Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor.
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PMID:Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon. 767 74

The translocation of protein kinase C (PKC) isoforms PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from soluble to particulate fractions was studied in ventricular cardiomyocytes cultured from neonatal rats. Endothelin-1 (ET-1) caused a rapid ETA receptor-mediated translocation of PKC-delta and PKC-epsilon (complete in 0.5-1 min). By 3-5 min, both isoforms were returning to the soluble fraction, but a greater proportion of PKC-epsilon remained associated with the particulate fraction. The EC50 of translocation for PKC-delta was 11-15 nM ET-1 whereas that for PKC-epsilon was 1.4-1.7 nM. Phenylephrine caused a rapid translocation of PKC-epsilon (EC50 = 0.9 microM) but the proportion lost from the soluble fraction was less than with ET-1. Translocation of PKC-delta was barely detectable with phenylephrine. Neither agonist caused any consistent translocation of PKC-alpha or PKC-zeta. Activation of p42 and p44 mitogen-activated protein kinase (MAPK) by ET-1 or phenylephrine followed more slowly (complete in 3-5 min). Phosphorylation of p42-MAPK occurred simultaneously with its activation. The proportion of the total p42-MAPK pool phosphorylated in response to ET-1 (50%) was greater than with phenylephrine (20%). In addition to activation of MAPK, an unidentified p85 protein kinase was activated by ET-1 in the soluble fraction whereas an unidentified p58 protein kinase was activated in the particulate fraction.
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PMID:Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts. 780 10

We have identified the beta (beta) isoform of the 14-3-3 family of proteins as an activator of the Raf-1 protein kinase. 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins. Purified bovine brain 14-3-3 interacted specifically with both c-Raf-1 and the isolated Raf-1 kinase domain. Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells. 14-3-3 was not a preferred Raf-1 substrate in vitro and did not significantly affect Raf-1 kinase activity in a purified system. However, in cell-free extracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain. The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase domain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation of PC12 cells. We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain.
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PMID:Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins. 788 72

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 activities and expression of protein kinase C isoenzymes were examined in glomerular mesangial cells cultured under high glucose conditions. Exposure of cells to high glucose concentrations (27.8 mmol/l) for more than 3 days resulted in a significant elevation of protein kinase C activities in the membrane fraction. Of the protein kinase C isoenzymes, the levels of protein kinase C alpha significantly increased in the membrane fraction after 3 days of exposure to glucose, and protein kinase C zeta increased after 5 days of exposure. Levels of protein kinase C delta and epsilon remained unchanged and protein kinase, C beta and gamma were not detected. These results indicate that protein kinase C alpha and zeta are translocated under high glucose conditions possibly through different mechanisms.
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PMID:Translocation of protein kinase C alpha and zeta in rat glomerular mesangial cells cultured under high glucose conditions. 798 87

The involvement of protein kinase C (PKC) in the mechanism of chemotaxis and invasiveness of human melanoma has been studied in 6 clones of 665/2 cell line characterized by a different integrin profile, differentiation grade and in vitro invasive ability. The levels of total protein kinase C activity revealed a direct correlation with the chemotactic and invasive ability of these clones. Protein kinase C inhibitors, sphingosine and staurosporine, reduced chemotaxis and invasiveness of the highly invasive clone 2/60, while 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) was ineffective. Immunofluorescence analysis revealed high levels of protein kinase C alpha in clone 2/60, while the less invasive clone 2/21 expressed low levels of protein kinase C alpha and beta, but surprisingly appreciable levels of protein kinase C gamma. Downregulation with phorbol 12-myristate 13-acetate (TPA) did not affect invasiveness of clone 2/60 unless the compound was present during the assay. H7 strongly increased invasiveness of clone 2/21 and was able to reverse the inhibitory effect of TPA on clone 2/60. Preliminary experiments showed higher levels of diacylglycerol in clones with lower protein kinase C, suggesting a constitutive downregulation of the enzyme in low invasive clones. Our results support a role for protein kinase C in the invasion process, but point out the complexity of the mechanism which might involve the proteolytic fragment of the enzyme, protein kinase M.
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PMID:Effect of protein kinase C inhibitors on invasiveness of human melanoma clones expressing different levels of protein kinase C isoenzymes. 815 65

Platelet-derived growth factor (PDGF) is a potent mitogen for a variety of cells. The calcium/phospholipid-dependent protein kinase C (PKC) represents a major signal transduction pathway for many growth stimuli including PDGF. Various isoforms of PKC are differentially expressed in the same or in different cells and tissues, and diverse stimuli may selectively activate one or more PKC isoforms. We studied the effect of PDGF on DNA synthesis and on the activity of PKC in human mesangial cells and vascular pericytes in the glomerular microvascular bed. PKC activity was measured as the amount of phosphorylated myelin basic protein-derived peptide substrate in the absence and presence of an inhibitor, a peptide spanning the pseudosubstrate region of PKC. PDGF (15 ng/ml) stimulated PKC activity within 5 min, and the effect was sustained for 60 min. Pretreatment of mesangial cells with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of PKC, abolished the stimulation of PKC and DNA synthesis in response to PDGF. This effect of H-7 was specific, because H-7 did not inhibit the tyrosine phosphorylation of the PDGF receptor in vivo when added to the cells or the in vitro kinase activity in the PDGF beta-receptor immunoprecipitates. Utilizing isotype-specific antibodies against PKC-alpha, -beta, or -gamma for immunoprecipitation of PDGF-treated mesangial cell extracts, followed by assay of PKC activity, we demonstrated the activation of PKC-alpha only. Northern blot analysis of mRNA prepared from mesangial cells also revealed two transcripts, 3.7 kb and 1.8 kb, that hybridized with cDNA specific for PKC-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Involvement of PKC-alpha in PDGF-mediated mitogenic signaling in human mesangial cells. 823 43

Protein kinase C (PKC) family members were examined in PC-12 rat pheochromocytoma cells to evaluate their role in the action of nerve growth factor (NGF). Immunoblot analysis of whole cell lysates using antibodies against various PKC isoforms revealed that PC-12 cells contained PKC-alpha, -delta, -epsilon and zeta. Assay of the protein kinase activity in these different anti-PKC immunoprecipitates demonstrated that NGF stimulated the kinase activity of PKC-epsilon, but not PKC-alpha, -delta and -zeta. Both histone phosphorylation and autophosphorylation of PKC-epsilon were increased by treatment of PC-12 cells with NGF. This increased phosphorylation observed in vitro is rapid, occurring maximally at 2.5 min and declining thereafter. Moreover, this effect of NGF is dose-dependent over physiological concentrations of the growth factor. Although the mechanistic basis for this specificity in PKC activation is not clear, NGF acutely stimulated the production of diacylglycerol without causing corresponding changes in intracellular Ca2+ concentrations. These results suggest that NGF may selectively stimulate the Ca(2+)-insensitive epsilon isoform of PKC by a phosphatidylinositol-independent mechanism.
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PMID:Nerve growth factor activates calcium-insensitive protein kinase C-epsilon in PC-12 rat pheochromocytoma cells. 824 Feb 90

Stimulation of human peripheral blood lymphocytes via T cell receptor/CD3 complex resulted in a bimodal activation of protein kinase(s) C (PKC). Within 10 min of stimulation PKC-alpha was translocated to, and thus activated in, the plasma membranes of human lymphocytes, followed by a fast dissociation of this isotype from the plasma membrane. This short term activation and translocation PKC-alpha proved to be cyclosporin A (CsA) insensitive. After 90 min of stimulation PKC-beta was translocated to and remained bound to the plasma membranes for up to 4 h. Preincubation of human lymphocytes with 200 ng/ml CsA specifically and completely abolished the sustained activation of PKC-beta. Neither the phorbol ester-induced direct activation of PKC nor the specific activity of the plasma membrane-bound enzyme was influenced by CsA, suggesting that a signal transduction pathway leading to sustained activation of PKC-beta was influenced by the immunosuppressive agent. In fact, CsA inhibited, in a concentration-dependent manner, the activation of lysophosphatid acyltransferase-catalyzed elevated incorporation of cis-polyunsaturated fatty acids into plasma membrane phospholipids. While interleukin-2 (IL-2) synthesis and cellular proliferation were completely inhibited by 200 ng/ml CsA in BMA 030- or BMA 031-stimulated cells, expression of high-affinity IL-2 receptors was not influenced by the immunosuppressive drug. These results suggest that synthesis and expression of high-affinity IL-2 receptors might be regulated by a signal-transducing pathway involving activation and translocation of PKC-alpha. Lysophosphatid acyltransferase-catalyzed incorporation of cis-polyunsaturated fatty acids might represent another mechanism of signal transduction implicated in the activation and translocation of PKC-beta, which is specifically inhibited by CsA. Neutralization of PKC-beta by introducing anti-PKC-beta antibodies prevented IL-2 synthesis and proliferation in stimulated human lymphocytes. The results suggest a possible link between activation of PKC-beta and regulation of IL-2 synthesis in activated human lymphocytes. Thus, inhibition of the activation and translocation of PKC-beta by CsA may result in inhibition of IL-2 gene expression in human lymphocytes.
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PMID:Cyclosporin A inhibits T cell receptor-induced interleukin-2 synthesis of human T lymphocytes by selectively preventing a transmembrane signal transduction pathway leading to sustained activation of a protein kinase C isoenzyme, protein kinase C-beta. 825 20

Skeletal muscle is one of the first tissues to become insulin resistant in genetically obese rodents. The activation of protein kinase-C (PKC) in rat skeletal muscle is mediated by insulin stimulation of diacylglycerol (DAG) levels. Defects in the activation of PKC in the heart and liver of obese Zucker rats indicate that an abnormality in either stimulation of DAG or PKC occurs in obese tissues. DAG levels were significantly increased in soleus muscle from 15- to 19-week-old obese (fa/fa) Zucker rats. PKC activity was diminished in soleus muscle from fa/fa rats. Decreased levels of PKC alpha and -beta activity wer enoted after resolution of common PKC isozymes (Ca2+ and phospholipid dependent) by hydroxyapatite chromatography. Immunoreactivity of PKC-alpha, -beta, and -epsilon also indicated that their levels are diminished in fa/fa soleus muscle by 70-90%. To determine at which level down-regulation occurs (i.e. gene expression or protein turnover), mRNA levels were examined by Northern blot analysis of total RNA. PKC alpha and -beta levels were diminished in Zucker obese soleus muscle compared to soleus from Zucker lean control (fa/-) animals, and PKC epsilon mRNA was not detected on the same blots. The transcript size for PKC beta mRNA in Zucker soleus muscle was unique. Both lean and obese Zucker muscle tissues expressed three transcripts that hybridized with the full-length PKC beta cDNA probes, with sizes ranging between 2.5-1.7 kilobases. Levels of all PKC beta transcripts were significantly decreased in obese Zucker tissues. Thus, levels of common PKC isozyme mRNA, protein, and enzyme activity in soleus muscle of the obese Zucker rat are decreased even though levels of the endogenous PKC activator DAG are elevated. The decreased levels of PKC may be related to the etiology of insulin resistance in skeletal muscle.
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PMID:Decreased expression of protein kinase-C alpha, beta, and epsilon in soleus muscle of Zucker obese (fa/fa) rats. 840 76


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