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 cAMP phosphodiesterase (PDE) activity of CHO cells was unaffected by the addition of Ca2+ +calmodulin (CaM), indicating the absence of any PDE1 (Ca2+/CaM-stimulated PDE) activity. Treatment with the tumour promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) led to the rapid transient induction of PDE1 activity which attained a maximum value after about 13 h before slowly decreasing. Such induction was attenuated by actinomycin D. PCR primers were designed to hybridize with two regions identified as being characteristic of PDE1 forms found in various species and predicted to amplify a 601 bp fragment. RT-PCR using degenerate primers allowed an approx. 600 bp fragment to be amplified from RNA preparations of rat brain but not from CHO cells unless they had been treated with PMA. CHO cells transfected to overexpress protein kinase C (PKC)-alpha and PKC-epsilon, but not those transfected to overexpress PKC-beta I or PKC-gamma, exhibited a twofold higher PDE activity. They also expressed a PDE1 activity, with Ca2+/CaM effecting a 1.8-2.8-fold increase in total PDE activity. RT-PCR, with PDE1-specific primers, identified an approx. 600 bp product in CHO cells transfected to overexpress PKC-alpha and PKC-epsilon, but not in those overexpressing PKC-beta I or PKC-gamma. Treatment of PKC-alpha transfected cells with PMA caused a rapid, albeit transient, increase in PDE1 activity, which reached a maximum some 1 h after PMA challenge, before returning to resting levels some 2 h later. The residual isobutylmethylxanthine (IBMX)-insensitive PDE activity was dramatically reduced (approx. 4-fold) in the PKC-gamma transfectants, suggesting that the activity of the cyclic AMP-specific IBMX-insensitive PDE7 activity was selectively reduced by overexpression of this particular PKC isoform. These data identify a novel point of 'cross-talk' between the lipid and cyclic AMP signalling systems where the action of specific PKC isoforms is shown to cause the induction of Ca2+/CaM-stimulated PDE (PDE1) activity. It is suggested that this protein kinase C-mediated process might involve regulation of PDE1 gene expression by the AP-1 (fos/jun) system.
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PMID:Induction of Ca2+/calmodulin-stimulated cyclic AMP phosphodiesterase (PDE1) activity in Chinese hamster ovary cells (CHO) by phorbol 12-myristate 13-acetate and by the selective overexpression of protein kinase C isoforms. 757 35

Several lines of evidence from our laboratory and others indicate that epigenetic alterations in protein kinase C (PKC) are involved in colonic carcinogenesis in both man and experimental animals. Furthermore, bile salts, known activators of PKC, have also been implicated in colonic tumor development. Recently, however, our laboratory has demonstrated that, whereas dietary cholic acid increased the occurrence of azoxymethane (AOM)-induced rat colonic tumors, ursodeoxycholic acid was associated with a significant protective effect. In the present studies, we therefore examined changes in PKC isoforms that accompanied AOM-induced tumor formation and investigated whether the chemopromotional and/or chemopreventional actions of these supplemental dietary bile salts involved changes in specific isoforms of PKC. Rats treated with vehicle (saline) or AOM and maintained on bile salt unsupplemented or supplemented diets were used to isolate control colonocytes and carcinogen-induced tumors, which were then subjected to subcellular fractionation. The homogenates and subcellular fractions were then probed for individual PKC isoforms by quantitative Western blotting using isoform-specific antibodies. Normal rat colonocytes expressed PKC-alpha, -beta II, -delta, -epilson, and -zeta. AOM, in unsupplemented or cholate-supplemented groups, caused significant down-regulation of PKC-alpha, -delta and -zeta and up-regulation of PKC-beta II, while increasing particulate PKC-alpha, -beta II, and -zeta in carcinogen-induced tumors compared to normal colonocytes. Dietary supplementation with ursodeoxycholic acid, in marked contrast to these groups, prevented the changes in the subcellular distributions of PKC-alpha, -beta II, and -zeta, and preserved the expression of PKC-zeta in AOM-induced tumors. These studies suggest that changes in specific isoforms of PKC (particularly, PKC-alpha, -beta II, -delta, and/or -zeta) are involved in colonic malignant transformation in the AOM model but do not account for the chemopromotional actions of cholic acid in this model. Furthermore, the ability of ursodeoxycholic acid to block AOM-induced increases in particulate PKC-alpha, -beta II, and -zeta, and/or inhibit down-regulation of PKC-zeta, may contribute to the chemopreventive effects of this bile acid.
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PMID:Mechanism of action of chemoprotective ursodeoxycholate in the azoxymethane model of rat colonic carcinogenesis: potential roles of protein kinase C-alpha, -beta II, and -zeta. 758 85

Intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma R is known to be a phospholipase C (PLC)-dependent process. Activation of PLC leads to the formation of second messengers that synergistically activate protein kinase C (PKC). The aim of this study was to obtain more insight into the role of PKC in Fc gamma R-mediated killing process. PKC inhibitors H-7 and staurosporine markedly suppressed the killing of S. aureus by monocytes stimulated by cross-linking Fc gamma RI or -II. Cross-linking Fc gamma R caused a transient increase in PKC activity in the membranes of monocytes, as measured by Ca2+/phospholipid-dependent phosphorylation of histone. Western blot analysis revealed that cross-linking Fc gamma R stimulated a transient increase in PKC-beta in the membranes of monocytes with kinetics that correlated closely with the translocation of PKC activity. Cross-linking Fc gamma R on monocytes also stimulated the translocation of PKC-epsilon but not PKC-alpha. PMA and 1-oleoyl-2-acetylglycerol (OAG), which caused translocation of PKC-alpha, -beta, and -epsilon, did not stimulate the killing process. Incubation with these PKC activators for 10 min rendered monocytes unresponsive to stimulation of killing of S. aureus via Fc gamma R. It could be that activation of certain PKC isozymes, probably PKC-alpha and -epsilon, by these activators causes feedback inhibition of PLC and, consequently, the killing in monocytes, because PMA blocks the Fc gamma R-mediated intracellular inositol(1,4,5)P3 formation and PKC translocation. Together, our results indicate that PKC isozymes play an important role in both stimulation and inhibition of the Fc gamma R-mediated intracellular killing of bacteria by monocytes.
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PMID:Role of protein kinase C isozymes in Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes. 760 54

Granulocyte macrophage colony-forming cells (GM-CFC) are bipotential progenitor cells that can proliferate and develop into macrophages in response to macrophage CSF or into neutrophils in response to stem cell factor or granulocyte CSF. These cytokines promoted growth and development in highly enriched GM-CFC. In [3H]thymidine suicide assays, IL-4 was shown to stimulate proliferation of GM-CFC to the same degree as IL-3 and other potent mitogens for GM-CFC. IL-4 also maintained the clonogenic potential of enriched GM-CFC over a 2-day period. However, after several days in the presence of IL-4, the GM-CFC began to die and retained blast cell morphology characteristic of the isolated GM-CFC. When a high concentration of IL-4 was added to GM-CFC with neutrophilic stimuli, the response of these cells was altered because macrophages were formed. This effect was achieved by a 4-h preincubation with IL-4, suggesting that an early signal produced by IL-4 promotes lineage restriction, although IL-4 itself cannot promote development. IL-4, like macrophage CSF, translocates PKC-alpha to the nucleus in GM-CFC, this redistribution of protein kinase C alpha (PKC-alpha) being inhibited by calphostin C (a PKC inhibitor). Calphostin C also blocked IL-4-mediated development of macrophages in stem cell factor- and granulocyte-CSF-treated cells. This is further evidence that PKC-alpha translocation is involved in the commitment of GM-CFC to macrophage development. This data also suggests that agonist-stimulated lineage commitment can be uncoupled from development in normal hematopoietic cells.
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PMID:IL-4 promotes macrophage development by rapidly stimulating lineage restriction of bipotent granulocyte-macrophage colony-forming cells. 760 62

Cultured astrocytes express bradykinin (BK) receptors coupled to phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis. Short term (10- or 90-min) treatment of cells with 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) decreased BK-induced PI breakdown, but this inhibitory action was lost after 3-hr TPA treatment. Extended (6- or 24-hr) pretreatment resulted in marked potentiation of the BK response. Western blot analysis using protein kinase C (PKC) isozyme-specific antibodies indicated that astrocytes express PKC-alpha, PKC-delta, and PKC-zeta. With TPA treatment of the cells for various times (10 min, 90 min, 3 hr, 6 hr, or 24 hr), translocation of PKC-alpha and PKC-delta from the cytosol to the membrane was seen after 10- or 90-min treatment and restoration to basal levels in the membrane fraction was seen after 3-hr treatment. However, partial or complete down-regulation of PKC-alpha and PKC-delta was seen after 6- or 24-hr treatment, respectively. No translocation or down-regulation of PKC-zeta was seen after either short term or long term TPA treatment. The inactive phorbol ester alpha-TPA had no effect on BK-induced PI hydrolysis or on the translocation or down-regulation of PKC-alpha and PKC-delta. These results suggest that, in unstimulated astrocytes, both PKC-alpha and PKC-delta, but not PKC-zeta, may exert tonic inhibition of BK-mediated PI turnover. After 10- or 90-min TPA treatment, AIF4(-)--but not Ca2+ ionophore-induced PI hydrolysis was inhibited, whereas [3H]BK binding was unaffected, indicating that the site of action of PKC-alpha and PKC-delta in the BK receptor/G protein/PLC pathway is after the receptor and before PLC, i.e., the G protein. After down-regulation of PKC-alpha and -delta, increases in both AIF4(-)-induced inositol phosphate formation and [3H]BK binding contributed to marked potentiation of BK-induced PI responses. Scatchard plot analysis showed an increase in both the maximal number of binding sites and the binding affinity. Both the up-regulation of [3H]BK binding and the subsequent BK-induced PI turnover were blocked by 0.5 microM cycloheximide, a protein synthesis inhibitor. The increase in AIF4(-)-induced PI hydrolysis after 24-hr TPA treatment was also inhibited by cycloheximide, indicating that new synthesis of BK receptors and G proteins was required after down-regulation of PKC-alpha and PKC-delta.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of protein kinase C subtypes alpha and delta in the regulation of bradykinin-stimulated phosphoinositide breakdown in astrocytes. 762 73

The three-dimensional structure of the second cysteine-rich domain of protein kinase C alpha (residues 95-159) was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing based calculations. On the basis of 687 distance constraints derived from assigned nuclear Overhauser effect (NOE) connectivities, a total of 10 converged structures were obtained from 40 runs of calculations. The atomic root-mean-square (RMS) difference about the mean coordinate positions (excluding residues 1-7, 16-17, 30-34, and 55-65) is 0.55 A for backbone atoms (N, C alpha, C') and 1.07 A for all non-hydrogen atoms. The molecular scaffold is maintained by triple-stranded and double-stranded twisted beta-sheets packed against an alpha-helix and two independent zincs are coordinated by His8, Cys38, Cys41, Cys57 and Cys21, Cys24, His46, Cys49, respectively. It should be noted that the metal ligands from the two sites are interleaved and this is thought to be a new structural motif of a zinc finger domain. Based on the resultant structure, we propose an interaction site of the cysteine-rich domain of protein kinase C with diacylglycerols and phorbol esters.
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PMID:Solution structure of cysteine-rich domain of protein kinase C alpha. 762 23

The present study examined the effect of phorbol esters, Ca2+, and angiotensin II (ANG II) on protein kinase C (PKC) isoforms in the rat proximal tubule. The immunoblot analysis of PKC isoforms of particulate and cytosolic fractions of proximal tubules revealed immunoreactive proteins when antibodies against PKC-alpha, -delta, -epsilon, and -zeta, but not -beta and -gamma were used. Phorbol dibutyrate (PDBU) induced the translocation of PKC-alpha, -delta, and -epsilon, whereas an inactive phorbol ester had no effect. PDBU and ionomycin increased particulate PKC specific activity from 0.67 +/- 0.09 to 1.56 +/- 0.18 and 0.96 +/- 0.04 pmol.microgram protein-1.2 min-1, respectively. ANG II (10(-7) M) induced a time-dependent increase in particulate PKC-alpha immunoreactivity observed after 2 min and maintained for 12 min. Particulate PKC-epsilon immunoreactivity increased after 4 min. Meanwhile, PKC-delta and -zeta were not modified by ANG II. Accordingly, ANG II elicited a rise in the specific activity of the particulate PKC, which increased to 0.89 +/- 0.09 pmol.micrograms protein-1.2 min-1 after 2 min. This was inhibited by a preincubation in the presence of 10(-5) M losartan, specific inhibitor of angiotensin subtype 1 receptors. These data indicate that PKC-alpha and -epsilon are potential candidates to regulate the activity of Na+/H+ and Na(+)-HCO3- transporters because they are translocated with a time course fitting with that of the reported effect of ANG II on those transporters.
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PMID:Protein kinase C isoforms in rat kidney proximal tubule: acute effect of angiotensin II. 763 40

The role of protein kinase C alpha 2-adrenoceptor-induced contractions of rabbit saphenous vein was investigated. Contractions induced by the alpha 2-adrenoceptor-selective agonist 5-bromo-6-[2-imidazolin-2-ylamino]-quinoline (UK14304) were inhibited by prior treatment with pertussis toxin and by Ca2+ removal, confirming a Gi/Go-dependent coupling pathway which was highly dependent upon Ca2+ influx. Protein kinase C inhibitors calphostin-C and staurosporine each caused a non-competitive inhibition of UK14304 response. Down-regulation of protein kinase C by pretreatment with tetradecanoylphorbol acetate reduced UK14304 response by almost 90% with no effect on contractions induced by elevated KCl. The ineffectiveness of L-type Ca2+ channel blockers and the absence of stimulated 45Ca2+ uptake or efflux by UK14304 indicated that phospholipid-derived products were most likely responsible for protein kinase C activation. alpha 2-Adrenoceptor stimulation failed to increase [3H]myoinositol phosphate formation, but caused a significant increase in the formation of both [32P]phosphatidic acid and diacylglycerol, indicating the possible activation of phospholipase D activity. These results suggest that protein kinase C is important for the vasoconstriction induced by alpha 2-adrenoceptors and that diacylglycerol derived from receptor-initiated phospholipase D activity may provide protein kinase C stimulation.
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PMID:Involvement of protein kinase C activation in alpha 2-adrenoceptor-mediated contractions of rabbit saphenous vein. 763 71

Rapid and long term effects of protein kinase C alpha activation on receptor tyrosine kinase signaling parameters were investigated in human 293 embryonic fibroblasts and mouse NIH 3T3 cells. Within minutes of phorbol 12-myristate 13-acetate treatment, epidermal growth factor receptor and HER2 tyrosine phosphorylation was decreased, while platelet-derived growth factor receptor and insulin receptor autophosphorylation was upregulated. These effects are not mediated by protein kinase C-dependent receptor tyrosine kinase phosphorylation but apparently by activation or inactivation of receptor tyrosine kinase-specific phosphatases, as indicated by neutralization of these phenomena upon treatment of cells with sodium orthovanadate. In contrast to these short term effects, sustained activation of protein kinase C alpha by phorbol 12-myristate 13-acetate results in translocation of protein kinase C from the cytosol to the membrane fraction where it forms stable complexes with all receptor tyrosine kinases investigated. Ligand-induced receptor tyrosine kinase/protein kinase C association in NIH 3T3 fibroblasts is accompanied by a mobility shift of the receptor, indicating phosphorylation by activated protein kinase C. This phenomenon correlates with the disappearance of receptor tyrosine kinases from the cell surface, implying that this interaction plays a role in the process of receptor internalization and degradation. Interestingly, ligand-stimulated receptor down-regulation is also enhanced by overexpression of phospholipase C gamma, which strongly indicates a role for this common receptor tyrosine kinase substrate in negative regulation of growth factor signals.
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PMID:Rapid and long-term effects on protein kinase C on receptor tyrosine kinase phosphorylation and degradation. 764 54

A mixed micellar assay was used to study the in vitro binding of [3H]phorbol-12, 13-dibutyrate ([3H]PDBu) to pure recombinant protein kinase C (PKC)-alpha, -beta 1, -beta 2, -gamma, -delta, -epsilon, and -zeta isotypes expressed in the baculovirus/insect cell system. Scatchard analysis revealed that all isotypes except PKC-zeta were able to specifically bind PDBu, with Kd values ranging from 1.6 to 18 nM in the presence of calcium. In the absence of calcium PKC-alpha, -beta 1, -beta 2, and -delta were observed to have a 2-3-fold drop in affinity, although Bmax values remained unchanged, at a stoichiometry of 1.4-2.8 mol of PDBu/mol of enzyme. Competition with specific [3H]PDBu binding was assessed for the phorbol esters PDBu, 12-tetradecanoylphorbol-13-O-acetate, 12-deoxyphorbol-13-O-phenylacetate, 12-deoxyphorbol-13-O-phenylacetate-20-acetate, thymeleatoxin, resiniferatoxin, and sapintoxin A. Resiniferatoxin and 12-deoxyphorbol-13-O-phenylacetate-20-acetate were found to compete effectively only with PDBu bound to the PKC-beta 1 and -beta 2 isotypes and were the least potent of the phorbol esters tested (IC50, > 5 microM). The phorbol esters sapintoxin A, 12-deoxyphorbol-13-O-phenylacetate, 12-tetradecanoylphorbol-13-O-acetate, and PDBu (in order of potency) competed for binding to all isotypes (IC50 values ranging from 2 to 70 nM), with unchanged or slightly decreased potency when calcium was replaced by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Thymeleatoxin, which was similar in other respects to these potent phorbol esters, was found to be less able to compete with binding to PKC-alpha and -epsilon isotypes (IC50, 3-5 microM). It appears that, whereas the binding of phorbol esters to PKC depends primarily on the C20 substituent, other areas of the molecule have an influence on this interaction and the PKC isotypes themselves display heterogeneity in their phorbol ester-binding characteristics.
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PMID:Characterization of phorbol ester binding to protein kinase C isotypes. 765 59


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