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)

Diacylglycerol (DAG) occupies a central position in the synthesis of complex lipids and also has important signaling roles. For example, DAG is an allosteric regulator of protein kinase C, and the cellular levels of DAG may influence a variety of processes including growth and differentiation. We previously demonstrated that human endothelial cells derived from umbilical vein express growth-dependent changes in their basal levels of diacylglycerol and diacylglycerol kinase activity (Whatley, R. E., Stroud, E. D., Bunting, M., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1993) J. Biol. Chem. 268, 16130-16138). To further explore the role of diacylglycerol metabolism in endothelial responses, we used a degenerate reverse transcription-polymerase chain reaction method to identify diacylglycerol kinase isozymes expressed by human endothelial cells. We report the isolation of a 3.5-kilobase cDNA encoding a novel diacylglycerol kinase (hDGKzeta) with a predicted molecular mass of 103.9 kDa. Human DGK zeta contains two zinc fingers, an ATP binding site, and four ankyrin repeats near the carboxyl terminus. A unique feature, as compared with other diacylglycerol kinases, is the presence of a sequence homologous to the MARCKS phosphorylation site domain. From Northern blot analysis of multiple tissues, we observed that hDGKzeta mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGKzeta cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived from a unique sequence in hDGK zeta. The transfected cells also express increased diacylglycerol kinase activity, which is not altered in the presence of R59949, an inhibitor of human platelet DGK activity. The hDGKzeta displays stereoselectivity for 1,2-diacylglycerol species in comparison to 1,3-diacylglycerol, but does not exhibit any specificity for molecular species of long chain diacylglycerols.
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PMID:Molecular cloning and characterization of a novel human diacylglycerol kinase zeta. 862 88

Diacylglycerol (DAG) is a second messenger that activates protein kinase C and also occupies a central role in phospholipid biosynthesis. Conversion of DAG to phosphatidic acid by DAG kinase regulates the amount of DAG and the route it takes. We used degenerate primers to amplify polymerase chain reaction products from cDNA derived from human endothelial cells. A product with a novel sequence was identified and used to clone a 2.6-kilobase cDNA from an endothelial cell library. When transfected with a truncated version of this cDNA, COS-7 cells had a marked increase in DAG kinase activity, which demonstrated clear selectivity for arachidonoyl-containing species of diacylglycerol. The open reading frame of this clone has 567 residues with a predicted protein of 64 kDa. This enzyme, which we designated DGK epsilon, has two distinctive zinc finger-like structures in its N-terminal region, but does not contain the E-F hand motifs found in several other mammalian DGKs. The catalytic domain of DGK epsilon, which is related to other DGKs, contains two ATP-binding motifs. Northern blotting demonstrated that DGK epsilon is expressed predominantly in testis. This unique diacylglycerol kinase may terminate signals transmitted through arachidonoyl-DAG or may contribute to the synthesis of phospholipids with defined fatty acid composition.
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PMID:Molecular cloning of a novel human diacylglycerol kinase highly selective for arachidonate-containing substrates. 862 89

In Dictyostelium, an ordered actin and myosin assembly-disassembly process is necessary for proper development, differentiation, and motility (Yumura S, Fukui F, 1985, Nature 314(6007): 194-196; Ravid S, Spudich JA, 1989, J Biol Chem 264(25): 15144-15150), and phosphorylation of myosin heavy chains has been implicated in the myosin assembly-disassembly process (Egelhoff TT, Lee RJ, Spudich JA, 1993, Cell 75(2):363-371). The developmentally expressed 84-kDa myosin heavy-chain kinase (MHCK) from Dictyostelium (Ravid S, Spudich JA, 1992, Proc Natl Acad Sci USA 89(13):5877-5881) is known to be a member of the protein kinase C (PKC) family. We have observed a rather striking homology between the large central domain of MHCK and the catalytic domain of diacylglycerol kinase (DGK), indicating that MHCK is in fact a gene fusion between a DGK and a PKC, possessing two separate kinase domains. The combined diacylglycerol kinase/myosin heavy-chain kinase (DGK/MHCK) may therefore have dual functionality, possessing the ability to phosphorylate both protein and lipid. We present a hypothesis that DGK/MHCK can antagonize both actin and myosin assembly, as well as other cellular processes, by coordinated down regulation of signaling via myosin heavy-chain kinase activity and diacylglycerol kinase activity.
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PMID:Developmentally expressed myosin heavy-chain kinase possesses a diacylglycerol kinase domain. 884 69

The involvement of protein kinase C (PKC) in the regulation of Na(+)-dependent and -independent hypoxanthine transport was investigated by exposing confluent monolayers of LLC-PK1 renal epithelia cells to the PKC activator, phorbol 12-myristate 13-acetate (PMA). Chronic exposure (> 2 h) of LLC-PK1 monolayers to 16 nM PMA resulted in approximately 75% inhibition of Na(+)-dependent hypoxanthine influx occurring maximally at 8 h and persisting for 72 h. In contrast, PMA had little effect on Na(+)-independent hypoxanthine influx at 8 h, but longer exposure resulted in stimulation of influx (approximately 3-fold) that peaked at 24 h and thereafter declined to control levels at 72 h. The effects of PMA were dose-dependent and were associated with changes in Vmax of transport (2-4-fold) with no significant change in apparent K(m). 4 alpha-Phorbol, a phorbol ester that does not activate PKC, had no effect on hypoxanthine transport by LLC-PK1 cells. The diacylglycerol kinase inhibitor, R59022 (10 microM), partially inhibited (28%) Na(+)-dependent hypoxanthine influx. In addition, the PMA-induced effects on hypoxanthine transport were reversed by Ro-31-8220 (1 and 5 microM) and calphostin C (50 nM), potent and selective inhibitors of PKC. The increase in Na(+)-independent hypoxanthine influx following exposure to PMA was blocked by the protein synthesis inhibitor, cycloheximide (20 microM), and correlated with an increase in LLC-PK1 cell proliferation. The PMA-induced decrease in Na(+)-dependent hypoxanthine transport was independent of PMA effects on cell proliferation and not dependent on protein synthesis. These results are consistent with the proposal that the PMA-induced effects on hypoxanthine transport are due to PKC activation.
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PMID:Regulation of nucleobase transport in LLC-PK1 renal epithelia by protein kinase C. 891 86

Phorbol 12-myristate 13-acetate, a potential stimulator of protein kinase C (PKC), inhibited taurine uptake in rat astrocytes. This effect was mimicked by 1-oleoyl-2-acetyl-sn-glycerol, an endogenous stimulator of PKC, and by r-59949, an inhibitor of diacylglycerol kinase. Maximal inhibition was obtained at microM phorbol 12-myristate 13-acetate (PMA) after 1 h of treatment. This effect was prevented by pretreatment of the cells with chelerythrine, a potent and selective inhibitor of PKC. The transport of beta-alanine, an amino acid that shares the same transporter as taurine, was inhibited to a comparable extent. The effect of PMA was potentiated by cotreatment of the cells with thapsigargin or the Ca2+ ionophore A-23187. However, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N1,N1-tetraacetic acid and verapamil did not prevent the PMA effect. Pretreatment of the cells with calmodulin antagonists W-13 or calmidazolium, prevented the PMA-induced inhibition of taurine uptake. This inhibition was not affected by cycloheximide, actinomycin D, colchicine, or cytochalasin D. The Na(+)-to-Cl(-)-to-taurine coupling ratio was unaffected. Dimethyl amiloride, a selective inhibitor of Na+/H+ antiport, was unable to prevent the effects of PMA. These effects were associated with a decrease in the maximal velocity and an increase in the Michaelis-Menten constant.
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PMID:Regulation of taurine transport in rat astrocytes by protein kinase C: role of calcium and calmodulin. 892 29

We have previously shown that 24,25-(OH)2D3 plays a major role in resting zone (RC) chondrocyte differentiation and that this vitamin D metabolite regulates protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 24,25-(OH)2D3 to stimulate PKC activation. Confluent, fourth passage RC cells from rat costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of RC cultures with 24,25-(OH)2D3 for 90 min produced a dose-dependent increase in diacylglycerol (DAG). Addition of R59022, a diacylglycerol kinase inhibitor, significantly increased PKC activity in cultures treated with 24,25-(OH)2D3. Addition of dioctanoylglycerol (DOG) to plasma membranes isolated from RC increased PKC activity 447-fold. Addition of pertussis toxin or cholera toxin to control cultures elevated basal PKC activity. When added together with 10(-9) M 24,25-(OH)2D3, there was an additive effect on PKC activity but in cultures treated with 10(-8) M 24,25-(OH)2D3, only the hormone-dependent stimulation of PKC was observed. The phospholipase C inhibitor, U73-122, had no effect on PKC activity, indicating that the DAG produced in response to 24,25-(OH)2D3 is not derived from phosphatidylinositol. Addition of the tyrosine kinase inhibitor, genistein, also had no effect on 24,25-(OH)2D3-stimulated PKC, further supporting the hypothesis that phospholipase C is not involved in the mechanism and that phospholipase D is responsible for the increase in DAG production. Phospholipase A2 inhibitors, quinacrine and AACOCF3, and the cyclooxygenase inhibitor indomethacin increased PKC activity in the RC cultures. Exogenous PGE2, one of the downstream products of phospholipase A2 action, inhibited PKC activity. These results suggest that 24,25-(OH)2D3 regulates PKC activity by two distinct phospholipid-dependent mechanisms: production of DAG via phospholipase D and inhibition of the production of PGE2 via inhibition of phospholipase A2 and cyclooxygenase.
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PMID:24,25-(OH)2D3 regulates protein kinase C through two distinct phospholipid-dependent mechanisms. 895

Treatment of aspirinated platelets with the electroneutral K+/H+ exchanger nigericin induces a decrease in intraplatelet pH as measured with the intracellular fluorescent indicator BCECF. Under these conditions, the proton permeability of the plasma membrane is unaffected. The addition of thrombin induces a rapid partial recovery of pH(i), which is completely abolished by the Na+/H+ exchanger inhibitor NHA. The effect is also evident in the presence of the PKC inhibitors GF 109203X or staurosporine and in the absence of both external (EGTA-chelated) and internal (BAPTA-chelated) Ca2+. This makes the thrombin-induced activation of the exchanger independent of the involvement of the hitherto described activators, namely PKC and the increase in [Ca2+]i, as well of the recently reported activator arachidonic acid [Cavallini, L., Coassin, M., Borean, A., and Alexandre, A. (1996) Biochem. J. 319, 567-574], whose production requires a high [Ca2+]i. The thrombin-dependent recovery of pH(i) is prevented by the phospholipase C inhibitor ET 18 O-CH3 and is mimicked by the addition of the permeable diglyceride dioctanoyl glycerol (DiC8) exogenously supplied. The effect of thrombin and DiC8 is unaffected by inhibition of diacylglycerol lipase and diacylglycerol kinase. These experiments identify diglyceride as a novel activator of the Na+/H+ exchanger in platelets.
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PMID:Diacylglycerol mediates the thrombin-induced, protein kinase C and Ca2+ independent activation of the Na+/H+ exchanger in platelets. 900 May 21

The presence and subcellular localization of the Ca2+-dependent protein kinase C (PKC) isoforms alpha and beta were investigated in freshly isolated adult rat cardiac ventricular myocytes. PKC activity was measured in cytosolic and particulate fractions prepared from control myocytes and those treated with either phorbol ester (phorbol 12-myristate 13-acetate, PMA) or a permeant synthetic diacylglycerol analog (1-oleoyl-2-acetylglycerol, OAG) in the absence or presence of an inhibitor of diacylglycerol kinase activity, compound R59022. Preliminary studies detected no Ca2+-/phospholipid-dependent histone kinase activity in either subcellular fraction. To reproducibly observe Ca2+-/phospholipid-dependent protein kinase activity, partial purification using a MonoQ HR 5/5 column and the presence of the peptide inhibitor of the cAMP-dependent protein kinase were essential. MonoQ chromatography of cytosolic and particulate fractions resulted in three peaks of Ca2+/phospholipid-dependent protein kinase activity. In the cytosolic fraction a large peak of activity eluted at 230-300 mM NaCl. Isoform-specific antisera indicated both PKC alpha and PKC beta were present. In the particulate fraction two peaks of Ca2+-/phospholipid-dependent protein kinase activity, both containing PKCa immunoreactivity, were observed. The larger peak eluted at 230-300 mM NaCl. In addition, a peak eluting at lower salt concentrations contained a Ca2+-/phospholipid-independent histone kinase activity. This peak of kinase activity contained PKC alpha immunoreactive bands of 80- and 50-kDa. The 80-kDa band was the holoenzyme of PKC alpha whereas the band of lower molecular mass was likely a proteolytic fragment. In both cytosolic and particulate fractions, the peak of kinase activity eluting at 230-300 mM NaCl contained PKC alpha in the form of an 80-kDa doublet; this suggested the presence of autophosphorylated PKC. Incubation of the myocytes with PMA, but not OAG, resulted in translocation of PKC from the cytosolic to the particulate fraction. Curiously, a transient decrease in PKC activity was observed in both subcellular fractions following treatment with either OAG or ethanol (1%). Results from this study show that freshly isolated adult rat cardiac ventricular myocytes contain both PKC alpha and PKC beta, and that these isoforms translocate to the particulate fraction in response to treatment with PMA, but not OAG.
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PMID:Characterization of calcium-dependent forms of protein kinase C in adult rat ventricular myocytes. 904 17

beta-Hexosaminidases (Hex) A and B promote mitogenesis via airway smooth muscle (ASM) mannose receptor. The objective of this study was to elucidate the contribution of protein kinase C (PKC) in Hex-induced mitogenesis in ASM cells (ASMC). Exposure of ASMC to Hex caused increases in both the calcium-dependent and the calcium-independent PKC activities. Both downregulation of PKC and PKC inhibitors staurosporine and calphostin C diminished Hex-induced DNA synthesis and cell number. Hex-induced DNA synthesis was enhanced by a diacylglycerol kinase inhibitor, R-59022, which was blocked by calphostin C. These data suggest that activation of PKC in part mediates Hex-induced mitogenesis in ASMC.
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PMID:Contribution of PKC to beta-hexosaminidase-induced airway smooth muscle proliferation. 914 36

Pleckstrin, originally described as a major substrate of protein kinase C (PKC) in platelets, was found to be highly expressed in human neutrophils (intracellular concentration, approximately 15 microM). As PKC isoforms play an important role in mediating neutrophil antimicrobial responses, we studied the regulation of pleckstrin phosphorylation in response to inflammatory stimuli. Following treatment of neutrophils with FMLP, 12-O-tetradecanoylphorbol-13-acetate, or opsonized zymosan, pleckstrin was rapidly phosphorylated, which resulted in a shift in its electrophoretic mobility. Several lines of evidence suggest that pleckstrin is phosphorylated in part by a nonconventional PKC following stimulation by FMLP: 1) chelation of intracellular Ca2+ had only a partial inhibitory effect; 2) diacylglycerol kinase inhibitors shortened the duration of phosphorylation, while the phosphatidic acid phosphohydrolase antagonist propranolol extended it; and 3) wortmannin and erbstatin blocked the phosphorylation of pleckstrin. These results suggest that nonconventional PKC isoforms, possibly delta or zeta, mediate the phosphorylation of pleckstrin. Both PKCdelta and -zeta are expressed in human neutrophils. Increased association of pleckstrin with both microsomes and with the cytoskeleton was observed in stimulated cells. These findings suggest that phosphorylation by nonconventional PKC isoforms induces a conformational change in pleckstrin that promotes its interaction with membranes and/or with the cytoskeleton. Such a translocation may serve to target proteins or lipids recognized by pleckstrin homology domains to sites where they can contribute to the microbicidal response.
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PMID:Phosphorylation and subcellular redistribution of pleckstrin in human neutrophils. 914 2


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