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)

The addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.
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PMID:Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity. 132 19

Activation of the respiratory burst in the monocytic cell line U937 by cross-linking human 40-kDa FcR for IgG (Fc gamma RII) with the IgG1 mAb, CIKM5, is dependent on the maturation state of the cell. Addition of anti-Fc gamma RII to undifferentiated cells does not activate the respiratory burst but differentiation with human rIFN-gamma (200 U/ml) for 13 to 15 days results in maximal stimulation by this agonist, with half-maximal responses in cells incubated for 10 to 12 days. During maturation the development of responsiveness to cross-linking Fc gamma RII occurs later than the development of responsiveness to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (maximal responses at 7 to 9 days), or the chemotactic peptide FMLP (half-maximal responses at 7 to 9 days). The late development of maximal Fc gamma RII responses is not associated with either increased Fc gamma RII expression, enhanced calcium mobilization induced by anti-Fc gamma RII, changes in protein kinase C activity (PKC) or a switch in PKC isotype expression. Activation of the respiratory burst via Fc gamma RII may not be mediated by activation of PKC as the kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride inhibited the Fc gamma RII response by less than 20% at concentrations which inhibit the 12-O-tetradecanoylphorbol-13-acetate-induced respiratory burst by more than 80%. IFN-gamma U937 cells did not metabolize incorporated arachidonate into eicosanoids when stimulated with anti-Fc gamma RII, suggesting that eicosanoids do not mediate activation of the respiratory burst, and this was confirmed by the lack of inhibition by the specific 5'-lipoxygenase and glutathione S-transferase inhibitor, piriprost, and the cyclo-oxygenase inhibitor, indomethacin. In addition there was no significant release of radiolabeled arachidonate in response to anti-Fc gamma RII. The response to anti-Fc gamma RII is inhibited by pertussis toxin, suggesting that signal transduction is via a GTP-binding protein. Agents that elevate intracellular cAMP increased the magnitude of the cAMP transients stimulated by anti-Fc gamma RII and also inhibited the respiratory burst. FMLP responses showed a similar pattern of sensitivity to this range of inhibitors, suggesting that both Fc gamma RII and FMLP receptor share common regulatory mechanisms. However, the termination of the respiratory burst activated via Fc gamma RII and FMLP receptor is independently regulated, in that after FMLP-induced activation there is no subsequent inhibition of the Fc gamma RII-mediated response and vice versa.
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PMID:Differentiation-linked activation of the respiratory burst in a monocytic cell line (U937) via Fc gamma RII. A study of activation pathways and their regulation. 165 5

Diacylglycerol (DG) and its analogue phorbol 12-myristate 13-acetate (PMA) activate the ubiquitous phospholipid/Ca2(+)-dependent protein kinase, protein kinase C (PKC), and cause it to become tightly associated with membranes. DG is produced transiently as it is rapidly metabolized by DG kinase (DGK) to phosphatidic acid. Phorbol esters such as PMA are not metabolized and induced a prolonged membrane association of PKC. Until recently, PKC was the only known phorbol ester receptor. We have shown that a novel brain-specific cDNA, neuronal chimaerin (NC), expressed in Escherichia coli, binds phorbol ester with high affinity, stereospecificity and a phospholipid requirement [Ahmed, Kozma, Monfries, Hall, Lim, Smith & Lim (1990) Biochem. J. 272, 767-773]. The proteins NC, PKC and DGK possess a cysteine-rich domain with the motif HX11/12CX2CXnCX2CX4HX2CX6/7C (where n varies between 12 and 14). The partial motif, CX2CX13CX2C, is present in a number of transcription factors including the steroid hormone receptors and the yeast protein, GAL4, in which zinc plays a structural role of co-ordinating cysteine residues and is essential for DNA binding (protein-nucleic acid interactions). The cysteine-rich domain of NC and PKC is required for phospholipid-dependent phorbol is required for phospholipid-dependent phorbol ester binding, suggesting an involvement of this domain in protein-lipid interactions. We have expressed recombinant NC, PKC and DGK glutathione S-transferase and TrpE fusion proteins in E. coli to investigate the relationship between the cysteine-rich motif, HX11/12CX2CX10-14CX2CX4HX2CX6/7C, zinc and phorbol ester binding. The cysteine-rich domain of NC, PKC and DGK bound 65Zn2+ but only NC and PKC bound [3H]phorbol 12,13-dibutyrate. When NC and PKC were subjected to treatments known to remove metal ions from GAL4 and the human glucocorticoid receptor, phorbol ester binding was inhibited. These data provide evidence for the role of a zinc-dependent structure in phorbol ester binding.
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PMID:The cysteine-rich domain of human proteins, neuronal chimaerin, protein kinase C and diacylglycerol kinase binds zinc. Evidence for the involvement of a zinc-dependent structure in phorbol ester binding. 166 Feb 66

The protein predicted by the sequence of the human pim-1 proto-oncogene shares extensive homology with known serine/threonine protein kinases, and yet the human Pim-1 enzyme has previously been reported to exhibit protein tyrosine kinase activity both in vitro and in vivo. Recently a new class of protein kinases has been identified which exhibits both protein-serine/threonine and protein-tyrosine kinase activities. We therefore investigated the possibility that the human Pim-1 kinase likewise possesses such bifunctional enzymatic phosphorylating activities. A full-length human pim-1 cDNA was subcloned into the bacterial vector pGEX-2T and the Pim-1 protein expressed as a fusion product with bacterial glutathione S-transferase (GST). The hybrid GST-Pim-1 fusion protein was affinity purified on a glutathione-Sepharose column prior to treatment with thrombin for cleavage of the Pim-1 protein from the transferase. Pim-1 was purified and the identity of recombinant protein confirmed by amino-terminal sequence analysis. Pim-1 was tested for kinase activity with a variety of proteins and peptides known to be substrates for either mammalian protein-serine/threonine or protein-tyrosine kinases and was found to phosphorylate serine/threonine residues exclusively in vitro. Both the Pim-1-GST fusion protein and the isolated Pim-1 protein exhibited only serine/threonine phosphorylating activity under all in vitro conditions tested. Pim-1 phosphorylated purified mammalian histone H1 with a Km of approximately 51 microM. Additionally, Pim-1 exhibited low levels of serine/threonine autophosphorylating activity. These observations place the human Pim-1 in a small select group of cytoplasmic transforming oncogenic kinases, including the protein kinase C, the Raf/Mil, and the Mos subfamilies, exhibiting serine/threonine phosphorylating activity.
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PMID:Recombinant human pim-1 protein exhibits serine/threonine kinase activity. 171 13

An electrophile-responsive element (EpRE) in the 5' flanking region of the mouse glutathione S-transferase Ya subunit gene was recently found to be responsible for the induction of gene expression by xenobiotics that contain or acquire by metabolism an electrophilic center. We now find that this EpRE is composed of two adjacent 9-base-pair motifs related in sequence to the AP-1 binding site, a transcriptional enhancer originally identified as the phorbol 12-myristate 13-acetate (PMA) response element and known to be regulated by the binding of protein products of c-jun and c-fos genes. Synthetic oligonucleotides representing each of the AP-1-like binding sites of the EpRE and the AP-1 site consensus sequence were prepared and assayed for their enhancer activity and inducibility by tert-butylhydroquinone, beta-naphthoflavone, and PMA. Single AP-1-like sequences showed a lower enhancer activity than an AP-1 consensus sequence and no inducibility. Two adjacent AP-1-like sites were found to act synergistically and to confer inducibility beyond that observed for a single AP-1 consensus sequence. Examination of the PMA-responsive region of a number of genes shows the presence of adjacent AP-1-like sites and indicates that the structure of the EpRE found in the Ya gene may occur more generally and may be important in regulating the magnitude of the electrophilic response. The present study demonstrates the binding and transactivation of the EpRE by Jun and Fos and indicates that the AP-1 site is part of the EpRE. The induction by PMA or tert-butylhydroquinone appears to be independent of protein kinase C activity since it is not affected by inhibitors of this enzyme.
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PMID:Two adjacent AP-1-like binding sites form the electrophile-responsive element of the murine glutathione S-transferase Ya subunit gene. 173 39

1. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) produces atrophy, morphological changes, impaired spermatogenesis, and epididymal lesions in testis of experimental animals. The effects of TCDD administration to male rats on various parameters in the testes were examined. 2. Nine days after TCDD administration, significant decreases in body and testes weights occurred. However, the testes weight as a percent of body weight was higher in treated than control animals. 3. An increase in lipid peroxidation (content of thiobarbituric acid reactive substances) occurred in conjunction with the decrease in testicular weights. 4. TCDD administration produced a 3-fold increase in protein kinase C activity, small but significant decrease is superoxide dismutase and glutathione peroxidase activities, and no effect on catalase, glutathione reductase or glutathione S-transferase activities in the testes. 5. Nine days after treatment with TCDD, in the testes the iron content of whole tissue and cytosol increased while a decrease in microsomal iron was observed. The copper content of mitochondria and microsomes decreased with a corresponding increase in cytosol copper content. A small increase in the zinc content of whole testes occurred. 6. The data indicate that testicular atrophy due to TCDD may be associated with lipid mobilization and peroxidation.
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PMID:2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced alterations in lipid peroxidation, enzymes, and divalent cations in rat testis. 324 26

cDNA clones encoding the third member of the RAC protein kinase family, termed RAC-PK gamma, were isolated from a rat brain cDNA library. The deduced amino acid sequence of RAC-PK gamma was highly related to those of previously identified family members, RAC-PK alpha and beta, that have a pleckstrin homology domain and a protein-serine/threonine kinase catalytic domain at the amino- and carboxyl-terminal regions, respectively. Northern blot analysis indicated that RAC-PK gamma was expressed abundantly in brain and testis. Specific activities of RAC-PK alpha, beta, and gamma purified from transfected COS-7 cells were similar when measured by using myelin basic protein as a phosphate acceptor. Analysis using fusion proteins of glutathione S-transferase revealed that the pleckstrin homology domain of the three subtypes of RAC-PK associate with both protein kinase C subspecies and beta gamma subunits of G proteins. These results suggest that the pleckstrin homology domains of RAC protein kinase family could associate more than one protein to regulate the activity and/or intracellular distribution of this enzyme family by different ways.
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PMID:Molecular cloning and characterization of a new member of the RAC protein kinase family: association of the pleckstrin homology domain of three types of RAC protein kinase with protein kinase C subspecies and beta gamma subunits of G proteins. 748 43

Although the human immunodeficiency virus type 1 (HIV-1) nef gene still has no precisely defined function, in vivo studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to identify cellular proteins capable of binding to Nef, the HIV-1LAI nef gene product was expressed in the bacterial vector pGEX-2T as a glutathione S-transferase (GST)-Nef fusion protein. Deletion mutants corresponding to 86 and 35 N-terminal residues of the Nef protein were prepared. The GST-Nef constructs were used to identify cellular kinases capable of interacting with Nef. After incubation with a Jurkat cell lysate, the GST-Nef constructs immobilized on glutathione-agarose beads bound to cellular kinase(s) and were phosphorylated at three sites in vitro: one on threonine at position 15, one on serine between residues 1 and 35, and one on threonine between residues 36 and 86. The Nef-phosphorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase was mainly in the membrane-associated fraction. These results suggest that kinase(s) of the PKC family are specifically bound to and phosphorylate Nef in vitro. The interaction of Nef with cellular kinases and its phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis.
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PMID:In vitro binding and phosphorylation of human immunodeficiency virus type 1 Nef protein by serine/threonine protein kinase. 754 Jan 94

Phosphorylation may play a role in modulating multidrug resistance by P-glycoprotein (P-gp). The linker region between the two homologous halves of human P-gp harbors several serine residues which are phosphorylated by protein kinase C (PKC) in vitro. We used the glutathione S-transferase gene fusion system to express and purify a series of fusion proteins containing the relevant portion (residues 644-689) of the linker region of the human MDR1 gene product. The fusion proteins were subjected to in vitro phosphorylation and phosphopeptide mapping analysis to identify specific phosphorylation sites. On the basis of a mutational strategy in which individual serine residues were systematically replaced with nonphosphorylatable alanine residues, Ser-661 and Ser-667 were identified as major PKC sites and Ser-683 was identified as a minor PKC site. Ser-661 and Ser-667 were also found to be the primary sites of phosphorylation for a novel membrane-associated P-gp specific kinase isolated from the multidrug-resistant KB-V1 cell line. Individual phosphorylation sites were recognized independently of each other. These data show that the linker region of P-gp represents a target for multisite phosphorylation not only for PKC but also for the P-gp specific V1 kinase. Specific serine phosphorylation sites are identified, and evidence is presented that the V1 kinase has a specificity which overlaps, but is more restricted than, that of PKC. In addition, these studies also suggest that the use of GST fusion peptides may be applicable for the analysis of multisite and ordered protein phosphorylation in other systems.
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PMID:Bacterial expression of the linker region of human MDR1 P-glycoprotein and mutational analysis of phosphorylation sites. 757 13

Cholecystokinin (CCK) has recently been shown to activate mitogen-activated protein (MAP) kinase in rat pancreatic acini [Duan and Williams, Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G401-G408, 1994]. To evaluate the mechanism of MAP kinase activation, we studied the effects of CCK on MAP kinase kinase (MEK) in rat pancreatic acini. Two forms of MEK were identified by immunoblotting, using antibodies specific to MEK1 and MEK2. MEK activity in acinar extracts and after immunoprecipitation with anti-MEK was detected using a recombinant fusion protein, glutathione S-transferase-MAP kinase, as a substrate. MEK activity rapidly increased after stimulation of acini by CCK, with significant stimulation at 1 min and a maximal effect at 5 min, followed by a slow decline to slightly above control levels after 30 min. The threshold concentration of CCK was approximately 10 pM, and the maximal effect was induced by 1 nM CCK, which increased MEK activity by 120%. In addition to CCK, bombesin and carbachol, but not secretin or vasoactive intestinal peptide, enhanced MEK activity. Phorbol ester mimicked the effect of CCK, whereas ionomycin and thapsigargin failed to activate MEK. We further studied the activation of Ras, an important component leading to activation of MEK by growth factors. Ras in acini was immunoprecipitated and identified by Western blotting. CCK and 12-O-tetradecanoylphorbol-13-acetate stimulated the incorporation of GTP into Ras, a requirement for its activation, reaching a maximum at 10 min of approximately 120% over control. In conclusion, the activation of MAP kinase by CCK can be explained by activation of MEK and may involve the activation of Ras by a protein kinase C-dependent mechanism.
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PMID:Activation of MAP kinase kinase (MEK) and Ras by cholecystokinin in rat pancreatic acini. 761 6


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