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)

Utilizing a digitonin-permeabilized cell system, we have studied the release of calcium from a non-mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (+/- 6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3-sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP-dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down-regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.
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PMID:Calcium mobilization in permeabilized fibroblasts: effects of inositol trisphosphate, orthovanadate, mitogens, phorbol ester, and guanosine triphosphate. 349 99

1. Bradykinin caused a transient reduction of about 25% in the cyclic AMP level in forskolin prestimulated DDT1 MF-2 smooth muscle cells (IC50: 36.4 +/- 4.9 nM) and a pronounced, sustained inhibition (40%) of the isoprenaline-stimulated cyclic AMP level (IC50: 37.5 +/- 1.1 nM). 2. The Ca2+ ionophore, ionomycin, mimicked both the bradykinin-induced transient reduction in the forskolin-stimulated cyclic AMP level and the sustained reduction in the isoprenaline-stimulated cyclic AMP level. 3. The Ca(2+)-dependent effect on cyclic AMP induced by bradykinin was mediated solely by Ca2+ release from internal stores, since inhibition of Ca2+ entry with LaCl3 did not reduce the response to bradykinin. 4. The involvement of calmodulin-dependent enzyme activities, protein kinase C or an inhibitory GTP binding protein in the bradykinin-induced responses was excluded since a calmodulin inhibitor, calmidazolium, a PKC inhibitor, staurosporine and pertussis toxin, respectively did not affect the decline in the cyclic AMP level. 5. Bradykinin enhanced the rate of cyclic AMP breakdown in intact cells, which effect was not mimicked by ionomycin. This suggested a Ca(2+)-independent activation of phosphodiesterase activity by bradykinin in DDT1 MF-2 cells. 6. The bradykinin B1 receptor agonist, desArg9-bradykinin, did not affect cyclic AMP formation in isoprenaline prestimulated cells, while the bradykinin B2 receptor antagonists, Hoe 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]-BK) and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK completely abolished the bradykinin response in both forskolin and isoprenaline prestimulated cells. 7. Bradykinin caused an increase in intracellular Ca2+, which was antagonized by the bradykinin B2 receptor antagonists, Hoe 140 and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK. The bradykinin B2 receptor agonist,desArg9-bradykinin, did not evoke a rise in cytoplasmic Ca2 .8. It is concluded, that stimulation of bradykinin B2 receptors causes a reduction in cellular cyclic AMP in DDT1, MF-2 cells. This decline in cyclic AMP is partly mediated by a Ca2+/calmodulin independent activation of phosphodiesterase activity. The increase in [Ca2+], mediated by bradykinin B2 receptors inhibited forskolin- and isoprenaline-activated adenylyl cyclase differently, most likely by interfering with different components of the adenylyl cyclase signalling pathway.
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PMID:Ca(2+)-dependent and -independent mechanism of cyclic-AMP reduction: mediation by bradykinin B2 receptors. 758 24

We have previously established that 21-day-old postnatal rat oligodendrocytes, maintained in monolayer culture and subjected to 6 h of hypoxia, show reversible inhibition of synthesis of alpha-hydroxy fatty acid and myelin basic protein but a dramatic induction of a 22-kDa protein, suggesting that this is a good model to study the mechanism of CNS demyelination caused by hypoxic injury. We now report that hypoxia also dramatically inhibits the basal protein kinase C-mediated phosphorylation of myelin basic protein and myelin 2',3'-cyclic nucleotide phosphohydrolase by 80%, but that the inhibition of phosphorylation can be reversed by addition of a protein kinase C activator, phorbol 12-myristate 13-acetate. The mechanism of action appears to involve the uncoupling of signal transduction at a site before phospholipase C, because hypoxia did not affect protein kinase C activity or its translocation to the membrane fraction. The most potent activator of phospholipase C (as measured by inositol phosphate release) was carbachol (muscarinic M1 receptor agonist), followed by L-phenylephrine (alpha 1-adrenergic receptor agonist) in normal oligodendrocytes. Excitatory amino acids and histamine were ineffective. Hypoxia for 6 h completely inhibited both muscarinic and alpha 1-adrenergic receptor-mediated inositol monophosphate release but did not affect phospholipase D-coupled phosphatidylethanol production in response to carbachol. We therefore conclude from this and earlier work that early, reversible changes in oligodendrocyte metabolism result not simply from ATP depletion, but may specifically target GTP binding protein-mediated processes.
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PMID:Effects of hypoxia on oligodendrocyte signal transduction. 768 39

Mechanisms of Ca2+ sensitization of both myosin light chain (MLC) phosphorylation and force development by protein kinase C (PKC) were studied in permeabilized tonic smooth muscle obtained from the rabbit femoral artery. For comparison, the Ca2+ sensitizing effect of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) was examined, which had been previously shown to inhibit MLC phosphatase in phasic vascular smooth muscle. We now report that PKC activators (phorbol esters, short chain synthetic diacylglycerols and a diacylglycerol kinase inhibitor) and GTP gamma S significantly increase both MLC phosphorylation and force development at constant [Ca2+]. Major phosphorylation site occurring in the presence of phorbol-12,13-dibutyrate (PDBu) or GTP gamma S at constant [Ca2+] is the same serine residue (Ser-19) as that phosphorylated by MLC kinase in response to increased Ca2+ concentrations. In an ATP- and Ca(2+)-free solution containing 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), to avoid the kinase activity, both PDBu and GTP gamma S significantly decreased the rate of MLC dephosphorylation to half its control value. However, PDBu inhibited the relaxation rate more than did GTP gamma S. In the presence of microcystin-LR to inhibit the phosphatase activity, neither PDBu nor GTP gamma S affected MLC phosphorylation and force development. These results indicate that PKC, like activation of GTP binding protein, increases Ca2+ sensitivity of both MLC phosphorylation and force production through inhibition of MLC phosphatase.
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PMID:A novel mechanism for the Ca(2+)-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase. 780 49

1. 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time- and dose-dependent with a half-maximal response (EC50) and a maximal response at 0.38 +/- 0.05 and 10 microM, respectively. 2. Ketanserin and mianserin (10 and 100 nM), 5-HT2 receptor antagonists, were equipotent in blocking the 5-HT-induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose-response curves of 5-HT-induced IPs accumulation were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased up to 10 microM. 3. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5-HT-induced IPs accumulation, but partially inhibited the AlF(4-)-induced IPs response. 4. Stimulation of IPs accumulation by 5-HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. A further Ca(2+)-dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphoshate) (GTP gamma S) or 5-HT. The combination of GTP gamma S and 5-HT elicited an additive effect on IPs accumulation. 5. Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM, 30 min) abolished the 5-HT-induced IPs accumulation. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 +/- 0.4 nM and 1 microM, n = 3, respectively. The protein kinase C (PKC) activator, 4 alpha-phorbol 12,13-didecanoate, at 1 microM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the effect of PMA is distal to the 5-HT receptor. 7. Acetylcholine-induced IPs accumulation was completely inhibited by atropine, but not affected by ketanserin or mianserin, suggesting that 5-HT-induced IPs accumulation is not due to release of acetylcholine.8. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis via a pertussis toxin- and cholera toxin-insensitive GTP binding protein in canine TSMCs and that this coupling process is negatively regulated by PKC. 5-HT2 receptors may be predominantly mediating IPs accumulation and presumably IP-induced Ca2+ release may function as the transducing mechanism for 5-HT stimulated contraction of tracheal smooth muscle.
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PMID:5-Hydroxytryptamine receptor-mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells. 801 56

The calcium requirement for serotonin (5-HT)- and the 5-HT3 receptor agonists, 2-Me-5-HT- and PBG-dependent breakdown of phosphatidyl inositol has been examined in the rat fronto-cingulate cortex. The omission of added Ca2+ from the Kreb's incubation medium reduced the [3H]inositol phosphate accumulation from pre-labelled phospholipids. Removal of Ca2+ by pre-incubation with EGTA (0.5 mM), as well as the addition of the calcium channel blocker, lanthanum (10 microM), abolished the 5-HT- and the 5-HT3 receptor agonists'-stimulated phosphoinositide (PI) response. By contrast, the calcium ionophores, A 23187 and Ionomycin (both at 30 microM) stimulated PI hydrolysis, and this effect was additive to the increased PI turnover induced by 5-HT, 2-Me-5-HT and PBG. The increase in phosphoinositide hydrolysis induced by 5-HT and 2-Me-5-HT was significantly inhibited by phorbol dibutyrate (PDBu) and phorbol myristate acetate, indicating that the activation of protein kinase C (PKC) may provide negative feedback to the PI response induced by 5-HT and 2-Me-5-HT-stimulated PI metabolism was reversed by the PKC inhibitors, staurosporine, calphostin C and chelerythrine (all at 10 microM), however, Pertussis toxin (0.5 and 1 microgram) had no effect on either 5-HT's or 2-Me-5-HT's increased stimulation of PI hydrolysis, suggesting that this response is not associated to a Gi GTP binding protein.
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PMID:Further characterization of 5-HT- and 5-HT3 receptor agonists'-stimulated phosphoinositol phosphates accumulation. 839 45

1. Effects of arachidonic acid (AA) on voltage-dependent Ca channel currents were investigated by whole-cell-clamp methods in single smooth muscle cells freshly isolated from vas deferens of the guinea-pig. 2. Ca channel current was decreased by application of 1-30 microM AA in a concentration-dependent manner. When Ca2+ or Ba2+ was the charge carrier, Ca channel current (ICa or IBa) was reduced by AA to a similar extent (IC50 = 10 and 6 microM, respectively). Addition of 15 mM BAPTA to the pipette solution did not affect the reduction of IBa by 10 microM AA. 3. The effect of AA on IBa was not prevented by internal application of 1 mM nordihydroguaiaretic acid (NDGA) and 1 mM indomethacin (Indo). When the pipette solution contained 0.1 mM guanosine-5'-triphosphate (GTP), IBa was decreased slightly but significantly by application of 30 microM prostaglandin F2 alpha (PGF2 alpha) but not by PGE2. This effect of PGF2 alpha was irreversible or not observed when the pipette solution contained 0.3 mM guanosine-5'-(3-thiotriphosphate) (GTP gamma S) or both GTP or guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), respectively. 4. External application of 100 units ml-1 superoxide dismutase slightly but significantly attenuated the inhibition of IBa by 1-30 microM AA. Intracellular application of 1 mM GDP beta S or 0.3 mM GTP gamma S did not significantly change the effect of AA. Intracellular application of 0.1 mM 1-(5-isoquinolinesulphonyl)-2-methylepiperazine (H-7) also did not change the effect of AA. 5. These results indicate that the decrease in Ca channel currents in vas deferens smooth muscle cells is mainly due to AA itself, as opposed to its metabolites. The effect of AA may be due to AA itself, as opposed to its metabolites. The effect of AA may be due to its direct action on Ca channels or membrane phospholipids, but may not be mediated by activation of GTP binding proteins or protein kinase C. The inhibition of Ca channel current by AA may be partly induced by superoxide radicals derived from AA oxidation. PGF2A also reduces Ca channel currents but probably by a separate mechanism via activation of a GTP binding protein.
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PMID:Modulation of calcium channel currents by arachidonic acid in single smooth muscle cells from vas deferens of the guinea-pig. 852 75

The enzyme that catalyzes the synthesis of the major structural component of the yeast cell wall, beta(1-->3)-D-glucan synthase (also known as 1,3-beta-glucan synthase), requires a guanosine triphosphate (GTP) binding protein for activity. The GTP binding protein was identified as Rho1p. The rho1 mutants were defective in GTP stimulation of glucan synthase, and the defect was corrected by addition of purified or recombinant Rho1p. A protein missing in purified preparations from a rho1 strain was identified as Rho1p. Rho1p also regulates protein kinase C, which controls a mitogen-activated protein kinase cascade. Experiments with a dominant positive PKC1 gene showed that the two effects of Rho1p are independent of each other. The colocalization of Rho1p with actin patches at the site of bud emergence and the role of Rho1p in cell wall synthesis emphasize the importance of Rho1p in polarized growth and morphogenesis.
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PMID:Rho1p, a yeast protein at the interface between cell polarization and morphogenesis. 860 5

GTP binding proteins, heterotrimeric molecules composed of alpha-, beta-, and gamma-subunits, are known to serve as transducers of information from seven-transmembrane receptors. Activation of G-proteins has been generally considered to involve subunit dissociation, with G(alpha) separating from G(betagamma). However, we have found a receptor activation of G(i) in proliferating cells that differs from these models and involves the subcellular translocation of the alpha-subunit from the cell periphery to the nucleus where G(i alpha) binds to chromatin for the duration of mitosis. This report describes the mechanism of G(i) activation in Swiss 3T3 cells in response to serum, thrombin, and epidermal growth factor, and describes a role for G(i2) in the cell cycle. Agonists were found to be unable to induce the physical dissociation of G(i2) subunits. The alpha- and beta-subunits of G(i2) could be coimmunoprecipitated with a G(i alpha) antibody from both the membrane and nuclear fractions of long-term activated cultures, showing that G(i alpha 2) and G(i beta) are induced to comigrate to the nucleus in response to growth factor receptor activation. G(i2) appears to be activated in part by a postreceptor signal that can be mimicked by protein kinase C activation; this signal may be responsible for the convergence of the signaling mechanisms of these distinct seven-transmembrane and tyrosine kinase receptors. We suggest that translocation of G(i alpha) to the nucleus induced by either thrombin or EGF may occur without subunit dissociation. Functional studies of the role of G(i) showed that pertussis toxin does not block DNA synthesis in Swiss 3T3 fibroblasts induced by serum or thrombin, but that cell proliferation is retarded to each. These results provide direct evidence for a novel mechanism of GTP binding protein activation and for an essential role of G(i) in the induction of cell division by a variety of growth factor receptors. G(i) can carry out this role in control of cellular proliferation through its translocation to the nucleus of mitotic cells.
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PMID:The G-protein G(i) regulates mitosis but not DNA synthesis in growth factor-activated fibroblasts: a role for the nuclear translocation of G(i). 903 62

1. The action of GTP-binding proteins on ATP-sensitive potassium (KATP) channels was investigated. KATP channels were expressed in a mammalian cell line (COS-1 cells) by cotransfecting vectors carrying the sulphonylurea receptor (SUR1) and BIR (Kir6.2), a member of the inward rectifier K+ channel family. G proteins were also tested on KATP channels composed of an isoform of SUR1, SUR2A, in combination with Kir6.2. 2. The alpha and beta gamma subunits of the GTP binding protein G1 were tested separately in inside-out patches under continuous recording. G alpha-11 increases the activity of SUR1-Kir6.2 and SUR2A-Kir6.2 channels by 200 and by 30%, respectively. 3. G alpha-12 does not increase the activity of SUR1-Kir6.2 channels, but increase the activity of SUR2A-Kir6.2 channels by 30%. 4. Control experiments showed that GTP gamma S, a specific activator of G proteins, and heat-inactivated G alpha-11 do not increase the single channel activity. 5. No effects of the other subunits (beta gamma) from either G11 or G12 on the single channel activity were observed. 6. The protein kinase C inhibitors H7 and an inhibitory peptide (FARKGALRQKNV) had no effect on the modulatory action of G alpha-11 on SUR1-Kir6.2 channels. 7. We conclude that both types of reconstituted KATP channels are modulated by G proteins.
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PMID:Modulation of reconstituted ATP-sensitive K(+)-channels by GTP-binding proteins in a mammalian cell line. 951 95


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