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)

The human erythroleukemia cell line (HEL) has been used as a model system for studying signal transduction processes as they might relate to platelet/megakaryocyte function. We were interested in examining the role of thrombin in the regulation of adenylyl cyclase in this cell line. As opposed to its predominantly inhibitory effects on cyclic AMP production in platelets or in membranes from HEL cells, our initial experiments in intact HEL cells revealed that thrombin markedly potentiated the cyclic AMP response to prostaglandin E1 (2.9 +/- 0.2-fold), prostacyclin (1.9 +/- 0.2-fold) and carbacyclin (2.5 +/- 0.5-fold), measured either by radioimmunoassay or by the [3H]adenine preloading procedure. Thrombin, although ineffective alone, also potentiated cyclic AMP production stimulated by vasoactive intestinal peptide (1.6 +/- 0.2-fold), cholera toxin (3.0 +/- 0.6-fold) and AIF4- (2.3 +/- 0.6-fold), but not by forskolin (0.9 +/- 0.1-fold). The thrombin effect 1) produced an increase in the efficacy of the prostaglandins with no change in potency; 2) was long-lived; 3) required the proteolytic activity of thrombin; 4) was insensitive to pertussis toxin; and 5) was at least partially mimicked by trypsin, extracellular ATP and UTP, platelet activating factor and activators of protein kinase C. Down-regulation of protein kinase C or pre-exposure to the protein kinase inhibitor staurosporine blocked the potentiating effect. Together, these results suggest that in HEL cells, the mechanism of thrombin potentiation of cyclic AMP production may involve alterations in the interaction between stimulatory guanine nucleotide binding protein and the catalytic subunit of adenylyl cyclase, possibly involving protein kinase C-mediated phosphorylation.
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PMID:Potentiation of cyclic adenosine monophosphate production by thrombin in the human erythroleukemia cell line, HEL. 133 12

Platelets are released into the peripheral circulation from the bone marrow where they arise as fragments of megakaryocyte cytoplasm. To characterize the effects of platelet agonists on megakaryocytes, we examined calcium signaling and desensitization to thrombin, the thromboxane A2 (TxA2) mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), and platelet-activating factor (PAF) in cultured CHRF-288-11 megakaryocytic cells. Initially, we compared agonist-stimulated calcium transients in fura-2-loaded CHRF-288-11 cells and human platelets. The 50% effective concentration values for the agonists to increase free cytosolic calcium were as follows: thrombin (0.11 +/- 0.02 U/ml in CHRF, 0.19 +/- 0.03 U/ml in platelets), U46619 (147 +/- 33 nM in CHRF, 157 +/- 5 nM in platelets), and PAF [15 +/- 2 nM in CHRF, 16 +/- 2 nM in platelets (n = 4 each)]. CHRF-288-11 thrombin, TxA2, and PAF receptors were demonstrated to be coupled to phospholipase C because each of the agonists stimulated phosphatidylinositol hydrolysis in myo-[3H]inositol-loaded CHRF-288-11 cells and pharmacological inhibition of phospholipase C-blunted agonist-stimulated calcium signaling. CHRF-288-11 cells exposed to the three agonists for 1 h showed different patterns and extent of homologous and heterologous desensitization. Protein kinase C activation appeared to be necessary but not sufficient for desensitization because 1) activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited the calcium responses to all three agonists, 2) inhibition of protein kinase C with staurosporine attenuated subsequent desensitization to each agonist, and 3) each agonist increased protein kinase C activity in CHRF-288-11 cell homogenates.
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PMID:Differential megakaryocytic desensitization to platelet agonists. 141 71

A role for second messenger-regulated protein kinases in the early post-IL-3 receptor signal transduction pathway was investigated in the mast cell/megakaryocyte line R6-XE.4. The activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was assessed by the ability of the enzyme to phosphorylate histone H1 in the presence of calcium, diacylglycerol, and phosphatidylserine or after proteolytic activation of PKC with trypsin. In high serum-supplemented cells, but not in cells that were preincubated in serum-deficient media for 6 h, subsequent treatment for 15 min with synthetic IL-3 (10 micrograms/ml) caused up to a sixfold increase in the calcium- and lipid-stimulated histone H1 phosphorylating activity of particulate-associated PKC after fractionation on MonoQ. However, there was no corresponding reduction of cytosolic PKC activity. Therefore, IL-3 appeared to modify the activity of preexisting membrane-associated PKC rather than eliciting its recruitment from the cytoplasm in R6-XE.4 cells. This was in contrast to the situation with FDC-P1 cells, where IL-3 induced PKC translocation. IL-3 also stimulated a cytosolic protein kinase that phosphorylated a synthetic peptide patterned after a phosphorylation site in ribosomal protein S6, but this IL did not alter the activity of cAMP-dependent protein kinase.
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PMID:IL-3-induced activation of protein kinases in the mast cell/megakaryocyte R6-XE.4 line. 230 40

In the present study, we examined the effect of prostaglandin E1 (PGE1) on Ca2+ mobilization in a human megakaryocyte (the progenitor of platelets) leukemia cell line, designated as CMK. PGE1 caused a rapid and dose-dependent increase in the intracellular free calcium level ([Ca2+]i) associated with the elevation of cyclic AMP. The PGE1-induced elevation of [Ca2+]i was decreased by the prior addition of ethylene glycol bis(2-aminoethylether)tetraacetic acid to the medium by approximately 25% of the control. This result indicates that the PGE1-induced elevation of [Ca2+]i is due to influx of Ca2+ from the external medium and to mobilization of Ca2+ from intracellular stores. Pretreatment of CMK cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), a stimulus for protein kinase C, further enhanced the PGE1-induced increase in the cellular cyclic AMP level. Inversely, pretreatment of CMK cells with TPA (10 nM), prior to the addition of PGE1, inhibited the PGE1-induced elevation of [Ca2+]i. Dibutyryl cyclic AMP and forskolin did not elevate [Ca2+]i or affect the PGE1-induced Ca2+ mobilization. The inhibitory action of TPA in the PGE1-induced elevation of [Ca2+]i was mimicked by other protein kinase C-activating agents, such as 1-oleoyl-2-acetylglycerol and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, and was selectively restored by protein kinase C inhibitors, such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride. Thus, the inhibitory modulation of TPA on the PGE1-induced elevation of [Ca2+]i is mediated through protein kinase C activation. PGE1 had no inductional effect of megakaryocytic phenotypic changes in CMK cells. The biological role of PGE1, which increased [Ca2+]i and cyclic AMP levels in the CMK cells, remains to be determined.
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PMID:Elevation of intracellular calcium ion by prostaglandin E1 and its inhibition by protein kinase C in a human megakaryocyte leukemia cell line. 273 22

Understanding of the events following factor-mediated megakaryocyte development is hindered by a lack of adequate quantities of purified progenitor cells or progenitor cell lines. In order to study the intracellular processes activated during development, probes of various signal transduction systems are used to perturb megakaryocyte colony formation. Studies utilizing tumor promoting phorbol diesters and calcium ionophores show that protein kinase C and calcium mobilization (and/or influx) are activated during megakaryocyte development. Examination of the adenylate cyclase complex, with agonists such as cholera toxin etc., implicate cyclic AMP as another mediator of growth-factor responsiveness. Finally, synergistic interactions occur between the calcium-protein kinase C system and the adenylate cyclase complex. These observations corroborate the multifactorial regulation of megakaryocytopoiesis postulated by Williams and coworkers. The data further provide an intracellular mechanism(s) by which megakaryocytic growth factors regulate cellular development.
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PMID:Signal transduction events in in vitro megakaryocytopoiesis. 278 1

The hemopoietic growth factor, interleukin 3, has been shown to activate protein kinase C without causing hydrolysis of inositol phospholipids. The potential involvement of phosphatidylcholine hydrolysis as an alternative source of diacylglycerol was investigated in an interleukin 3-dependent murine mast/megakaryocyte cell line, R6-XE.4. Treatment of these cells with interleukin 3 rapidly stimulated both the release of water-soluble choline metabolites and the resynthesis of phosphatidylcholine. Therefore, a phosphatidylcholine cycle may operate as part of the signal transduction pathway in cells responding to interleukin 3.
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PMID:Interleukin 3 stimulates phosphatidylcholine turnover in a mast/megakaryocyte cell line. 281 89

Guinea pig bone marrow megakaryocytes were cultured on a type I rat tail collagen gel which stimulated proplatelet formation. Proplatelet formation was inhibited by monoclonal antibody LM609 to the alpha v beta 3 integrin (VnR), but not by monoclonal antibodies to the alpha 5, alpha 6, beta 1, or IIb beta 3(GPIIb-IIIa) integrin proteins. Megakaryocytes cultured on a plastic surface and stimulated with thrombin undergo a spreading and an adhesion reaction. This reaction is blocked in a dose-dependent manner by the tetrapeptide RGDS and by the monoclonal antibody PG2 to the GPIIb-IIIa integrin, but not by the monoclonal antibody LM609 to the VnR. Immunoprecipitation and affinity chromatography experiments demonstrate that guinea pig megakaryocytes have distinct GPIIb-IIIa and VnR integrins with similar electrophoretic mobility. Spreading was significantly inhibited in a dose-dependent fashion by drugs which elevate cellular cyclic AMP, including forskolin, dibutyryl cAMP, and isobutylmethylxanthine. In contrast to spreading, megakaryocyte proplatelet formation was stimulated by these agents in a dose-dependent manner. Megakaryocyte spreading was stimulated by the protein kinase C (PKC) activator phorbol myristate acetate (PMA) and inhibited by the PKC inhibitors Calphostin C and K5720 in a dose-dependent manner. PKC inhibitors did not inhibit megakaryocyte proplatelet formation. These results demonstrate that the closely related VnR and GPIIb-IIIa integrins regulate different aspects of megakaryocyte morphological change and appear to be associated with different second messenger systems.
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PMID:Differential regulation of integrin-mediated proplatelet formation and megakaryocyte spreading. 753 14

Thrombin initiates many physiological processes in platelets and other megakaryocyte-lineage cells by interacting with surface receptors and generating rises in cytoplasmic Ca2+; these rises result from both Ca2+ release from intracellular stores and receptor-mediated Ca2+ entry. Regulators that limit Ca2+ entry after its initiation by thrombin have not been identified. In this study, prevention of expression of a single protein kinase C isoenzyme (PKC beta) by antisense cDNA overexpressed in HEL cells, a human megakaryoblastic cell line that expresses thrombin receptors, promotes thrombin receptor-mediated Ca2+ entry without altering thrombin-induced intracellular release of Ca2+. The cytoplasmic Ca2+ rise initiated by endoperoxide analogs was not affected by inhibiting PKC beta. Overexpression of a cDNA encoding wild-type PKC beta mutated to prevent recognition by the antisense cDNA abolished the enhancement of Ca2+ influx following thrombin. Thus, PKC beta appears to be a specific negative regulator of thrombin receptor-mediated Ca2+ entry.
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PMID:Selective inhibition of thrombin receptor-mediated Ca2+ entry by protein kinase C beta. 759 74

Extracellular application of ATP and ADP evoked the oscillatory K+ currents (IKCa) resulting from the periodic rise in cytoplasmic Ca2+ concentration ([Ca2+]i) of megakaryocyte isolated from rat bone marrow (Uneyama, H., Uneyama, C., and Akaike, N. (1992) Jpn. J. Pharmacol. 58, 231). The intracellular mechanism of ATP-induced cytoplasmic Ca2+ oscillation was investigated by the use of nystatin-perforated patch-clamp technique. Caffeine and ryanodine, which release Ca2+ from the Ca(2+)-induced Ca2+ release pool (CICR), and procaine, a blocker of Ca2+ release from CICR, had no effect on the IKCa oscillation in megakaryocyte. Thapsigargin and A23187 activated IKCa irreversibly by mobilizing Ca2+, but under Ca(2+)-free conditions they activated IKCa transiently. Intracellular application of inositol 1,4,5-triphosphate (IP3) also induced IKCa oscillation. Phorbol myristate acetate (PMA), a strong activator of protein kinase C (PKC), inhibited the oscillation completely. The inhibitory action of PMA was reversed by an inhibitor of PKC, staurosporin. The oscillation of ATP-induced IKCa was disrupted by staurosporin or calmodulin (CaM) antagonists such as W-7 and trifluoperazine, resulting in a transient and successive plateau-like IKCa. These results suggest that Ca2+ oscillation in megakaryocyte is caused by the interaction of both the Ca2+ release from IP3-sensitive Ca2+ pool and the Ca2+ uptake stimulated by PKC and Ca2+/CaM complex. Furthermore, forskolin, an activator of adenylate cyclase, and isobutylmethylxanthin, an inhibitor of phosphodiesterase, inhibited the frequency, latency, and current amplitude of the oscillation in a concentration-dependent manner. These reagents inhibited IP3-induced oscillation as well as an ATP-induced oscillation. Thus, the ATP-induced [Ca2+]i oscillation of rat megakaryocyte is also modulated by cAMP.
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PMID:Intracellular mechanisms of cytoplasmic Ca2+ oscillation in rat megakaryocyte. 767 93

Colony formation of mouse primitive hemopoietic progenitors with interleukin-6 (IL-6) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and their signal transduction were studied. Although IL-6 or TPA alone could not form colonies, their combination gave rise to significant number of colonies from Day-2 post 5-FU bone marrow cells. When colony numbers were compared with those supported by IL-3, IL-6+TPA gave rise to 86 + 47% of colonies formed with IL-3. Time course of colony formation with IL-6+TPA run parallel with that of IL-3. These colonies included not only granulocyte/macrophage (GM) colonies, but also granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies and blast cell colonies. Delayed addition of IL-6 or TPA decreased colony numbers, suggesting that both IL-6 and TPA were needed from the start of cultures for maximal colony formation. When cultures were started with TPA, and IL-6 was added on Day 2 of culture or later, few colonies developed. These data suggested that IL-6 might be essential to the survival of the progenitors in culture. Chronic exposure of progenitors to TPA prior to the culture with IL-6+TPA suppressed colony formation. Addition of calphostin C, a specific protein kinase C (PKC) inhibitor or genistein and herbimycin A, specific tyrosine kinase (TK) inhibitors to the culture also decreased colony numbers formed with IL-6 and TPA. To clarify which effects of IL-6 or TPA on colony formation were blocked by the inhibitors, the inhibitors were added to preincubation of progenitors with IL-6. Both the PKC inhibitor and TK inhibitors blocked the increase of colonies resulted from a pre-incubation with IL-6. Although delayed addition of TPA enhanced IL-6-dependent colony formation, delayed addition of TPA with either the PKC inhibitor or TK inhibitors canceled the increase of colonies. These data suggested that both signals of IL-6 and TPA might be transduced via activation of PKC and TK, but further studies are needed to confirm that.
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PMID:[Colony formation of mouse primitive hemopoietic progenitors with interleukin-6 and phorbol ester, and their signal transduction]. 786 57


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