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)

Stimulation of hepatocytes by the tumor promoter phorbol 12-myristate 13-acetate (PMA) caused translocation of cytosolic Ca2+/phospholipid-dependent protein kinase C (PK-C). The major part of PK-C activity (greater than 80%) was associated with the membrane fraction after 30 min. During the following 6 h protein kinase C activity decreased to less than 10%. Minor amounts of Ca2+/phospholipid-independent PK-C activity were found in the cytosol fraction at all times; they temporarily increased 2.5-fold with PMA and decreased after 1 h. Cyclosporin A did not affect the translocation of PK-C from the cytoplasm to the membrane fraction, but the decrease of PK-C activity following translocation was blocked. No marked increase of Ca2+/phospholipid-independent PK-C activity was observed in the cytosol in the presence of cyclosporin A. Leupeptin, which is known to inhibit Ca2+-requiring non-lysosomal proteinases (e.g. calpain), showed an effect similar to cyclosporin A. Both agents reduced proteolytic degradation of cellular proteins observed in isolated hepatocytes after PMA treatment. Ca2+-ionophore A23187 in high doses (greater than 10(5) M) partly reversed cyclosporin A and leupeptin action.
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PMID:Cyclosporin A inhibits proteolytic cleavage and degradation of membrane-bound protein kinase C in hepatocytes after stimulation by phorbol ester. 312 91

Protein kinase C(PKC) activity in macrophages and polymorphonuclear leukocytes was assayed in beige mouse, the model of Chediak-Higashi syndrome, control C57BL/6 and the heterozygous (+/bg) mice. Regarding enzyme activity in the cytosolic and membrane fractions of these cells, there was no difference between beige mouse and the control. After short-term activation by TPA, the translocated membrane-bound PKC activity in beige mouse decreased rapidly compared with that in control mouse. However, the cytosolic PKC activity decreased at just the same pace as the control. The change in [3H] PDBu binding paralleled the changes in PKC activity. An increase in Ca2+/phospholipid-independent protein kinase by TPA was notable in the membrane fraction of beige mouse. The increase in the kinase activity was abolished and the PKC activity recovered to normal level by the addition of calpain inhibitor, leupeptin, to the incubation of cells along with TPA. Therefore, these findings suggest that a rapid decrease in membrane-bound PKC activity in beige mouse by TPA stimulation is associated with calpain.
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PMID:Rapid down-regulation of protein kinase C in (Chediak-Higashi syndrome) beige mouse by phorbol ester. 338 95

Stimulation of intact human neutrophils with phorbol 12-myristate 13-acetate results in the selective phosphorylation of two cytoskeletal protein components with molecular masses of 20 and 48 kDa. After phosphorylation the 48-kDa protein is no longer recovered as a component of the cytoskeletal fraction but is present as a fully soluble phosphoprotein. Phosphorylation of the 20-kDa protein (probably myosin light chains) signals a proteolytic conversion, catalyzed by calpain, to a smaller species having a molecular mass of approximately 15 kDa. Phosphorylation of both the 48- and 20-kDa proteins is related to the conversion of protein kinase C, also catalyzed by calpain, to the soluble fully active form. Leupeptin, an inhibitor of calpain, blocks both the phosphorylation of the target proteins and the proteolytic modification of the 20-kDa polypeptide. Thus, phosphorylation of cytoskeletal proteins and signal-directed proteolysis appear to be related processes that follow stimulation of human neutrophils by phorbol esters. The resulting changes in cytoskeletal organization may be involved in the expression of some neutrophil functions, such as exocytosis of specific granules.
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PMID:Phosphorylation and proteolytic modification of specific cytoskeletal proteins in human neutrophils stimulated by phorbol 12-myristate 13-acetate. 347 71

The activation of protein kinase C (PKC) is irreversibly regulated by limited proteolysis catalyzed by a calcium-activated neutral cysteine protease, calpain. Calpain cleaves PKC alpha at specific sites in the hinge region between the catalytic and the regulatory domains of this kinase. Here we show a novel method for production of antibodies that bind specifically to the catalytic fragment of PKC alpha but not to the unproteolyzed protein. To detect proteolyzed PKC alpha 'cleavage site-directed antibodies,' which recognize amino-terminal regions in the nascent catalytic fragments and do not cross-react with the unproteolyzed enzymes, were raised using synthetic peptides corresponding to the amino-terminal sequences. The synthetic peptides used in this study were the sequences of human PKC alpha at the cleavage sites by m- and mu-types of calpains (LGPAGNKV and VISPSEDRKQPSNNLDRVKLT, respectively) and they are designated as CF alpha 2, CF alpha 4, in this order. Each synthetic peptide was injected into rabbit after conjugation with a carrier protein. The antibodies thus obtained (anti-CF alpha 2 or -CF alpha 4) specifically reacted with either the 46- or 45-kDa catalytic fragment of PKC alpha, respectively, whereas they did not cross-react with other fragments. Furthermore, the antibodies did not bind to the unproteolyzed enzyme nor fragments of PKC alpha obtained by treatment with other proteinases unless the fragment carried the same amino-terminal sequence. When human platelets were treated with calcium ionophore, the catalytic fragments of PKC alpha (45- and 46-kDa) were detected in the cytosol by immunoblotting with the antibodies. However, these antibodies did not bind unproteolyzed 80-kDa PKC alpha, although this form was dominant in the cytosol of the calcium ionophore-treated human platelets. In addition, the 45-kDa catalytic fragment of PKC alpha was detected in an apoptotic human fibroblast TIG-3 cells cultured in serum-free medium. Our method is applicable for analysis of proteolysis in various cellular states.
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PMID:Antibodies specific for proteolyzed forms of protein kinase C alpha. 749 78

We have examined the mechanism for the selective down-regulation of protein kinase C epsilon (nPKC epsilon) in rat pituitary GH4C1 cells responding to thyrotropin-releasing hormone (TRH) stimulation. Among various low molecular weight protease inhibitors examined, only a cysteine protease inhibitor (calpain inhibitor I, N-acetyl-Leu-Leu-norleucinal) blocked the down-regulation of nPKC epsilon. Furthermore, the introduction of a synthetic calpastatin peptide, an exclusively specific inhibitor of calpain, into the cells also reduced the down-regulation, suggesting the involvement of calpain among all the intracellular cysteine proteases in this process. In accordance, we observed TRH-induced translocation of m-calpain from the cytosol to the membrane and the concomitant up-regulation of calpastatin isoforms; presumably, the former represents activation of the protease initiating the kinase degradation, while the latter constitutes a negative feedback system protecting the cells from activated calpain. These results suggest that in GH4C1 cells, TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKC epsilon pathway mediating TRH signals.
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PMID:The role of the calpain-calpastatin system in thyrotropin-releasing hormone-induced selective down-regulation of a protein kinase C isozyme, nPKC epsilon, in rat pituitary GH4C1 cells. 755 44

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

We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH-SY-5Y human neuroblastoma cells. SH-SY-5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum-containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell-permeant cysteine protease inhibitors N-acetyl-leucyl-leucyl-norleucinal ("C1") and calpeptin or by the phospholipid-mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti-calpain antisera. Calpain inhibition in intact cells was confirmed by immunoblot analysis showing inhibition of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule-associated protein 1. The above inhibitory peptides and antiserum did not induce neurites in medium containing serum but lacking hirudin, suggesting that increased surface protein adhesiveness is a prerequisite for enhancement of neurite outgrowth by calpain inhibition. Treatment of cells with the PKC inhibitor H7, staurosporine, or sphingosine induced neurite outgrowth independently of serum concentration. Because calpain is thought to regulate PKC activity, we examined this potential interrelationship during neurite outgrowth. Simultaneous treatment with calpain and PKC inhibitors did not produce additive or synergistic effects on neurite outgrowth. PKC activation by 2-O-tetradecanoylphorbol 13-acetate (TPA) prevented and reversed both neurite initiation by serum deprivation and its enhancement by calpain inhibitors. Treatment of cells with the calpain inhibitor C1 retarded PKC down-regulation following TPA treatment. Cell-free analyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of millimolar calcium-requiring calpain to cleave the SH-SY-5Y PKC regulatory subunit from the catalytic subunit, yielding a free catalytic subunit (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that of surface adhesiveness and calpain activity.
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PMID:Enhancement of neurite outgrowth following calpain inhibition is mediated by protein kinase C. 761 5

Butylated hydroxytoluene (BHT)-induced lung damage in mice is an excellent model system for studying mechanisms of chemically-induced, reversible alveolar injury. Changes in the pulmonary contents of protein kinase C (PKC) and the calcium-dependent protease, calpain, were previously noted during the repair phase following BHT-induced pneumotoxicity. Calpain is believed to initiate PKC down-regulation. PKC-alpha is the major PKC isozyme and calpain II the major calpain isozyme in mouse lung. We have now studied the time course of these enzymatic changes in detail. Pulmonary PKC-alpha concentrations decreased as early as 45 min after an i.p. injection of 200 mg/kg BHT. Calpain II levels rose within the first 40 min after BHT injection, and then declined below control levels. The rapidity of these changes implies a role of these enzymes in mediating the onset of injury. Lung damage and repair, as estimated by measuring the lung weight/body weight ratio, is maximal 6 days after administration of this dose of BHT. The extent of the decreased PKC-alpha and calpain II concentrations at this time was linearly related to the estimated degree of injury based on increased lung weight. This correlation suggests the value of monitoring these enzymes as putative early biomarkers of alveolar injury.
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PMID:Very early changes in pulmonary protein kinase C-alpha and calpain II contents following injection of butylated hydroxytoluene (BHT) into mice. 771 80

The degradation of troponin (Tn) subunits by calpain was studied by incubating either isolated cardiac Tns or myocardial cryosections with two different calpain isoenzymes isolated from rat skeletal muscle. Western-blot analysis with monoclonal antibodies against TnI and TnT showed that mu-calpain was at least ten times more active than m-calpain in degrading TnI and TnT both in vitro and in situ. TnC was completely resistant to both proteinase forms. Phosphorylation by cyclic AMP-dependent protein kinase (PKA) isolated from rat skeletal muscle reduced the sensitivity of TnI to degradation. This effect in combination with an increased efficiency of the endogenous inhibitor [Salamino, De Tullio, Michetti, Mengotti, Melloni and Pontremoli (1994) Biochem. Biophys. Res. Commun. 199, 1326-1332] probably reduces the proteolytic activity of calpain in cells on PKA stimulation. Conversely, phosphorylation by protein kinase C (PKC) resulted in a twofold increase in the degradation of TnI. Degradation by m-calpain was not modified by Tn phosphorylation. The different sensitivity to mu-calpain might be related to changes in TnI oligomeric structure. Indeed, on PKC phosphorylation, the apparent molecular mass of TnI calculated from the distribution coefficient of Tn complex in Sephadex G-100 matrix was reduced from 90 to 30 kDa suggesting dissociation of the Tn complex.
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PMID:Specific degradation of troponin T and I by mu-calpain and its modulation by substrate phosphorylation. 775 88

The relationship between platelet aggregation, calpain activation, PKC activities and the secretory response have been examined in PMA-and ionomycin-stimulated platelets. Co-addition of PMA and ionomycin resulted in a maximal synergistic secretion of [14C]5-hydroxytryptamine ([14C]5-HT) from platelet dense granules. However, prior addition of PMA for 5 or 10 min resulted in a reduction of this secretory response. Inclusion of either RGDS (to inhibit platelet aggregation) or E64-d (to inhibit calpain activity) resulted in full restoration of the secretory response. In experiments to determine the activity status of PKC, PMA was found to induce a loss in cytosolic and total PKC activity without an increase in membrane-associated activities during this time period. Inhibition of either platelet aggregation or calpain activity resulted in preservation of total and cytosolic activities with a measurable increase in membrane translocated activity. PMA-induced phosphorylation of a number of PKC substrates was measured in 32P-labelled platelets. PMA induced potent phosphorylation of the 45 and 20 kDa species and also proteins of the molecular masses 66, 80, 97 and 119 kDa. Phosphorylation was maximal at either 1 or 2 min after which dephosphorylation occurred. Inclusion of either RGDS or E64-d resulted in a reduction of the dephosphorylation rates, and sustained phosphorylation of the 66, 80, 97 and 119 kDa proteins. These studies suggest that the activity status of PKC is an important factor in the level of secretion obtained and that platelet aggregation is involved in calpain-initiated down-regulation of PKC.
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PMID:Calpain-induced down-regulation of protein kinase C inhibits dense-granule secretion in human platelets. Inhibition of platelet aggregation or calpain activity preserves protein kinase C and restores full secretion. 780 7


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