID:Inhibition of the calpain-mediated proteolysis of protein kinase C enhances lytic activity in human NK cells. 191 39

Phorbol esters such as phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBU) are generally considered to have similar effects through a similar mechanism, i.e. protein kinase C (PKC) activation. We recently suggested that this was not the case in human neutrophils. To identify further differences between the two phorbol esters, we compared their priming effects on fMet-Leu-Phe-induced superoxide anion (O2-) production, cytosolic PKC activity and binding of fMet-Leu-Phe. Priming could be initiated with a low (0.2 nM) concentration of both PDBU and PMA. Their effects on the pattern of fMet-Leu-Phe-induced superoxide production were similar in both Ca2(+)-containing and Ca2(+)-free medium. PDBU, like PMA, abolished the Ca2+ dependency of fMet-Leu-Phe-induced O2- production in a dose-dependent manner. In cytochalasin B-treated cells and in the presence of Ca2+, priming with PDBU or PMA did not alter the enhancing effect of cytochalasin B on fMet-Leu-Phe-induced O2- production. In Ca2(+)-free medium, priming abolished the Ca2+ dependency of fMet-Leu-Phe stimulation in cytochalasin B-treated cells. Cytochalasin B, however, enhanced the effect of PMA but not that of PDBU. Priming with PDBU was not associated under any experimental conditions with a decrease in cytosolic PKC activity, or an increase in PKM activity before or after fMet-Leu-Phe stimulation. Furthermore, priming effects were abolished by cell washing but not by H-7 or staurosporine, which are potent PKC inhibitors. PDBU, in contrast to PMA, increased fMet-Leu-Phe binding to PMNs through a decrease in the dissociation constant and induced degranulation of specific granules as measured by the release of vitamin B12 binding protein. These findings show that the priming effects of PDBU differ in certain respects from those of PMA, namely with regard to its synergism with cytochalasin B and the expression of fMet-Leu-Phe receptors. In addition, priming concentrations of PDBU, like PMA, did not alter cytosolic PKC activity in fMet-Leu-Phe-stimulated neutrophils.
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PMID:A comparison of the priming effect of phorbol myristate acetate and phorbol dibutyrate on fMet-Leu-Phe-induced oxidative burst in human neutrophils. 217 83

Protein kinase C activity of the human myeloma cell line, RPMI 8226, was studied after prepurification on DEAE-cellulose. The total protein kinase activity, eluted at 0.12 M NaCl, was 493 nmol/min/10(10) cells, but 38% was associated with membranes. The lipid dependence of cytosolic and membrane activities was only 52% and 21%, respectively. This activity increased with time, to as much as 200% for the membrane fraction after 7 days, whereas lipid dependence and the PDBu binding properties were lost. This modified activity was not due to the extinction of a copurifying endogenous inhibitor nor to classical PKC proteolysis. TPA-treatment of these cels is accompanied by a rapid, selective and complete loss of lipid-dependent activity of the cytosol, thus benefiting co-migrating lipid independent activity, with no membrane fraction recovery or PKM formation.
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PMID:Abnormal behavior of protein kinase C in the human myeloma cell line, RPMI 8226. 240 58

In 32Pi-loaded bovine neutrophils stimulated with phorbol myristate acetate (PMA), radioactivity was preferentially incorporated into a protein of low molecular mass, suggesting a PKC-dependent phosphorylation. This protein, termed 23-kDa protein, was predominantly localized in the cytosol. It was purified from bovine neutrophil cytosol by a series of chromatographic steps, including ion exchange on DE-52 cellulose and Mono Q, and filtration on Bio-Gel P60 in the presence of mercaptoethanol and urea. The apparent molecular mass of the purified protein, assessed by SDS-PAGE and mercaptoethanol by reference to protein markers, ranged between 20 and 23 kDa, depending on the percentage of polyacrylamide and conditions of migration. In the absence of mercaptoethanol, a dimer accumulated. Homogeneity of the 23-kDa protein was verified by 2D-PAGE analysis. Some properties of the 23-kDa protein, including its amino acid composition, were determined. Gel isoelectric focusing (IEF) of the purified 23-kDa protein followed by Coomassie blue staining allowed the visualization of four discrete protein bands with isoelectric points ranging between pH 6.3 and 6.7. Phosphorylation of the 23-kDa protein by [gamma-32P]ATP in the presence of bovine neutrophil PKC supplemented with Ca2+, phosphatidylserine, and diacylglycerol or with PMA occurred on serine and required the presence of mercaptoethanol. The apparent KM of ATP was 9 microM. The 23-kDa protein was also phosphorylated by PKM, the catalytic fragment of PKC obtained after removal of the regulatory domain, but not by cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization. 251 5

In marked contrast to ligands which activate B cells via their physiological receptors for antigen, transforming Epstein-Barr virus (EBV) was found to be mitogenic for human B lymphocytes without increasing inositol phospholipid hydrolysis. B-cell stimulation by EBV showed similar characteristics to those achieved by the tumour-promoting phorbol ester TPA, in terms of the temporal appearance of surface activation antigens, the induction of RNA and DNA synthesis and the lower requirement for medium Ca++ in comparison to agonists that lead to an increase in inositol phospholipid hydrolysis. The calcium- and phospholipid-dependent kinase, protein kinase C (PKC), is activated by TPA and a proteolytically cleaved fragment (PKM) results. EBV induced the appearance of a calcium- and phospholipid-independent activity that was chromatographically inseparable from PKM and this activity was capable of phosphorylating vimentin, a cell component that is thought to participate in the signal transduction cascade. These findings are discussed with special reference to the biochemical signalling pathways on which EBV might impinge to usurp growth control in B lymphocytes.
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PMID:Epstein-Barr virus and a tumour-promoting phorbol ester use similar mechanisms in the stimulation of human B-cell proliferation. 253 33

The potential involvement of platelet activating factor (PAF, 1-O-alkyl 2-O-acetyl-sn-glycero-3-phosphocholine) in aggravation of ischemic brain injury has been recently postulated. Reported evidences in support of this thesis include increases of brain PAF concentration during ischemia and the neuroprotective effect exerted by PAF antagonists. In this article, we demonstrate that several PAF-mediated biochemical responses in synaptoneurosomes in vitro resemble these observed previously in ischemic brain and are widely acknowledged as the potentially causal factors in this pathology. In synaptoneurosomes prepared from rat hippocampus, 10 nM PAF caused an observable elevation of intracellular calcium as measured by fluorescence Fura-2A probe. A similar elevation of synaptoneurosomal [Ca2+]i was evoked by 1 mM glutamate treatment. As an effect of calcium entry after PAF application, a translocation of protein kinase C (PKC) toward plasma membranes was demonstrated by 3H-labeled phorbol-binding method. It was followed by an increase of 50 kDa proteolytic fragment of the enzyme (PKM) recognized on Western blots with anti-PKC antibody. Incubation of synaptoneurosomes in the presence of calcium chelators abolished these effects of PAF and significantly decreased the content of PKC in the membranes. Furthermore, PAF treatment markedly attenuated the receptor- and postreceptor-activated cAMP accumulation in synaptoneurosomes. The decrease of cAMP level seems to be secondary to the PAF-induced calcium entry with subsequent activation of cAMP-specific phosphodiesterase, since it was completely blocked by IBMX, a potent inhibitor of this enzyme. Our observations indicate that PAF in a concentration found in ischemic brain can elevate [Ca2+]i and potentiate calcium-dependent intracellular signalling in synaptoneurosomes in vitro, including PKC translocation/activation and proteolysis, followed by IBMX-sensitive inhibition of cAMP production. The relative contribution of these events to ischemic brain injury is currently under extensive investigation.
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PMID:Modulation of signal transduction in rat synaptoneurosomes by platelet activating factor. 754 18

Limited proteolysis of protein kinase C (PKC) by calpain under cell free conditions cleaves the regulatory and catalytic PKC subunits, generating a free, co-factor independent catalytic subunit, termed PKM. In the present study, we demonstrate distinct differences in the rate, nature, and lipid-sensitivity of PKC and PKM proteolysis by microM and mM calcium-requiring calpain isozymes (mu calpain or m calpain, respectively). PKC is a preferred substrate for m calpain; not even a 100-fold increase in mu calpain was capable of degrading PKC as fast as in calpain. PKM was generated by both m and mu calpains, but was itself rapidly degraded by m calpain and therefore was only transiently detectable. By contrast, PKM was formed but not degraded by mu calpain, and persisted in the presence of mu calpain long after all PKC had been degraded. Phosphatidyl serine (PS) inhibited PKC hydrolysis by m calpain yet enhanced PKC hydrolysis by mu calpain. The ability of either calpain isoenzyme to degrade [14C]azocasein was unaffected by PS, suggesting that the influence of PS was on PKC conformation. These findings point towards distinct roles for mu and m calpain in PKC regulation.
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PMID:Proteolysis of protein kinase C: mM and microM calcium-requiring calpains have different abilities to generate, and degrade the free catalytic subunit, protein kinase M. 760 11

Protein kinase C has been implicated in the modulation of calcium channel function. However, controversy exists concerning the actions of agents such as phorbol esters or diacylglycerol (DAG) that activate endogenous PKC, with both enhancement and inhibition of Ca2+ currents described. In this article we report the effects of direct intracellular application of a constitutively active form of PKC (PKM) on whole cell calcium currents in acutely dissociated rat dorsal root ganglion neurons. PKM application significantly enhanced high threshold voltage-activated calcium currents elicited from holding potentials of -80 mV and -40 mV. The rate of current rundown in PKM-treated cells was not significantly different from controls. The enhancement observed with PKM was not due to a shift in the voltage dependence of the peak current. Synthetic PKC inhibitor peptide (PKC-I) added to recording solutions containing PKM (PKM+PKC-I) abolished the PKM-associated enhancement. The rate of current rundown was significantly increased in the presence of PKM+PKC-I, and PKC-I alone, suggesting that substantial enhancement of voltage-activated calcium currents by endogenous PKC occurred in this preparation of rat dorsal root ganglion neurons. The portions of current attributable to N-, L-, and non-N,L-type currents [determined by applying the N- and L-type calcium antagonists omega-conotoxin GVIA and nifedipine (3-10 microM)] were not affected by PKM, suggesting that both N and L current components were enhanced by PKM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enhancement of high threshold calcium currents in rat primary afferent neurons by constitutively active protein kinase C. 766 90


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