Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activation of protein kinase enzyme activity by Ca2+ and diacylglycerol or phorbol esters is a feature of certain isoforms of protein kinase C (PKC). Although the binding sites of phorbol ester on the regulatory domain of PKC have been extensively studied, little is known about the actual mechanisms of Ca2+ binding and how this leads to enzyme activation. We previously reported that high affinity binding of 45Ca2+ to the regulatory domain of PKC beta 1, expressed as a GST fusion protein in Escherichia coli, is dependent on the presence of phosphatidylserine (PS) or 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study we have used this system to further analyze Ca2+ binding. Using various deletions, we found that different domains in the regulatory domain of PKC beta 1 are involved in TPA-induced Ca2+ binding, depending on whether or not PS was also present in the binding assay. In addition, Ca2+ binding in the presence of TPA alone displayed very different kinetics than Ca2+ binding in the presence of TPA and PS. Scatchard analysis indicated that in the presence of TPA, the Kd value for Ca2+ binding was 51.9 microM. However, in the presence of both TPA and PS, the Kd value dropped to 0.23 microM. These results provide direct evidence that TPA activates certain isoforms of PKC by enhancing PS-dependent Ca2+ binding, thus decreasing the Kd value for Ca2+ binding to a physiological level.
Carcinogenesis 1995 Apr
PMID:The phorbol ester TPA markedly enhances the binding of calcium to the regulatory domain of protein kinase C beta 1 in the presence of phosphatidylserine. 772 72

It has recently become clear that cyclin-dependent kinase (cdk) complex regulates the cell cycle by phosphorylating Rb protein, a tumor suppressor protein. It is likely that this complex is a target of various growth factors and anti-growth factors (UV, TGF-beta etc.) in keratinocyte (KC). It has also been suggested that abnormalities in the cell cycle regulating mechanism such as increased activity of cyclin-cdk due to mutation of p53, a tumor suppressor gene, and overexpression of cyclin D may be concerned with carcinogenesis of KC. Thus, recent studies indicate that the cyclin-cdk complex is a common target of proliferation and carcinogenesis in KC.
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PMID:Cell cycle regulators in the keratinocyte (cyclin-cdk). 775 27

The profiles of the calcium-dependent protein kinase C (PKC) isozymes alpha, beta, and gamma were examined in subcellular fractions from Fischer 344 rat liver during the early stages (48 h, 96 h, 7 d, and 60 d) of diethylnitrosamine (DEN)-induced carcinogenesis, using the Solt-Farber "resistant hepatocyte" model (DEN-2-acetylaminofluorene-partial hepatectomy; DEN-AAF-PH), and then related to the presence of focal or nodular gamma-glutamyl transpeptidase (GGT)-positive morphologic changes in the liver. After DEAE and hydroxyapatite column chromatography, two peaks, immunologically identified as PKC-alpha and -beta isoforms, were detected in the liver of normal (alpha/beta ratio = 4.0) and treated rats. In DEN-AAF-PH hepatocarcinogenesis an increase in PKC-alpha expression was found after PH (+43 +/- 19% at 48 h, alpha/beta ratio = 5.1; +125 +/- 25% at 96 h, alpha/beta ratio = 4.8), whereas the PKC-beta isoform appeared less significantly modified (+11 +/- 3% at 48 h and +89 +/- 17% at 96 h). Seven and 60 days after PH, a marked increase in the PKC-alpha (+96 +/- 20% and +150 +/- 48%, respectively) and PKC-beta isoforms (+158 +/- 41%, alpha/beta ratio = 3.1 and +130 +/- 26%, alpha/beta ratio = 4.4, respectively), occurred along with the appearance of GGT-positive altered hepatic foci and nodules in the liver sections. Sham hepatectomy caused PKC-alpha and -beta isoform activities similar to those of normal controls. In contrast, saline-AAF-PH-treated rats had downregulation of PKC-alpha after PH (alpha/beta ratio = 1.8 at 96 h), possibly due to the mitoinhibitory effect of the carcinogen AAF on normal uninitiated hepatocytes. Immunohistochemical analysis with monoclonal antibodies to PKC-alpha and -beta revealed diffuse positive cytoplasmic signals in GGT-positive foci and nodules in rat liver. Taken together, these preliminary results, using the Solt-Farber model of liver carcinogenesis, suggest a role for PKC in tumor promotion. They also suggest that the PKC-alpha isoform may play a specific role in clonal expansion of DEN-initiated hepatocytes after PH.
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PMID:Analysis of calcium-dependent protein kinase C isoforms in the early stages of diethylnitrosamine-induced rat hepatocarcinogenesis. 790 65

The ability of chemicals with tumor-promoting or tumor-inhibiting activity to modulate gap junctional intercellular communication is reviewed. The two most extensively used types of assays for screening tests are (1) metabolic cooperation assays involving exchange between cells of precursors of nucleic acid synthesis and (2) dye-transfer assays that measure exchange of fluorescent dye from loaded cells to adjacent cells. About 300 substances of different biological activities have been studied using various assays. For tumor promoters/epigenetic carcinogens, metabolic cooperation assays have a sensitivity of 62% and dye-transfer assays 60%. Thirty percent of DNA-reactive carcinogens also possess the ability to uncouple cells. The complete estimation of the predictive power of these assays could not be made because the majority of the substances studied for intercellular communication effects in vitro have not yet been studied for promoting activity in vivo. Both metabolic cooperation assays and dye transfer assays respond well to the following classes of substances: phorbol esters, organochlorine pesticides, polybrominated biphenyls, promoters for urinary bladder, some biological toxins, peroxisome proliferators, and some complex mixtures. Results of in vitro assays for such tumor promoters/nongenotoxic carcinogens, such as some bile acids, some peroxides, alkanes, some hormones, mineral dusts, ascorbic acid, okadaic acid, and benz(e)pyrene, do not correlate with the data of in vivo two-stage or complete carcinogenesis. Enhancement of intercellular communication was found for 18 chemicals. Among these, cAMP, retinoids, and carotenoids have demonstrated inhibition of carcinogenesis. We examine a number of factors that are important for routine screening, including the requirement for biotransformation for some agents to exert effects on gap junctions. We also discuss the mechanisms of tumor promoter and tumor inhibitor effects on gap junctional permeability, including influences of protein kinase activation, changes in proton and Ca2+ intracellular concentrations, and effects of oxy radical production.
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PMID:Cell culture assays for chemicals with tumor-promoting or tumor-inhibiting activity based on the modulation of intercellular communication. 795 12

The amino-terminal regulatory domain portion of each protein kinase C (PKC) family member (which in the case of PKC beta 1 includes the pseudosubstrate, C1, V1 and C2 domains) plays an important role in regulating the kinase activity of the carboxyl-terminal catalytic domain. To examine the possibility that this regulatory domain region (designated 'PAT') might have biological functions independent of the catalytic domain, we have developed derivatives of R6 cells which stably express a truncated PKC beta 1 cDNA that encodes the amino-terminal 317 amino acids, including the entire regulatory domain. These R6-plPAT cells express abundant amounts of a 38 kDa protein which binds a labeled phorbol ester, but lacks protein kinase activity. In contrast to the 79 kDa PKC beta 1 holoenzyme which, when overexpressed in R6 cells, is found mostly in the cytosol, the 38 kDa PAT protein is predominantly associated with the particulate subcellular fraction. Furthermore, the PAT protein fails to show down-regulation following treatment of R6-plPAT cells with 12-O-tetradecanoylphorbol-13-acetate (TPA). Evidence is also presented that TPA-stimulated growth is suppressed in R6-plPAT cells. These findings suggest that the PKC beta 1 regulatory domain could be involved in the suppression of mitogenic signaling.
Carcinogenesis 1994 Dec
PMID:Suppression of mitogenic activity by stable expression of the regulatory domain of PKC beta. 800 Dec 56

The involvement of protein kinase C (PKC), a 12-O-tetradecanoylphorbol-13-acetate (TPA) receptor, in the transcriptional regulation of TPA-inducible genes was determined. Expression plasmids harboring full-length or kinase domain of PKC alpha and PKC delta (PKC alpha K and PKC delta K) were constructed. Transient transfection of PKC alpha K and PKC delta K into COS cells resulted in approximately 20- and 16-fold increase in phospholipid-, calcium-independent protein kinase activity. To determine the effects of overexpression of PKC alpha K and PKC delta K on the AP-1-mediated TPA-inducible genes, we transfected into COS cells the PKC alpha K or PKC delta K expression plasmids with collagenase chloramphenicol acetyltransferase (CAT) reporter construct containing one TPA responsive element (TRE), or a construct containing five synthetic TRE linked to a thymidine kinase promoter. PKC alpha K or PKC delta K overexpression resulted in a comparable increase (approximately 4-fold) in CAT activity. However, CAT activity was not increased after transfection of PKC constructs with non-TPA responsive thyroid hormone responsive elements CAT construct (delta MTV-TyRE-pCAT). We also found that deletion of the AP-1-like motif in the SV40 promoter abolished the PKC alpha K or PKC delta K-induced activity of luciferase (luc) reporter constructs. Overexpression of full-length PKC delta in COS cells also increased the activity of the CAT construct with TRE after TPA treatment. We determined the effects of overexpression of PKC alpha K and PKC delta K on transcription of the ornithine decarboxylase (ODC) gene, which has a non-AP-1 TRE. Cotransfection of PKC alpha K or PKC delta K expression plasmids with a TPA-inducible ODC luc construct (-72/+130-ODC-luc) into HeLa cells resulted in an increased luc activity. These results indicate that both PKC alpha (calcium dependent) and PKC delta (calcium independent) may mediate the transcription of TPA-inducible genes through both AP-1 and non-AP-1 sequences.
Carcinogenesis 1994 Apr
PMID:Involvement of protein kinase C in the transcriptional regulation of 12-O-tetradecanoylphorbol-13-acetate-inducible genes modulated by AP-1 or non-AP-1 transacting factors. 814 84

The Met proto-oncogene product is a tyrosine kinase receptor whose ligand is hepatocyte growth factor (HGF). The Met protein is first synthesized in the hepatocytes as a single chain precursor, or p170MET proreceptor, and is then processed to a mature heterodimer receptor consisting of an extracellular alpha subunit (p50 alpha MET) and a transmembrane beta subunit (p 145 beta MET). The beta subunit has a protein kinase domain which is activated through phosphorylation on tyrosine residue by the binding of HGF to the receptor. In order to elucidate the function of the Met gene product in hepatic disorders, we analyzed the expression and tyrosine phosphorylation of the Met protein on regeneration and carcinogenesis of the liver. For studies on carcinogenesis we used human hepatoma tissues, and for studies on regeneration we used rat hepatectomy. Two antibodies were used for western blotting; a mouse monoclonal anti-phosphotyrosine antibody, which recognizes phosphorylated tyrosine residue in proteins, and a polyclonal rabbit anti-Met antibody, which recognizes the C-terminus of both the Met beta chains and proreceptor. To compare the amount of protein in each experiment, the results of western blotting were evaluated using an image analyzing system. In experiments involving rats with partial hepatectomy, a decreased expression of the proreceptor with a decreased amount of tyrosine phosphorylation was observed within 12 hours of hepatectomy. However, there were no significant changes of the Met beta subunit during the experiment. These data suggest that the Met proreceptor is decreased in the early stages of liver regeneration. In experiments on human samples surgically removed from 18 patients with hepatocellular carcinoma, the met proteins, p 145 beta MET and p 160 MET proreceptor, were expressed both in cancer tissues (12/18, and 10/18, respectively) and in non-cancer tissues (8/18, and 15/18, respectively). From the comparative analyses of the intensity of the signals in cancerous region against those of non-cancerous region in the 18 individual cases, it was demonstrated that expression of p 160 MET proreceptor was increased in non-cancerous region more significantly than in cancerous region (p < 0.05). On the contrary, expression of p145 beta MET was increased in cancerous region more significantly than in non-cancerous region (p < 0.05), except for a few cases of poorly differentiated carcinomas in which p 145 beta MET signal was not detected. These findings suggested that a processing pathway from the proreceptor to the mature Met receptor is amplified in carcinogenesis of the liver.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Analysis of the hepatocyte growth factor receptor in regeneration and oncogenesis of hepatocytes]. 815 55

The effects of K2CrO4, H2O2, benzoyl peroxide, menadione, KBrO3 and UV365nm on gap junctional intercellular communication (GJIC) have been studied in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-sensitive Syrian hamster embryo (SHE) cell line BPNi. All agents were found to increase the level of GJIC by 50-100%. Also, in early passage SHE cells, a tendency for increased GJIC was found for the oxidative agents studied. Hydrogen peroxide was used as a model compound in the subsequent studies. The increase in GJIC was reversible, and it was not due to an increased non-junctional permeability. Hydrogen peroxide counteracted the TPA-induced decrease in GJIC, regardless of whether the cells were exposed to the compounds simultaneously or the cells were pre-exposed to TPA before addition of H2O2. The GJIC enhancement by H2O2 was slightly reduced by the addition of the hydroxyl radical scavenger dimethylsulphoxide or by the inhibition of catalase by amitrole. The cAMP/protein kinase A system is the only characterized signal transduction system that is known to increase GJIC in most cell types. Hydrogen peroxide did not increase the amount of cAMP (or cGMP) in BPNi cells, while forskolin and a phosphodiesterase inhibitor had to increase the cAMP level several-fold to affect GJIC to the same degree as the oxidative agents. Some inhibitors of protein kinase A were assayed for their ability to inhibit the increases in GJIC caused by H2O2 and forskolin. Staurosporine inhibited the forskolin-induced increase in GJIC, with much less effect on the H2O2-induced increase. H8, H88 and H89 had less effect than staurosporine on the forskolin-induced increase in GJIC. The results suggest that the cAMP/protein kinase A system may not be involved in the increase in GJIC caused by H2O2, although this cannot be completely ruled out.
Carcinogenesis 1994 Feb
PMID:Increased gap junctional intercellular communication in Syrian hamster embryo cells treated with oxidative agents. 831 32

Epidemiological and laboratory animal model studies suggest that the effect of dietary fat on colon carcinogenesis depends on the amount and its type. In the present study, we investigated the modulating effect of high-fat diets rich in omega-3, omega-6 and omega-9 fatty acids on liver, colon and small intestine mucosal ornithine decarboxylase (ODC) and tyrosine-specific protein kinase (TPK) activities and plasma, liver and colon mucosal prostaglandin E2 (PGE2) and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) levels in male F344 rats. At 6 weeks of age, groups of animals were fed the low-fat diet containing 5% corn oil (LFCO), or high-fat diets containing 23.5% corn oil (HFCO), 23.5% olive oil (HFOO) and 20.5% fish oil + 3% corn oil (HFFO). Two weeks later, all animals except the vehicle-treated groups received azoxymethane (AOM) s.c. once weekly for 2 weeks at a dose rate of 15 mg/kg body wt. All animals were killed 5 days later and liver, colon and small intestine mucosa were analyzed for ODC, TPK and PGs and plasma for PGs. Carcinogen treatment enhanced the ODC and TPK activities (P < 0.0001) in the liver and colon of animals, irrespective of dietary treatment. Dietary HFCO compared with LFCO significantly increased the ODC (P < 0.01) and membrane TPK (P < 0.05) activities in the liver and colon of carcinogen-treated animals, whereas the HFOO and HFFO diets significantly (P < 0.002) suppressed the ODC and membrane TPK (P < 0.05) activities in the liver and colon mucosa compared with the HFCO diet. Carcinogen treatment also significantly (P < 0.01) increased the PG levels in plasma, liver and colon. Feeding of the HFFO diet significantly suppressed both the basal levels and ex vivo production of PGE2 and 6-keto PGF1 alpha levels compared with the HFCO diet, whereas the HFOO diet only decreased PGE2 in liver and colon. These results thus demonstrate that high levels of corn oil in the diet increase colon and liver ODC, TPK and PGs whereas high dietary levels of fish oil and olive oil suppress these activities.
Carcinogenesis 1993 Jul
PMID:Modulating effect of amount and types of dietary fat on ornithine decarboxylase, tyrosine protein kinase and prostaglandins production during colon carcinogenesis in male F344 rats. 833 Mar 45

The response to a number of agents has been compared in two short-term assays used for the detection of virus inducers and tumor promoters: (i) induction of the EBV-DR-promoter in Raji cells, as measured by DR-CAT induction (DR-CAT test) and (ii) induction of the oxidative burst in human PMN, as measured by chemiluminescence in the presence of luminol or lucigenin (CL test). In order to validate the two assays, we have investigated the responses to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 1,2-dioctanoylglycerol (DAG), phospholipase C (PLC EC-3-1-4-30) and ionophore A23187, which are active in both systems: arachidonic acid, linoleic acid and NaCl were found active only in the CL test. Staurosporine (protein kinase inhibitor), tamoxifen (estrogen antagonist and protein kinase C inhibitor), forskolin (protein kinase A activator), R59949 (diacylglycerol kinase inhibitor), curcumin and N-acetyl-L-cysteine (scavengers of reactive oxygen species) and NaCl acted as inhibitors. A good concordance of the EC50 values of inducing substances was found between the two assays, except for A23187 and DAG, which were required at much higher concentrations in the DR-CAT test. The inhibition patterns by the panel of inhibitors revealed similarities and discrepancies in the induction pathways between the two systems, providing information on their mode of action. The two assays, which complement each other, were shown to detect a number of known or suspected EBV inducers or tumor promoters, and thus appear useful for screening of new compounds or mixtures as well as of potential antiviral and antipromoting substances.
Carcinogenesis 1993 Aug
PMID:Validation of two test systems for detecting tumor promoters and EBV inducers: comparative responses of several agents in DR-CAT Raji cells and in human granulocytes. 839 78


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