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)

Synaptotagmin (Syt) II, a synaptic vesicle protein containing two copies of highly conserved protein kinase C homology regions known as the C2A and C2B domains, acts as a Ca2+ sensor and provides both phospholipid and inositol polyphosphate (IPn) recognition domains important in endo- and exocytosis. Four photoaffinity analogues of IP3, IP4, and IP6 containing a P-1- or P-2-linked 4-benzoyldihydrocinnamidyl (BZDC) photophore were used to label glutathione S-transferase (GST) fusion constructs of the Syt II-C2A and C2B domains. The P-2-linked [3H]BZDC-IP6 showed efficient, IP6-displaceable labeling of the GST-Syt II-C2B. The rank order of photocovalent modification paralleled the order of competitive displacement: IP6 (P-2-linked) > IP4 > IP3. The P-1-linked [3H]BZDC-IP6 failed to label the C2B domains. The GST-Syt III-C2B domain, which lacks IP6 binding affinity, also failed to undergo labeling by P-2-linked [3H]BZDC-IP6. When mixtures of the 32-amino acid basic peptide corresponding to the essential IPn binding region of the Syt II-C2B domain and GST-Syt II-C2B were labeled by a stoichiometric amount of P-2-linked [3H]BZDC-IP6, the two polypeptides showed equivalent affinity for the photolabel. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP6 appeared to induce a binding-compatible structure in the short peptide.
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PMID:Selective photoaffinity labeling of the inositol polyphosphate binding C2B domains of synaptotagmins. 902 Jan 39

Regulation of the bradykinin-evoked increase in intracellular Ca2+ concentration by protein kinase C (PKC)-alpha was investigated in A549 human lung carcinoma cells. Bradykinin, a potent and selective kinin B2 receptor agonist, induces calcium mobilization in a concentration-dependent fashion in this cell line. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, is known to reduce the amplitude of agonist-induced calcium mobilization in various cell lines. Because PKC-alpha is a major PKC isozyme in A549 cells, we investigated whether this isozyme plays a role in this process. A 20-mer phosphorothioate oligonucleotide targeting the 3'-untranslated region of the human PKC-alpha mRNA, which contains 2'-methoxyethyl modifications incorporated into the 5' and 3' segments of the oligonucleotide, was used to assess the putative role of PKC-alpha in the receptor regulation. ISIS 9606 reduced PKC-alpha mRNA for > or = 72 hr after the initial treatment and the reduction was concentration dependent, whereas the mismatch control, ISIS 13009, had no effect. Concentrations of ISIS 9606 of 150 nM specifically reduced the level of immunoreactive PKC-alpha protein by 66.3 +/- 2.5% at 72 hr after treatment, without an effect on immunoreactive PKC-delta protein. This reduction in PKC-alpha was sufficient to inhibit the reduction of bradykinin-induced calcium mobilization by TPA. This finding is corroborated by the use of staurosporine, a nonselective PKC inhibitor, that prevented the effect of TPA. These results suggest that PKC-alpha is involved in kinin B2 receptor regulation by phorbol esters in A549 cells.
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PMID:Antisense oligonucleotides targeting human protein kinase C-alpha inhibit phorbol ester-induced reduction of bradykinin-evoked calcium mobilization in A549 cells. 920 25

Transforming growth factor beta (TGFbeta) induces the expression of a wide variety of genes in many cell types. Our previous studies have shown that TGFbeta stimulates both clusterin mRNA and protein levels, and induces its accumulation in the nucleus of CCL64 cells. To further investigate the molecular mechanism of clusterin mRNA induction by TGFbeta, we created a 1.3-kilobase rat clusterin promoter/luciferase reporter construct. We demonstrate that TGFbeta enhances luciferase activity 2.5-6-fold in transient transfection assays of epithelial, endothelial, and fibroblast cell lines. Deletional analysis reveals that an AP-1-binding site (5'-TGAGTCA) in the minimal promoter region is necessary for initiating transactivation by TGFbeta. A single T to G base mutation in the AP-1 site (5'-TGAGGCA) abolishes TGFbeta-induced clusterin promoter transactivation. In transcription factor decoy experiments, 23-mer oligonucleotides of wild type AP-1 reduce TGFbeta induction of clusterin mRNA levels and promoter transactivation, while an oligonucleotide containing the mutated AP-1 site has no effect. Two specific protein kinase C inhibitors, GF109203X and calphostin C, block TGFbeta-induced clusterin mRNA levels and promoter transactivation. Together these results indicate that TGFbeta regulates clusterin gene expression through an AP-1 site and its cognate transcription factor AP-1, and requires the involvement of protein kinase C.
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PMID:Regulation of clusterin gene expression by transforming growth factor beta. 933 43

A 20-mer phosphorothioate oligodeoxynucleotide (ODN) directed against human protein kinase C-alpha (CGP 64128A = ISIS 3521) was analyzed for its antitumor activity either alone or in combination therapy. Combination studies with CGP 64128A and standard chemotherapeutic agents (cisplatin, mitomycin-C, vinblastine, estracyt and adriamycin) were performed in nude mice that had been transplanted s.c. with a variety of human tumors (breast, prostate, large cell lung and small cell lung carcinomas, and melanomas). Additive antitumor effects with CGP 64128A and the cytotoxins were found for half of the combinations studied. The combination of CGP 64128A with vinblastine or cisplatin showed superadditive antitumor activities against MCF-7 human breast carcinomas and PC3 prostate carcinomas with complete responses. CGP 64128A in combination with adriamycin resulted in superadditive antitumor effects against BT20 human breast carcinomas with complete tumor responses, and in combination with mitomycin-C superadditive antitumor effects with cures were observed against NCI-H460 human large cell carcinomas. The antitumor activity of CGP 64128A appeared to be due to a sequence-dependent mechanism of action as two 20-mer control ODNs were completely inactive as single agents against A549 and NCI-H69 human lung carcinomas. The antitumor activity of cisplatin against NCI-H69 human small cell lung carcinomas was slightly inhibited by one of the control ODNs, indicating that the superadditive antitumor activities of CGP 64128A in combination with cisplatin are the result of a sequence-dependent mechanism of action.
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PMID:Antitumor activity of a PKC-alpha antisense oligonucleotide in combination with standard chemotherapeutic agents against various human tumors transplanted into nude mice. 947 41

Myotrophin, a novel protein that has been shown to stimulate myocyte growth, has been isolated, purified, and sequenced from the hearts of spontaneously hypertensive rats and dilated cardiomyopathic human tissue. Recently, the cDNA clones encoding myotrophin have been isolated and expressed in Escherichia coli, and the recombinant myotrophin was found to be as biologically and immunologically active as natural myotrophin. The mechanism by which myotrophin stimulates protein synthesis and initiates myocardial hypertrophy is not known. To evaluate the involvement of protein kinase C (PKC) in myotrophin-induced hypertrophy, PKC activity and its distribution in the subcellular fraction were determined in cultured neonatal and adult myocytes. PKC activity was determined by measuring the incorporation of 32P into histone type III-S and PKCepsilon substrate peptide (epsilon(pep)) from [gamma-32P]ATP in neonatal myocytes. Myotrophin significantly stimulated PKC activity in neonatal myocytes and was associated with a significant increase in protein synthesis. The effect of myotrophin on the stimulation of PKC activity and [3H]leucine incorporation was abolished by pretreatment with either staurosporine or H-7, two selective, pharmacological PKC inhibitors. Pretreatment of myocytes with staurosporine also reduced the myotrophin-induced mRNA levels of c-fos and beta-myosin heavy chain. To evaluate the subcellular events whose occurrence was due to myotrophin and translocation of PKC, we studied the effect of genistein, a tyrosine kinase inhibitor, on myotrophin-induced neonatal myocyte growth. Genistein attenuated the [3H]leucine incorporation induced by myotrophin. To define the specificity of the PKC isoform(s) involved in myotrophin-stimulated myocyte growth, both neonatal and adult myocytes were treated with myotrophin, and Western blot analyses were performed by using the antibodies of different PKC isoforms. Results showed that both PKCalpha and PKCepsilon isoforms participated in the myotrophin-induced neonatal myocyte growth, whereas only the PKCepsilon isoform was involved in myotrophin-induced adult myocyte hypertrophy. PKCdelta and PKCzeta do not seem to participate in either neonatal or adult myocyte growth induced by myotrophin. Treatment with antisense oligonucleotides specific for PKCalpha and PKCepsilon isoforms further supported this result. PKCalpha is the major PKC isoform in neonatal myocytes and needs Ca2+ and phospholipids for its activation, and PKCepsilon (the Ca2+-independent PKC isoform) is present in both neonatal and adult myocytes; the 15-mer antisense oligodeoxynucleotides of each were used for this study. Treatment of neonatal myocytes with the PKCalpha and PKCepsilon antisense oligodeoxynucleotides for 5 days significantly reduced Ca2+-dependent and Ca2+-independent PKC activity, respectively, as well as the [3H]leucine incorporation induced by myotrophin. Furthermore, myotrophin-induced PKC activity was primarily located in the particulate fraction and did not result in a concomitant decrease in the cytosolic fraction. Myotrophin does not change PKC isoform expression (both Ca2+ dependent and independent PKC isoforms used in this study) in rat neonatal cardiac fibroblasts. Our data suggest that myotrophin exerts its action on protein synthesis, possibly through a tyrosine kinase-coupled pathway and translocation of PKC from the cytosol to the cell membrane.
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PMID:Increased protein kinase C activity in myotrophin-induced myocyte growth. 963 17

The Rab2 protein is a resident of pre-Golgi intermediates and required for vesicular transport in the early secretory pathway. We have previously shown that a peptide corresponding to the amino terminus of Rab2 (residues 2-14) arrests protein traffic prior to a rate-limiting event in VSV-G movement through pre-Golgi structures (Tisdale, E. J., and Balch, W. E. (1996) J. Biol. Chem. 271, 29372-29379). To determine the mechanism by which this peptide inhibits transport, we investigated the effect of the Rab2 peptide on the distribution of the beta-COP subunit of coatomer because COPI partially localizes to pre-Golgi intermediates. We found that the peptide caused a dramatic change in the distribution of pre-Golgi intermediates containing beta-COP. A quantitative binding assay was employed to measure recruitment of beta-COP to membrane when incubated with the Rab2 (13-mer). Peptide-treated microsomes showed a 25-70% increase in the level of membrane-associated beta-COP. The enhanced recruitment of coatomer to membrane was specific to the Rab2 (13-mer) and required guanosine 5'-3-O-(thio)triphosphate, ADP ribosylation factor, and protein kinase C-like activity. The ability to enhance beta-COP membrane binding was not limited to the peptide. Similarly, the addition of recombinant Rab2 protein to the assay promoted beta-COP membrane association. Our results suggest that the Rab2 peptide causes the persistent recruitment of COPI to pre-Golgi intermediates which ultimately arrests protein transport due to the inability of membranes to uncoat.
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PMID:Rab2 protein enhances coatomer recruitment to pre-Golgi intermediates. 964 98

Beta-adrenergic agonists are well known to increase the activity of adenylate cyclase, yielding increases of the intracellular concentration of cAMP. It has been reported that activation of protein kinase C (PKC) by phorbol esters reduces the amplitude of isoproterenol-induced cAMP production in a 3T3-L1 cell line. In this study, we investigated whether PKC-alpha is involved in this process in murine Swiss 3T3 fibroblasts. A 20-mer phosphorothioate oligonucleotide designed to hybridize to the AUG initiation codon of the murine PKC-alpha mRNA, which contains 2'-O-methoxyethyl modifications incorporated into the 5' and 3' segments of the oligonucleotide, was used to assess the putative role of PKC-alpha in the beta-adrenergic receptor regulation. ISIS 14012 reduced PKC-alpha mRNA for over 72 hr after the initial treatment and the reduction was concentration dependent, whereas the mismatch control, ISIS 13818, had no effect. This depletion was found to be selective; ISIS 14012 had no effect on the mRNA expression of PKC-delta and PKC-zeta. ISIS 14012 reduced in a time and concentration-dependent fashion the levels of immunoreactive PKC-alpha protein by over 85% at 72 hr after treatment. Depletion of PKC-alpha inhibited the effect of isoproterenol-induced cAMP production by phorbol dibutyrate (PdBu). This finding is corroborated by the use of a nonspecific inhibitor of PKC, GF-109203x, which also prevented the effect of PdBu. Depletion of PKC-alpha by ISIS 14012 potentiated isoproterenol-induced cAMP production in cells untreated with PdBu. However, neither depletion of PKC-alpha nor PKC activation by a phorbol ester altered beta-adrenergic receptor affinity and density. PKC activation by PdBu did not alter forskolin-induced cAMP levels, but enhanced cAMP production by cholera toxin. PKC-alpha inhibition by ISIS 14012 had no effect on either cholera toxin-induced increases in cAMP or the acute effects of phorbol esters on cholera toxin in induction of cAMP. Thus, PKC-alpha appears to be involved in the regulation of beta-adrenergic receptor coupling to adenylate cyclase, possibly by phosphorylating the Gs protein, but other PKC isotypes must be involved in the effects observed when cells are treated with cholera toxin.
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PMID:Depletion of protein kinase C-alpha by antisense oligonucleotides alters beta-adrenergic function and reverses the phorbol ester-induced reduction of isoproterenol-induced adenosine 3'-5'-cyclic monophosphate accumulation in murine Swiss 3T3 fibroblasts. 976 65

The mechanism of the antineoplastic effects of suramin may involve interference with signal transduction, but in general is not well understood. We examined several polyanions to determine their effects on the kinase activity of the protein kinase C (PKC) beta1 and other PKC isoforms. Similar to suramin, a phosphorothioate oligodeoxynucleotide 28-mer homopolymer of cytidine (SdC28) inhibited the phosphatidylserine and Ca2+-dependent phosphorylation of an epidermal growth factor receptor octapeptide substrate. The inhibition by suramin was mixed competitive/noncompetitive with respect to ATP, but uncompetitive with respect to substrate. In contrast, the inhibition by SdC28 was competitive with respect to substrate (Ki = 5.4 microM) and not competitive with respect to ATP. The PKC alpha and beta1 isoforms were inhibited to the same extent with SdC28, while PKC epsilon was not inhibited. SdC28, in the absence of lipid cofactor, stimulated substrate phosphorylation, and in the absence of substrate induced PKC beta1 autophosphorylation. Similar behavior was seen with another polyanion, the polysulfated carbohydrate pentosan polysulfate (polyxylyl hydrogen sulfate). H4, a bis-naphthalene disulfonate tetraanion structurally related to suramin, also inhibited kinase activity but was not competitive with respect to ATP. Dianions closely related to H4 failed to inhibit PKC beta1, suggesting that multiple (>2) negative charges are required. The interactions of polyanions with PKC are complex, and are dependent on the molecular structure of the polyanion, the presence of cofactors, and the PKC isoform.
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PMID:Effects of suramin-related and other clinically therapeutic polyanions on protein kinase C activity. 981 93

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-alpha expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2' modifications have been incorporated into the protein kinase C-alpha targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5 degrees C per modification) and an increase in resistance toward both 3'-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-alpha in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-alpha protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-alpha expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.
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PMID:Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha expression. 988 May 52

Late-phase and sustained activation of p44/42(MAPK) has been reported to be a critical factor in cell mitogenesis. We therefore hypothesized that p44/42(MAPK) is involved in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth-muscle cells (ASMC). Treatment of adherent ASMC with beta-hexosaminidase A (Hex A, 50 nM), an endogenous mannosyl-rich glycoprotein, resulted in a late-onset (30-min) activation of p44/42(MAPK) that lasted for 4 h. Activation of p44/42(MAPK) induced by Hex A was inhibited by an 18-mer phosphorothioate-derivatized antisense oligonucleotide (1-5 microM) directed to human p44(MAPK); the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059 (5 microM); the p42(MAPK) inhibitor Tyrphostin AG-126 (0.2 microM); the farnesyl transferase inhibitors SCH-56582 (10 microM) and FPT III (10 miroM), which inhibit p21Ras activation; and Calphostin C (0.2 microM), an inhibitor of protein kinase C. These agents also inhibited Hex A-induced cell proliferation in bovine ASMC. These data suggest that Hex A activates p44/42(MAPK) in a p21Ras- and PKC-dependent manner and that this activation mediates Hex A- induced mitogenesis in bovine ASMC.
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PMID:beta-hexosaminidase-induced activation of p44/42 mitogen-activated protein kinase is dependent on p21Ras and protein kinase C and mediates bovine airway smooth-muscle proliferation. 1038 99


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