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)

Antigen binding to its specific receptor on T cells initiates a series of intracellular events that result in cell differentiation, activation, and clonal expansion. However, the mechanism by which these antigen-occupied receptors induce the transmembrane signal transduction needs clarification. Because this mechanism appears to involve an increase in intracellular free Ca2+ concentration and activation of protein kinase C (PKC), we tested the effect of Ca2+ ionophores and PKC activators on alloantigen-specific primary mixed leukocyte culture cells. Both calcium ionophores, A23187 and ionomycin, in conjunction with 12-O-tetradecanoylphorbol 13-acetate (TPA) mimicked the effect of antigen or interleukin 2 (IL 2) by inducing strong proliferative and alloantigen-specific cytotoxic responses. In addition, Ca2+ ionophore and TPA induced IL 2 receptor expression and IL 2 secretion. The capacity of other phorbol esters or a non-phorbol ester tumor promoter (teleocidin) to replace TPA in induction of cell activation correlated with their ability to bind to and to activate PKC. In addition, the synergistic effect of Ca2+ ionophore and TPA was blocked by either a Ca2+ chelator (EGTA) or cAMP, which is thought to inhibit phosphatidylinositol metabolism. To determine whether the induction of this cytotoxic activity was mediated by a direct effect of Ca2+ ionophore and TPA on cytotoxic T (Tc) cells or was secondary to IL 2 secretion by activated helper T (Th) cells, we tested the effect of Ca2+ ionophore and TPA on isolated populations of cloned, alloantigen-specific Th and Tc cells. Both agents induced cell proliferation and IL 2 production by Th cells, but not by Tc cells. Activation of mixed clones of Th and Tc cells, but not of Tc cells alone, resulted in cytotoxic activity, an effect that could be blocked by anti-IL 2 receptor antibodies. The results thus demonstrate that an increased concentration of intracellular Ca2+ in conjunction with PKC activation can bypass the signal provided by antigen-receptor interaction on Th cells, but does not substitute for IL 2 in activating cytotoxicity by isolated Tc cells.
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PMID:Tumor promoters in conjunction with calcium ionophores mimic antigenic stimulation by reactivation of alloantigen-primed murine T lymphocytes. 299 31

Phytohemagglutinin (PHA) and a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act synergistically to induce interleukin 2 (IL2) mRNA in human lymphocytes in vitro. The induction was inhibited by a potent inhibitor of protein kinase C (C-kinase), 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) at less than 10 microM. H-7 inhibited C-kinase activity itself in lymphocytes at the same range of the concentration but did not interfere with the translocation of C-kinase from the cytosol to the membrane fraction of the lymphocytes induced by TPA. H-7 is also known to inhibit cAMP-dependent protein kinase (A-kinase) and cGMP-dependent protein kinase (G-kinase). However, the lymphocytes cultured with dibutyryl cAMP or dibutyryl cGMP could not be activated to produce IL2 mRNA. These results show that activation of C-kinase but not A-kinase and G-kinase is necessary in signal transduction for IL2 gene expression. Prostaglandin E2, which is known to elevate intracellular cAMP level, also inhibited IL2 mRNA induction in the lymphocytes stimulated with PHA and TPA. Addition of alpha-methylornithine and methylglyoxal bis (guanyl hydrazone), which inhibit polyamine synthesis, did not affect the induction of IL2 mRNA in the lymphocytes stimulated with PHA and TPA, indicating that polyamine synthesis is not necessary for IL2 mRNA induction.
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PMID:Induction and regulation of human interleukin 2 gene expression: significance of protein kinase C activation. 302 5

Mouse thymocytes treated with the lectin Concanavalin A do not proliferate nor do they develop responsiveness to interleukin 2. Co-treatment with Concanavalin A and either lectin-activated splenocyte conditioned medium or phorbol ester caused increased interleukin 2 receptor expression and proliferation. Under these conditions, lectin alone stimulated a 3.4 fold increase in phosphatidylinositol turnover which was unaffected by the presence of conditioned medium. Phosphorylation of a 55 kD protein was stimulated in response to conditioned medium or phorbol ester, but not lectin. These results indicate that stimulation of phosphoinositide turnover is not sufficient to activate thymocytes, and suggest that costimulating factors activate a kinase which is distinct from protein kinase C, or alternatively, activate protein kinase C through a process which is not coupled to phosphoinositide turnover.
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PMID:Stimulation of phosphatidylinositol turnover by concanavalin A is not sufficient to activate mouse thymocytes. 302 88

The murine T lymphoma line, LBRM-33 1A5, requires synergistic signals delivered by phytohemagglutinin (PHA) and interleukin 1 (IL1) for activation to high level interleukin 2 production. The activation signals provided by PHA and IL1 were replaced by the Ca2+ ionophore, ionomycin, and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), respectively. These observations supported a two-signal model for T cell activation involving increases in intracellular Ca2+ concentration ([Ca2+]i) (signal 1) and activation of protein kinase C (signal 2) as necessary and sufficient events. However, biochemical analyses revealed that additional signals were involved in the activation of LBRM-33 cells by both receptor-dependent and -independent mediators. Both signal 1-type mediators, PHA and ionomycin, exerted pleiotropic effects at the concentrations required for synergy with signal 2-type mediators (IL1, TPA). Within 1-2 min of addition, PHA stimulated phospholipid turnover, including hydrolysis of phosphatidylinositol 4,5-bisphosphate, Ca2+ mobilization, and protein kinase C activation. The [Ca2+]i increase induced by PHA was due to influx from both intracellular and extracellular Ca2+ pools. Similarly, ionomycin increased phospholipid turnover, [Ca2+]i, and directly affected protein kinase C activity in LBRM-33 cells. In contrast, the signal 2-type mediators, TPA and IL1, appeared to act by distinct intracellular mechanisms. TPA induced a protracted association of cellular protein kinase C with the plasma membrane, consistent with the two-signal activation model. Furthermore, acute TPA treatment inhibited PHA-stimulated inositol phosphate release and Ca2+ mobilization, suggesting that this mediator partially antagonized signal 1 delivery. IL1, in contrast, neither activated protein kinase C directly nor did it positively modulate the coupling of signal 1-type mediators to [Ca2+]i or protein kinase C via the phosphoinositide pathway. The intracellular signal delivered by IL1 is, therefore, generated through a mechanism distinct from or distal to the activation of protein kinase C. These studies indicate that the two-signal hypothesis, in its simplest form, is inadequate to explain the signals required for the initiation of IL1-dependent T cell activation.
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PMID:Transmembrane signaling during interleukin 1-dependent T cell activation. Interactions of signal 1- and signal 2-type mediators with the phosphoinositide-dependent signal transduction mechanism. 302 93

Since evidence indicates that phorbol ester-induced production of interleukin 2 requires transcription, we investigated the possibility that the phorbol ester receptor acts directly in the nuclei of EL4 thymoma cells. Using a procedure that minimized plasma membrane contamination (as measured by 5'-nucleotidase activity) and maintained the integrity of the double nuclear membrane, we were unable to detect specific binding of [3H]phorbol 12,13-dibutyrate in nuclei of unstimulated cells. Treatment of cells with phorbol 12,13-dibutyrate (100 nM, 37 degrees C) for up to 6 h did not cause appearance of phorbol ester binding capacity in nuclei (4 +/- 8% of homogenate value; 5'-nucleotidase activity = 10 +/- 3%) despite translocation of 40% of the cytosolic binding capacity to the plasma membrane fraction. The failure to detect nuclear binding capacity in treated cells was not due to occupation of nuclear sites with unlabeled ligand; effective exchange binding was demonstrated by recovery of total homogenate binding capacity in treated cells of 82 +/- 13% of that in untreated cells. Treatment of isolated nuclei with DNase to liberate DNA binding proteins also failed to reveal any nuclear phorbol ester binding capacity. Assay of nuclei for protein kinase C enzymatic activity gave similar negative results. These data argue strongly against a direct action of the intact phorbol ester receptor (or the phorbol ester binding fragment) in the transcriptional activation of interleukin 2 in EL4 cells but cannot rule out the possibility of a role for the catalytic fragment.
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PMID:Absence of protein kinase C in nuclei of EL4 mouse thymoma cells. 303 47

We have investigated the inhibitory potential of prostaglandin E2 (PGE2) with respect to intracellular messengers implicated in the signaling system of T-lymphocyte activation pathway. Using the fluorescent indicator Quin 2, it is demonstrated that PGE2 inhibits the increase in cytosolic-free calcium concentration [Ca2+]i. Reconstitution of calcium mobilization in the presence of PGE2 by the calcium ionophore A23187 results in a partial restoration of both interleukin 2 (IL2) production and cell proliferation and has no effect on the inhibition of transferrin receptor expression. In contrast, the treatment of cell cultures with the tumor promotor 12.0 tetra decanoyl phorbol-13-acetate (TPA) abrogates the suppressor activity of PGE2. When T lymphocyte stimulation is provided by the combination of A23187 and TPA, the PGE2 inhibitory effect does not occur. These data also indicate that the down regulation of transferrin receptor by PGE2 is proximal to protein kinase C activation and is not associated with decreased expression of the functional IL2 receptor.
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PMID:Analysis of prostaglandin E2 effect on T lymphocyte activation. Abrogation of prostaglandin E2 inhibitory effect by the tumor promotor 12.0 tetradecanoyl phorbol-13 acetate. 303 54

We have studied the activation of interleukin 1 (IL 1)-dependent and IL 1-independent T cell lines, specifically their capacity to produce and secrete interleukin 2 (IL 2). The IL 1-dependent T cell lymphoma LBRM33-1A5.47, which requires phytohemagglutinin (PHA) and IL 1 to produce IL 2, was compared with the IL 1-independent T cell lymphoma LBRM33-5A4 and T cell hybridomas DO-11.10/S4.4 and 3DO-54.8. The latter hybridomas do not require exogenous IL 1 to produce IL 2 in response to mitogens or ovalbumin (OVA)/I-Ad. Even though IL 1 is not required by these IL 1-independent T cell lines, we tested whether IL 1 could modulate their response but found no significant effect of exogenous IL 1. We then studied the activation of these T cell lines by the calcium ionophore A23187 and phorbol myristate acetate (PMA). In the case of the IL 1-dependent line LBRM33-1A5.47, there was a strong response when both A23187 and PMA were used simultaneously. We subsequently found that A23187 can replace PHA, and PMA can replace IL 1 in the activation of this cell line to IL 2 production. These observations suggest that the signal(s) provided by PHA and IL 1 involve at least in part a calcium flux, and activation of protein kinase C. Parallel experiments with the use of the IL 1-independent T cell lines showed a strong response to both agents when used simultaneously. A modest response observed to A23187 alone was always enhanced by the addition of PMA. No response was observed to PMA alone. IL 1-rich P388D1 supernatant could replace the enhancing effect of PMA in the response of the IL 1-independent T cell lines. We suggest that the activating signals provided by A23187 and PMA are at least part of the sequence of events that lead to production of IL 2 in either IL 1-dependent or IL 1-independent T cell lines. In IL 1-independent T cell lines, however, both of the activating signals studied may be delivered through stimulation of the Antigen-MHC T cell receptor.
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PMID:Activation of IL 1-dependent and IL 1-independent T cell lines by calcium ionophore and phorbol ester. 307 93

The role of the accessory cell in optimizing T cell proliferative responses to mitogens is a well known but poorly understood phenomenon. To further dissect the function of the accessory cell in allowing T cell proliferation, we compared mitogen-induced c-myc, interleukin 2 (IL 2), and IL 2 receptor gene expression in peripheral blood mononuclear cells (PBMC) and in T cells rigorously depleted of accessory cells through differential adherence and anti-Dr (anti-class II major histocompatibility antigen) monoclonal antibody complement-directed cytotoxicity. In cultures stimulated with phytohemagglutinin (PHA), a mitogen that requires accessory cells to induce T cell proliferation, expression of all measured genes was accessory cell dependent, since accumulation of their mRNA in PBMC was greater than that in cultures depleted of accessory cells. These genes varied in their accessory cell dependence, with IL 2 expression most dependent, c-myc expression least dependent, and IL 2 receptor expression intermediate in dependency. Use of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or ionomycin, mitogens that stimulate T cell proliferation independent of accessory cells, induced equal levels of gene expression in PBMC and in T cells depleted of accessory cells. These results suggest that PHA-stimulated T cells are dependent on an accessory cell signal(s) for optimal expression of the genes for c-myc, IL 2, and IL 2 receptor, and for proliferation. In addition, this signal(s) appears to be delivered early in the course of T cell activation events, since it can be bypassed by mitogens that directly activate protein kinase C (TPA) or induce calcium translocation (ionomycin). In addition, these data provide further evidence that expression of the c-myc protooncogene is insufficient for T cell mitogenesis, since PHA-induced accumulation of c-myc mRNA was only partially accessory cell dependent, whereas proliferation was completely accessory-cell dependent.
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PMID:The role of the accessory cell in mitogen-stimulated human T cell gene expression. 308 9

Numerous biochemical events precede the proliferation of primary lymphocytes stimulated by mitogenic lectins in the presence of macrophages. Various compounds can activate parts of this response. Specifically the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, (TPA), can replace the requirement for macrophages, apparently by mimicking the macrophage product interleukin 1 (IL1). Wheat germ agglutinin (WGA), itself a non-mitogenic lectin, is reported to cause a calcium flux, phosphatidylinositol turnover, and enhance interleukin 2 (IL2) synthesis. In spite of these positive responses, WGA inhibits DNA synthesis caused by mitogenic lectins. Nevertheless, in this study, we tested the possibility that together TPA and WGA could complement and bring about DNA synthesis. This prediction turned out to be true. The combination of two non-mitogenic compounds resulted in lymphocyte proliferation. The TPA overcame the inhibitory effects of WGA. Moreover, macrophages were not required. The TPA also synergized with the calcium ionophores A23187 or ionomycin to cause lymphocyte proliferation in the absence of macrophages. WGA and the ionophores together did not cause proliferation, a finding which suggested that they fulfill the same roles. These observations led us to conclude that at least two signals were required for lymphocyte stimulation. One signal caused the mobilization of calcium and the other signal circumvented the need for macrophages or macrophage products possibly by mimicking diacylglycerol, the activator of protein kinase C.
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PMID:Phorbol ester circumvents the need for macrophages as well as for mitogenic lectins in the stimulation of lymphocytes with wheat germ agglutinin or the calcium ionophores A23187 or ionomycin. 309 74

Isolated and combined effects of the calcium ionophore A23187 and of the protein kinase C activator phorbol myristate acetate (PMA) on T cell activation parameters were analysed on unprimed Balb/c lymph node T lymphocytes (LNL). High doses of PMA were mitogenic for resting T cells, but non-mitogenic doses of PMA induced T cell proliferation in combination with A23187, which was non-mitogenic by itself. Mitogenesis induced by a combination of A23187 and PMA (A23187/PMA) showed the following characteristics: it was not abolished after extensive depletion of accessory cells; purified L3T4+, but not Lyt2+ T cells responded in the absence of accessory cells; mitogenesis was completely blocked by a mixture of two monoclonal antibodies directed to the murine interleukin 2 (IL-2) receptor (7D4/3C7mAbs); cyclosporin A, dibutyril cyclic AMP, and T cell K+ channel blockers quinine and verapamil all blocked mitogenesis. A marked synergism between A23187 and PMA was noted in induction of T cell enlargement, IL-2 release, and induction of IL-2 responsiveness. No synergism was noted in IL-2 receptor expression, A23187 and PMA being able to induce IL-2 receptors alone. Calcium ionophore induced IL-2 receptor expression, but failed to induce IL-2 release and IL-2 responsiveness. Addition of A23187/PMA to the IL-2-dependent CTL-L clone did not result in cell proliferation. Addition of A23187/PMA to Con A-activated T cell blasts leads to a vigorous proliferative response. This response is blocked by 7D4/3C7 mAbs, indicating a role for endogenously produced IL-2 in this case. The results indicate that T cell mitogenesis by A23187/PMA is IL-2-dependent, and suggest a critical role for protein kinase C in IL-2 release and induction of IL-2 responsiveness. In addition, the data suggest distinct, but co-operative pathways of IL-2 receptor induction, controlled by elevated Ca2+ alone and by protein kinase C. Subsequent intracellular events of T cell activation by A23187/PMA may be quite similar to those triggered by Con A, since both kinds of stimulation are blocked by agents such as cyclosporin A, dbcAMP and K+ channel blockers.
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PMID:Analysis of isolated and combined effects of calcium ionophore and phorbol ester on T lymphocyte activation. 309 19


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