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)

Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells. Phorbol 12-myristate 13-acetate treatment enhanced high potassium-induced [3H]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25. A phosphorylation is likely to occur at Ser187, as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins. SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co-immunoprecipitated with SNAP-25 was decreased by phosphorylation. These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.
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PMID:Phosphorylation of 25-kDa synaptosome-associated protein. Possible involvement in protein kinase C-mediated regulation of neurotransmitter release. 866 51

Nitric oxide (NO) has been known to induce programmed cell death or apoptosis in murine macrophages, mouse splenocytes, and thymocytes. We demonstrate here that phorbol ester, a protein kinase C (PKC) activator, synergistically augments the antileukemic actions of the NO in HL-60 human promyelocytic leukemia cells. Exposure of cells to sodium nitro-prusside (SNP; 0.5 to 2 mM), a NO-generating agent, induced time- and concentration-related increases in morphological changes, including condensation of nuclear chromatin, nuclear fragmentation, and the apoptotic peak of propidium iodide-stained nuclei by flow cytometry. Phorbol ester alone had a small effect on inducing DNA damage, whereas SNP in combination with phorbol ester at all concentrations tested markedly increased the extent of fragmentation. Maximal potentiation of fragmentation (e.g., four- to fivefold greater than that obtained with 0.5 mM SNP alone) was observed with simultaneous treatment of phorbol ester. Similar results were obtained with another commonly used NO donor agents such as SNAP (0.5 mM) and GSNO (0.5 mM). DNA fragmentation of HL-60 cells was also augmented by 100 U/ml human recombinant interferon-gamma but not by 1.5% (v/v) DMSO or 1 microM retinoic acid. The stage-2 tumor promotor mezerein also mimicked the effect of phorbol ester to induce NO-induced apoptosis. In contrast, PKC inhibitors such as staurosporine and 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine partially blocked high concentration of SNP (2-3 mM)-induced apoptosis, suggesting that activation of PKC closely relates to the potentiation of the activity of NO on HL-60 cell apoptosis. Under the same conditions, SNP in combination with phorbol ester caused apoptosis in another transformed cell line, U-937 cells, but was ineffective at inducing apoptosis in normal peripheral blood mononuclear cells. Taken together, these findings suggest that exposure of HL-60 cells to phorbol ester renders them more susceptible to NO-induced DNA damage and that this phenomenon contributes to the cytotoxic effects of the NO-PKC combination in myeloid leukemia cells.
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PMID:Potentiation of the activity of nitric oxide by the protein kinase C activator phorbol ester in human myeloid leukemic HL-60 cells: association with enhanced fragmentation of mature genomic DNA. 907 Mar 16

The synaptic vesicle exocytosis occurs by a highly regulated mechanism: syntaxin and 25 kDa synaptosome-associated protein (SNAP-25) are assembled with vesicle-associated membrane protein (VAMP) to form a synaptic core complex and then synaptotagmin participates as a Ca2+ sensor in the final step of membrane fusion. The 43 kDa growth-associated protein GAP-43 is a nerve-specific protein that is predominantly localized in the axonal growth cones and presynaptic terminal membrane. In the present study we have examined a possible interaction of GAP-43 with components involved in the exocytosis. GAP-43 was found to interact with syntaxin, SNAP-25 and VAMP in rat brain tissues and nerve growth factor-dependently differentiated PC12 cells, but not in undifferentiated PC12 cells. GAP-43 also interacted with synaptotagmin and calmodulin. These interactions of GAP-43 could be detected only when chemical cross-linking of proteins was performed before they were solubilized from the membranes with detergents, in contrast with the interaction of the synaptic core complex, which was detected without cross-linking. Experiments in vitro showed that the interaction of GAP-43 with these proteins occurred Ca2+-dependently; its maximum binding with the core complex was observed at 100 microM Ca2+, whereas that of syntaxin with synaptotagmin was at 200 microM Ca2+. These values of Ca2+ concentration are close to that required for the Ca2+-dependent release of neurotransmitters. Furthermore we observed that the interaction in vitro of GAP-43 with the synaptic core complex was coupled with protein kinase C-mediated phosphorylation of GAP-43. Taken together, our results suggest a novel function of GAP-43 that is involved in the Ca2+-dependent fusion of synaptic vesicles.
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PMID:Ca2+-dependent interaction of the growth-associated protein GAP-43 with the synaptic core complex. 923 Jan 28

The SNARE hypothesis proposes that synaptic vesicles dock at presynaptic membranes via interactions among the vesicular, integral membrane proteins VAMP (vesicle-associated membrane protein) and synaptotagmin and the target membrane proteins SNAP25 (synaptosome-associated protein with an Mr of 25 kDa) and syntaxin-1. Non-neuronal cells express isoforms of these proteins, believed to mediate secretory vesicle docking and/or fusion. Secretion in neuronal and non-neuronal systems differs in time course, Ca2+ dependence, and regulatory input. It is not known whether the non-neuronal protein isoforms form complexes akin to those of their neuronal counterparts. In this study, we defined the binding characteristics of three SNARE proteins: SNAP23, VAMP-2, and syntaxin-4. Binary, saturable interactions among all three partners (VAMP-2-syntaxin-4, VAMP-2-SNAP23, and SNAP23-syntaxin-4) were measured in vitro. Unlike its neuronal counterpart, SNAP23 did not potentiate VAMP-2 binding to its putative t-SNARE partner, syntaxin-4. The susceptibility of SNARE proteins to phosphorylation by exogenous kinases and their impact on binary interactions were explored. Syntaxin-4 was efficiently phosphorylated by casein kinase II (CKII) and cAMP-dependent protein kinase (PKA) (incorporating 0.8 and 3.9 mol of phosphate/mol of syntaxin-4, respectively), while syntaxin-1 was only strongly phosphorylated by CKII. Each of the syntaxin isoforms was weakly phosphorylated by protein kinase C (PKC) (<0.05 mol of phosphate/mol of syntaxin-4). Importantly, PKA but not casein kinase II phosphorylation of syntaxin-4 disrupted its binding to SNAP23. We hypothesize that PKA may modulate syntaxin-4-dependent SNARE complex formation to regulate exocytosis in non-neuronal cells.
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PMID:Binary interactions of the SNARE proteins syntaxin-4, SNAP23, and VAMP-2 and their regulation by phosphorylation. 969 5

N- and P/Q-type calcium channels are localized in high density in presynaptic nerve terminals and are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. As outlined in the preceding article, these calcium channels can be purified from brain as a complex with SNARE proteins which are involved in exocytosis. In addition, N-type and P/Q-type calcium channels are co-localized with syntaxin in high-density clusters in nerve terminals. Here we review the role of the synaptic protein interaction (synprint) sites in the intracellular loop II-III (L(II-III)) of both alpha1B and alpha1A subunits of N-type and P/Q-type calcium channels, which bind to syntaxin, SNAP-25, and synaptotagmin. Calcium has a biphasic effect on the interactions of N-type calcium channels with SNARE complexes, stimulating optimal binding in the range of 10-20 microM. PKC or CaM KII phosphorylation of the N-type synprint peptide inhibits interactions with native brain SNARE complexes containing syntaxin and SNAP-25. Introduction of the synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibits EPSPs from synchronous transmitter release by 42%. At physiological Ca2+ concentrations, synprint peptides cause an approximate 25% reduction in transmitter release of injected frog neuromuscular junction in cultures, consistent with detachment of 70% of the docked vesicles from calcium channels based on a theoretical model. Together, these studies suggest that presynaptic calcium channels not only provide the calcium signal required by the exocytotic machinery, but also contain structural elements that are integral to vesicle docking, priming, and fusion processes.
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PMID:Physical link and functional coupling of presynaptic calcium channels and the synaptic vesicle docking/fusion machinery. 975 30

Activated platelets and endothelium surface express the cell adhesion molecule P-selectin (CD62P), which plays an important role in mediating interactions with leukocytes. Increased levels of a functional soluble form of P-selectin (sP-selectin) have been reported in several pathological states but it is not clear whether this circulating sP-selectin originates from platelets and/or endothelial cells. Here we describe the concurrent kinetics of intracellular storage, surface expression and release of platelet P-selectin induced by thrombin or the protein kinase C activator PMA. Platelet activation with submaximal concentrations of thrombin (0.1 U/ml) resulted in a rapid decrease of intracellular P-selectin. This decrease of intracellular P-selectin concurred with a gradual increase of surface expression and an initial increase of sP-selectin. Our results indicate that intracellular stores of P-selectin were only partly mobilized upon activation with submaximal concentrations of thrombin. A high concentration of thrombin (1.0 U/ml) induced a rapid and nearly total decrease of intracellular stores and a more pronounced, but transient, increase of surface expression. The release of P-selectin was fast and occurred during the initial activation phase. The NO donor SNAP inhibited both surface expression and release of platelet P-selectin in a similar manner. PMA (0.1-1.0 microM) mediated a more slow, gradual and sustained surface expression and release of P-selectin than thrombin. Thus, surface expression and release of platelet P-selectin show different kinetics depending on the mode of activation.
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PMID:Kinetics of platelet P-selectin mobilization: concurrent surface expression and release induced by thrombin or PMA, and inhibition by the NO donor SNAP. 986 63

The effect of protein kinase C (PKC) on the release of neurotransmitters from a number preparations, including sympathetic nerve endings, brain slices, synaptosomes, and neuronally derived cell lines, is considered. A comparison is drawn between effects of activation of PKC on neurotransmitter release from small synaptic vesicles and large dense-cored vesicles. The enhancement of neurotransmitter release is discussed in relation to the effect of PKC on: 1. Rearrangement of the F-actin-based cytoskeleton, including the possible role of MARCKS in this process, to allow access of large dense-cored vesicles to release sites on the plasma membrane. 2. Phosphorylation of key components in the SNAP/SNARE complex associated with the docking and fusion of vesicles at site of secretion. 3. Ion channel activity, particularly Ca2+ channels.
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PMID:The regulation of neurotransmitter secretion by protein kinase C. 1006 77

In response to thrombin and other extracellular activators, platelets secrete molecules from large intracellular vesicles (granules) to initiate thrombosis. Little is known about the molecular machinery responsible for vesicle docking and secretion in platelets and the linkage of that machinery to cell activation. We found that platelet membranes contain a full complement of interacting proteins-VAMP, SNAP-25, and syntaxin 4-that are necessary for vesicle docking and fusion with the plasma membrane. Platelets also contain an uncharacterized homologue of the Sec1p family that appears to regulate vesicle docking through its binding with a cognate syntaxin. This platelet Sec1 protein (PSP) bound to syntaxin 4 and thereby excluded the binding of SNAP-25 with syntaxin 4, an interaction critical to vesicle docking. As predicted by its sequence, PSP was detected predominantly in the platelet cytosol and was phosphorylated in vitro by protein kinase C (PKC), a secretion-linked kinase, incorporating 0.87 +/- 0.11 mol of PO4 per mole of protein. PSP was also specifically phosphorylated in permeabilized platelets after cellular stimulation by phorbol esters or thrombin and this phosphorylation was blocked by the PKC inhibitor Ro-31-8220. Phosphorylation by PKC in vitro inhibited PSP from binding to syntaxin 4. Taken together, these studies indicate that platelets, like neurons and other cells capable of regulated secretion, contain a unique complement of interacting vesicle docking proteins and PSP, a putative regulator of vesicle docking. The PKC-dependent phosphorylation of PSP in activated platelets and its inhibitory effects on syntaxin 4 binding provide a novel functional link that may be important in coupling the processes of cell activation, intracellular signaling, and secretion.
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PMID:Human platelets contain SNARE proteins and a Sec1p homologue that interacts with syntaxin 4 and is phosphorylated after thrombin activation: implications for platelet secretion. 1019 41

Nitric oxide (NO) is an unstable free radical that functions as a cytotoxic agent secreted by macrophages to kill cancer cells. Here we report the effect of NO on the expression of intercellular adhesion molecule-1 (ICAM-1) on cancer cells. NO donors such as SNP, SNAP and SIN-1 up-regulated the expression of ICAM-1 on NA cells, a squamous cell carcinoma cell line. Northern blot analysis showed that the induction of ICAM-1 might be due to transcriptional induction of ICAM-1 mRNA. Up-regulation of ICAM-1 mRNA by NO donors was inhibited by carboxy-PTIO, a NO scavenger. Although NF-kappaB activity was induced by NO donors, AP-1 was not induced by them. Staurosporin, a protein kinase C (PKC) inhibitor, inhibited the induction of ICAM-1 on NA cells by NO, whereas genistein, a protein tyrosine kinase inhibitor, did not. These findings indicate that NO up-regulates ICAM-1 expression on cancer cells by a regulatory mechanism involving PKC and suggest that NF-kappaB, but not AP-1, might be involved in induction of ICAM-1 by NO in cancer cells.
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PMID:Nitric oxide up-regulates the expression of intercellular adhesion molecule-1 on cancer cells. 1019 24

Tricyclic antidepressants (e.g., imipramine, desipramine) are currently used in the treatment of mood disorders such as depression. At the cellular level they inhibit the re-uptake of the exocytosed monoamines serotonin and noradrenaline. However, they also stimulate phospholipase C activity and the production of the second messenger inositol 1,4,5-trisphosphate. Since phospholipase C activation can also lead to the production of the protein kinase C activator diacylglycerol, we have undertaken experiments to see whether acutely applied desipramine could change the synaptic strength of neurons in a protein kinase C-dependent manner. Experiments performed with cultured hippocampal neurons dissociated from neonatal rats revealed that desipramine rapidly enhanced the spontaneous vesicular release of glutamate. This was observed by measuring the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. Analysis of amplitude distribution histograms indicated a presynaptic site of action. The protein kinase inhibitor staurosporine and down-regulation of protein kinase C activity greatly reduced the desipramine-dependent enhancement of the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. This presynaptic modulation requires SNARE proteins because cleavage of SNAP-25 with the botulinum neurotoxin A strongly reduced the desipramine-induced glutamate release. Thus, acute applications of desipramine stimulated the ongoing neurotransmitter release pathway, probably by activating protein kinase C. Our data indicate that tricyclic antidepressant drugs not only act on serotoninergic and/or noradrenergic cells but can also modify the activity of glutamatergic neurons.
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PMID:Acute application of the tricyclic antidepressant desipramine presynaptically stimulates the exocytosis of glutamate in the hippocampus. 1021 74


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