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Proceedings of the National Academy of... May 1975Comparisons of the stimulation of normal lymphocytes and lymphoma cells by tetrameric concanavalin a (Con ta) and dimeric succinyl-Con A suggest that both stimulatory...
Comparisons of the stimulation of normal lymphocytes and lymphoma cells by tetrameric concanavalin a (Con ta) and dimeric succinyl-Con A suggest that both stimulatory and inhibitory signals operate to modulate mitogenesis. Synergistic effects can be obtained for the stimulatory event using lectins, phorbol esters, and calcium ionophores, all of which are independently mitogenic for lymphocytes. The inhibitory effects of high doses of Con A could be mimicked by the simultaneous addition of the phorbol ester and Con A under conditions in which both reagents are optimally mitogenic when used alone. No inhibition of stimulation was found, however, when succinyl-Con A was used with phorbol ester under the same conditions. Moreover, when lymphocytes were cultured with Con A in the presence of succinyl-Con A, the inhibitory effect of the native lectin was seen at lower doses than in the absence of the derivative. These observations suggest that the stimulatory and inhibitory portions of the dose-response curve can be manipulated independently and may be mediated by two distinct signals. It is likely the signal for the inhibition of cell proliferation is regulated by the same cell surface modulating assembly that controls the mobility of cell surface receptors.
Topics: Animals; Anti-Bacterial Agents; Carboxylic Acids; Cell Line; Concanavalin A; Dose-Response Relationship, Drug; Humans; Lymphocytes; Macromolecular Substances; Mice; Mitogens; Mitosis; Phorbol Esters; Protein Binding; Succinates; Thymidine
PubMed: 1057182
DOI: 10.1073/pnas.72.5.1917 -
Cellular and Molecular Neurobiology Oct 2020Excitatory neurotransmission relies on the precise targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors to the neuronal...
Excitatory neurotransmission relies on the precise targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors to the neuronal plasma membrane. Activity-dependent ubiquitination of AMPA receptor (AMPAR) subunits sorts internalised receptors to late endosomes for degradation, which ultimately determines the number of AMPARs on neuronal membrane. Our recent study has demonstrated a functional cross-talk between the phosphorylation and ubiquitination of the GluA1 subunit in mammalian central neurons. However, the existence of such a cross modulation for the GluA2 subunit remains unknown. Here, we have shown that bicuculline induced GluA2 ubiquitination on the same lysine residues (Lys-870 and Lys-882) in the C-terminal as those elicited by the AMPA treatment. Interestingly, bicuculline-induced ubiquitination was markedly enhanced by the phospho-mimetic GluA2 S880E mutant. Pharmacological activation of protein kinase C (PKC) by phorbol ester, which mediates the phosphorylation of GluA2 at Ser-880, augmented bicuculline-induced ubiquitination of GluA2 in cultured neurons. This effect was specific for the GluA2 subunit because phorbol ester did not alter the level of GluA1 ubiquitination. However, phorbol ester-induced enhancement of GluA2 ubiquitination did not require Ser-880 phosphorylation. This suggests that pseudo-phosphorylation of Ser-880 is sufficient but is not necessary for the augmentation of bicuculline-induced GluA2 ubiquitination. Collectively, these data provide the first demonstration of subunit-specific modulation of AMPAR ubiquitination by the PKC-dependent signalling pathway in mammalian central neurons.
Topics: Animals; Cells, Cultured; Central Nervous System; Hippocampus; Neurons; Phorbol Esters; Rats; Receptors, AMPA; Synaptic Transmission; Ubiquitination
PubMed: 32052226
DOI: 10.1007/s10571-020-00809-2 -
Proceedings of the National Academy of... Sep 1988The studies described here tested the hypothesis that the changes in synaptic efficacy produced by phorbol esters in hippocampal slices are equivalent to the long-term...
The studies described here tested the hypothesis that the changes in synaptic efficacy produced by phorbol esters in hippocampal slices are equivalent to the long-term potentiation (LTP) induced by high-frequency stimulation. In contrast to the extremely stable synaptic potentiation induced by electrical stimulation, the facilitatory effects of phorbol 12,13-diacetate and phorbol 12,13-dibutyrate were transient: washout of the drugs restored normal responses in approximately 1-2 and 2-4 hr for phorbol diacetate and phorbol dibutyrate, respectively. It is noteworthy that the more liposoluble of the phorbol esters required longer washout periods. Robust LTP still occurred in response to high-frequency stimulation after washout of phorbol esters and to a lesser degree during their application. Treatment of slices with H-7, an inhibitor of protein kinase C, did not prevent LTP induction although it significantly affected neuronal excitability and produced effects opposite to those of phorbol esters. Finally, phorbol esters altered responses to repetitive stimulation in a way that could account for the reduced LTP elicited in their presence. These results indicate that the increases in synaptic responses caused by phorbol esters and high-frequency electrical stimulation are quite different and thus do not support the hypothesis that activation of protein kinase C, the presumed target of the phorbol esters, triggers LTP.
Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Evoked Potentials; Hippocampus; Isoquinolines; Phorbol 12,13-Dibutyrate; Phorbol Esters; Piperazines; Protein Kinase C; Synapses
PubMed: 3166141
DOI: 10.1073/pnas.85.18.6997 -
Proceedings of the National Academy of... May 1983A specific high-affinity phorbol ester binding component has been identified in the cytosol of an EL4 mouse thymoma line by using conditions similar to those for...
A specific high-affinity phorbol ester binding component has been identified in the cytosol of an EL4 mouse thymoma line by using conditions similar to those for demonstrating activity of a calcium/phospholipid-dependent protein kinase. Specific binding is absolutely dependent on acidic phospholipids (maximal binding at 96 micrograms of phosphatidylserine per ml or 200 micrograms of phosphatidylinositol per ml) and is greatly enhanced by addition of calcium (0.5 mM) and magnesium (75 mM). Scatchard analysis of the cytosolic binding component indicated a Kd of 4.2 +/- 2.5 nM with 1.8 +/- 0.6 x 10(4) sites per cell compared to a Kd of 11.9 +/- 4.4 nM and 4.1 +/- 1.0 x 10(4) sites per cell for the membrane receptor. Consistent with the existence of at least two phorbol ester binding sites in EL4 cells was the observation of curvilinear Scatchard plots for binding to whole homogenates. The cytosolic binding showed the same order of potency for binding phorbol ester analogs as has been observed for intact cells. These results further support the similarity between phorbol ester receptors and the calcium/phospholipid-dependent protein kinase and suggest that the cytosolic receptor may be involved in subsequent phorbol ester effects in EL4 cells including loss of the kinase activity from the cytosol and production of T-cell growth factor.
Topics: Animals; Caenorhabditis elegans Proteins; Calcium; Carrier Proteins; Cell Line; Cytosol; Kinetics; Magnesium; Membranes; Mice; Phorbol Esters; Phospholipids; Protein Kinase C; Receptors, Cell Surface; Receptors, Drug; Thymoma; Thymus Neoplasms
PubMed: 6302698
DOI: 10.1073/pnas.80.9.2642 -
FEBS Letters Oct 1983A large amount of specific high affinity binding sites for tumor promoting phorbol esters as well as of a Ca2+- and phospholipid-dependent protein kinase is present in...
A large amount of specific high affinity binding sites for tumor promoting phorbol esters as well as of a Ca2+- and phospholipid-dependent protein kinase is present in cytosol of chick oviduct. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is able to replace either Ca2+ or the phospholipid phosphatidylserine as activators of the kinase to some extent. The maximum activity of the enzyme in the presence of Ca2+ and phosphatidylserine, however, cannot be increased further by TPA. Various second stage tumor promoters also exhibit the capacity to stimulate the protein kinase, whereas the non-promoting phorbol ester 4-O-methyl-TPA, as well as the non-promoting, but with respect to other responses TPA-like, calcium ionophore A23187, do not affect the kinase.
Topics: Animals; Binding Sites; Calcium; Chickens; Cytosol; Enzyme Activation; Female; Oviducts; Phorbols; Phosphatidylserines; Phospholipids; Protein Kinases; Tetradecanoylphorbol Acetate
PubMed: 6617887
DOI: 10.1016/0014-5793(83)81067-9 -
The Journal of Biological Chemistry Dec 1981An esterase which cleaves the 12-ester group of phorbol-12,13-diesters has been purified to electrophoretic homogeneity from murine liver cytosol using ammonium sulfate...
An esterase which cleaves the 12-ester group of phorbol-12,13-diesters has been purified to electrophoretic homogeneity from murine liver cytosol using ammonium sulfate fractionation, Sephadex G-200 gel filtration, Con A-Sepharose chromatography, and phenyl-Sepharose chromatography. The enzyme is a single chain, hydrophobic glycoprotein with a molecular weight of 60,000 and exhibits optimum activity at pH 7.5 to 8.5. The hydrolase has an isoelectric point (pI) of 5. The enzyme is heat- and acid-labile. Zinc, cobalt, and fluoride ions inhibit its activity. Phenylmethylsulfonyl fluoride is a potent inhibitor of the hydrolase. Sarkosyl also inhibits the enzyme at millimolar concentrations. The enzyme inactivates biologically active phorbol-12,13-diesters in a dose-, time-, and temperature-dependent manner. The enzyme inhibits phorbol-12,13-dibutyrate binding to its receptor in a noncompetitive manner. The inhibition constant (KI) has been found to be 6.6 X 10(-8) M.
Topics: Animals; Brain; Caenorhabditis elegans Proteins; Carcinogens; Carrier Proteins; Cell Membrane; Cytosol; Esterases; Kinetics; Liver; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Protein Kinase C; Receptors, Drug; Substrate Specificity
PubMed: 6946062
DOI: No ID Found -
The Journal of Biological Chemistry May 1995Human involucrin (hINV) is a cornified envelope precursor that is specifically expressed in the suprabasal epidermal layers. We previously demonstrated that 2500 base...
Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity.
Human involucrin (hINV) is a cornified envelope precursor that is specifically expressed in the suprabasal epidermal layers. We previously demonstrated that 2500 base pairs of the hINV gene upstream regulatory region confers differentiation appropriate regulation in transgenic mice. An analysis of the hINV gene sequence upstream of the transcription start site reveals five potential AP1 binding sites (AP1-1 to 5). Using reporter gene constructs in human keratinocytes, we show that the most distal (AP1-5) and most proximal (AP1-1) AP1 sites are essential for high level transcriptional activity. Simultaneous mutation of these sites reduces transcription by 80%. Gel supershift experiments indicate the interaction of these sites with Fra-1, junB, and junD. Involucrin mRNA levels increase 10-fold and promoter activity 5-11-fold when differentiation is induced by phorbol ester. Functional studies implicate AP1-1 and AP1-5 in mediating the phorbol ester-dependent increase in promoter activity. No involucrin promoter activity or involucrin mRNA was detected in 3T3 fibroblasts. We conclude that (i) two AP1 sites in the hINV promoter are important elements required for keratinocyte-specific expression, (ii) these AP1-1 sites mediate the phorbol ester-dependent increase in promoter activity, and (iii) Fra-1, junB, and junD may be important regulators of hINV expression in epidermis.
Topics: Base Sequence; DNA Mutational Analysis; Gene Expression Regulation; Genes, Reporter; Humans; Keratinocytes; Luciferases; Molecular Sequence Data; Nuclear Proteins; Phorbol Esters; Promoter Regions, Genetic; Protein Binding; Protein Precursors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Recombinant Fusion Proteins; Sequence Deletion; Transcription Factor AP-1; Transcription, Genetic
PubMed: 7759510
DOI: 10.1074/jbc.270.21.12614 -
African Health Sciences Sep 2017Neutrophil extracellular traps (NETs) constitute a network of chromatin fibres containing histone and antimicrobial peptides that are released by activated neutrophils....
BACKGROUND
Neutrophil extracellular traps (NETs) constitute a network of chromatin fibres containing histone and antimicrobial peptides that are released by activated neutrophils. NETs protect the host against infection by trapping and facilitating phagocytosis of potentially harmful pathogens.
OBJECTIVES
The aim of the current study was to investigate the effects of cigarette smoke condensate (CSC) on phorbol-ester (PMA)-mediated NETosis in vitro.
METHODS
Isolated human blood neutrophils were exposed to PMA (6.25 ng/ml) in the presence or absence of CSC (40-80 µg/ml) for 90 min at 37oC. NET formation was measured using a spectrofluorimetric procedure to detect extracellular DNA and fluorescence microscopy was used to visualize nets. Oxygen consumption by PMA-activated neutrophils was measured using an oxygen sensitive electrode.
RESULTS
Activation of neutrophils with PMA was associated with induction of NETosis that was significantly attenuated in the presence of CSC (40 and 80 µg/ml), with mean fluorescence intensities of 65% and 66% of that observed with untreated cells, respectively, and confirmed by fluorescence microscopy. The rate and magnitude of oxygen consumption by activated neutrophils pre-treated with CSC (80 µg/ml) was significantly less than that observed with untreated cells (73% of the control system), indicative of decreased production of reactive oxidants in the presence of CSC.
CONCLUSION
The inhibition of NETosis observed in the presence of CSC correlated with attenuation of oxygen consumption by PMA-activated neutrophils suggesting a mechanistic relationship between these events. If operative in vivo, smoking-related attenuation of NETosis may impair host immune responses and increase the risk of respiratory infections.
Topics: Extracellular Traps; Humans; Microscopy, Fluorescence; Neutrophils; Oxygen Consumption; Phorbol Esters; Reactive Oxygen Species; Smoking; Tobacco Smoke Pollution
PubMed: 29085418
DOI: 10.4314/ahs.v17i3.33 -
The Journal of Biological Chemistry May 1985In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of...
In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.
Topics: Astrocytoma; Calcium; Carbachol; Cells, Cultured; Humans; Hydrolysis; Phorbol Esters; Phorbols; Phosphatidylinositols; Receptors, Muscarinic; Tetradecanoylphorbol Acetate
PubMed: 2985584
DOI: No ID Found -
Journal of Neurochemistry Jun 2008We examined the mechanisms involved in protein kinase C (PKC)-dependent down-regulation of dopamine transporter (DAT) activity and cell surface expression by treating... (Comparative Study)
Comparative Study
We examined the mechanisms involved in protein kinase C (PKC)-dependent down-regulation of dopamine transporter (DAT) activity and cell surface expression by treating heterologously expressing cells with the clathrin-mediated endocytosis inhibitor concanavalin A (Con A) or the cholesterol depleter/membrane raft disrupter methyl-beta-cyclodextrin (MbetaC) prior to treatment with the PKC activator phorbol 12-myristate, 13-acetate (PMA). Con A blocked PMA-induced surface reductions of DAT but only partially inhibited down-regulation, while MbetaC partially blocked down-regulation but did not inhibit loss of cell surface DAT, demonstrating that PKC-induced DAT down-regulation occurs by a combination of trafficking and non-trafficking processes. Using density-gradient centrifugation, we found that DATs are distributed approximately equally between Triton-insoluble, cholesterol-rich membrane rafts and Triton-soluble non-raft membranes. DATs in both populations are present at the cell surface and are active for dopamine and cocaine binding. PMA-induced loss of cell surface DAT occurred only from non-raft populations, demonstrating that non-raft DATs are regulated by trafficking events and indicating the likelihood that the cholesterol-dependent non-trafficking regulatory mechanism occurs in rafts. PMA did not affect the DAT raft-non-raft distribution but stimulated the phosphorylation of DAT to a substantially greater level in rafts than non-rafts. These findings reveal a previously unknown role for cholesterol in DAT function and demonstrate the presence of distinct subcellular DAT populations that possess multiple regulatory differences that may impact dopaminergic neurotransmission.
Topics: Animals; Cholesterol; Dopamine Plasma Membrane Transport Proteins; Humans; LLC-PK1 Cells; Membrane Microdomains; Phorbol Esters; Phosphorylation; Protein Transport; Rats; Swine
PubMed: 18248623
DOI: 10.1111/j.1471-4159.2008.05262.x