Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.
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PMID:Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1. 768 43

The plasminogen activator inhibitor PAI-1 is markedly elevated in vivo and in vitro upon exposure to the inflammatory mediators tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and bacterial lipopolysaccharide. Here we report that the isoflavone compound genistein prevents the increase in synthesis of PAI-1 induced by these inflammatory mediators in human endothelial cells in vitro, and partially reduces the basal PAI-1 production by these cells. These effects of genistein were accompanied by a decrease in PAI-1 mRNA and in a suppression of the PAI-1 transcription rate as shown by run-on assay. A specific action of genistein, probably by inhibiting a tyrosine protein kinase, is likely, because the structural genistein analogue daidzein, which has a low tyrosine protein kinase inhibitor activity, did not inhibit PAI-1 synthesis. Vanadate, a tyrosine protein phosphatase inhibitor, increased PAI-1 production. The effect of genistein on PAI-1 synthesis was rather selective. Herbimycin A also reduced PAI-1 synthesis, but several other tyrosine protein kinase inhibitors, namely tyrphostin A47, methyl-2,5-dihydroxy-cinnamate, and compound 5, were unable to do so. All these tyrosine protein kinase inhibitors reduced basic fibroblast growth factor (b-FGF)-induced [3H]thymidine incorporation in endothelial cells. This indicates that the effect of genistein on PAI-1 transcription proceeds independently of its effect on mitogenesis. In contrast to TNF-alpha-induced PAI-1 production, the transcription and synthesis of urokinase-type plasminogen activator (u-PA) was not inhibited by genistein. A TNF-alpha-mutant (Trp32Thr86TNF alpha) that specifically recognizes the 55-kD TNF-receptor, mimicked the effects of TNF alpha on both PAI-1 and u-PA. Because genistein affected PAI-1, but not u-PA induced by this mutant, involvement of different TNF-receptors cannot underlie the difference in the effects of genistein on PAI-1 and u-PA synthesis. Because genistein also inhibited PAI-1 induction by thrombin and IL-4, it is likely that genistein does not act on a TNF alpha-receptor-coupled protein kinase but on the signal transduction pathway enhancing PAI-1 transcription. Our results suggest that the TNF alpha-induced signal transduction pathway of PAI-1 transcription involves a genistein-sensitive step that is not involved in the induction of u-PA by TNF alpha. Given the limited sensitivity to several other tyrosine protein kinase inhibitors, this genistein-sensitive step may be a potential target for pharmacologic intervention to reduce elevated plasma PAI-1 levels.
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PMID:Genistein reduces tumor necrosis factor alpha-induced plasminogen activator inhibitor-1 transcription but not urokinase expression in human endothelial cells. 794 70

Two potent inhibitors of protein phosphatase type 1 (PP1) and type 2A (PP2A), calyculin A (CAL-A) and okadaic acid (OKA), inhibited human platelet aggregation induced by thrombin, collagen and 9,11-epithio-11,12-methano-thromboxane A2 (STA2). IC50 values of CAL-A and OKA for STA2-induced aggregation were 53 nM and 3.5 microM, respectively. These drugs also inhibited thrombin-induced [14C]serotonin secretion of platelets. CAL-A and OKA elicited phosphorylation of certain proteins with an apparent M(r) (x 10(-3) of 200, 60, 50 and 20 light chain of myosin (MLC). Agonist-induced 47,000 M(r) protein phosphorylation was strongly inhibited by these compounds, whereas phosphorylation of 20,000 M(r) MLC was enhanced. The increase in 50,000 M(r) protein phosphorylation by CAL-A and OKA was observed in the presence of agonists, and the 50,000 M(r) phosphorylation may be involved in the inhibition of platelet activation by these compounds. Subcellular analysis of the phosphatase activity in human platelets showed that MLC phosphatase activity was present mainly (approx. 78%) in the cytosolic fraction. Chromatography of human platelet extract on heparin-Sepharose resolved two peaks of MLC phosphatase activity: PP2A in 0.1 M NaCl eluate and PP1 in 0.5 NaCl eluate. PP2A and PP1 isozymes (PP1 alpha, PP1 gamma and PP1 delta) have also been identified in human platelets, by cross-reactivity with polyclonal antibodies against PP2A and PP1 isozymes, respectively. These results suggest that PP1 and/or PP2A may play an important role in the process of platelet activation by regulating levels of phosphorylation of certain proteins.
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PMID:Calyculin A and okadiac acid inhibit human platelet aggregation by blocking protein phosphatases types 1 and 2A. 801 29

Platelets activated by various agonists produce vesicles (microparticles; MPs) from the plasma membrane. However, the mechanism of this MP formation remains to be elucidated. To investigate the possible involvement of protein phosphorylation and cytoskeletal reorganization in MP formation, the effects of various inhibitors on MP formation were investigated. Flow cytometry was employed to detect the amount of MP formation by using monoclonal antibodies against glycoprotein (GP) IIb-IIIa (NNKY 1-32) or GPIIb (Tab). The relationship between changes in cytoskeletal architecture and MP formation in the platelets activated by thrombin plus collagen was observed by scanning electron microscopy (SEM). MPs were observed in the vicinity of the terminals of pseudopods, suggesting that MPs may be related by budding of the pseudopods. Cytochalasin D (10 microM) inhibited MP formation from the activated platelets almost completely. Moreover, SEM of the cytochalasin D-treated platelets revealed the absence of shape change, pseudopod formation and MPs. These findings suggest that cytoskeletal reorganization is necessary for MP formation. Since cytoskeletal reorganization is considered to be regulated by a dynamic phosphorylation-dephosphorylation process, we investigated the effects of the protein phosphatase inhibitors, calyculin A (CLA) and okadaic acid (OA), on MP formation. Flow cytometry showed that these two inhibitors doubled MP formation in activated platelets. SEM of the platelets treated with CLA or OA demonstrated more prominent shape change and pseudopod formation in these platelets than in those without inhibitor. From these results, we conclude that cytoskeletal reorganization, which is controlled by phosphorylation, is involved in MP formation.
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PMID:The role of protein phosphorylation and cytoskeletal reorganization in microparticle formation from the platelet plasma membrane. 816 55

Thrombin dramatically activated p72syk in a time- and dose- dependent fashion in extracts of resting porcine platelets in the presence of EDTA. Separation analysis using Sephacryl S-300 column chromatography has demonstrated that p72syk may exist as large (complex) and small (monomer) forms in resting platelets, and activation of p72syk was only observed in the fraction of large form. Pretreatment with ATP scavenger, GDP beta S and protein phosphatase inhibitors had no effect on this activation. Furthermore, washed immuno-precipitates of large form p72syk were also activated by thrombin or fibrinogen. These results suggest that p72syk may associate with thrombin receptor or other agonist receptors and there may be a novel activation mechanism of non-receptor type protein-tyrosine kinase, which does not require the modification by other protein kinases, protein phosphatases and GTP binding proteins.
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PMID:Activation of p72syk by thrombin in a cell-free system. 816 76

Protein phosphatase 1 is considered to be involved in thrombin-induced platelet activation (Murata et al., Biochem Int 26:327-334, 1992). To clarify the mechanism, we examined the effects of protein phosphatase 1 and 2A inhibitors (calyculin A, tautomycin, okadaic acid) on Ca2+ influx. In the presence of 1 mM Ca2+, thrombin- (0.1 U/ml) induced platelet aggregation and ATP release were inhibited by calyculin A, while this inhibitory effect was abolished in the absence of Ca2+ (EGTA 1 mM). Furthermore, thrombin-induced Mn2+ influx but not intracellular Ca2+ mobilization was inhibited by calyculin A in a dose-related manner. Calyculin A also blocked the ongoing Ca2+ influx when added 3 min after thrombin stimulation. Similar inhibitory effects were observed with okadaic acid and tautomycin in the same potency sequence as the reported one for protein phosphatase 1 (calyculin A > tautomycin > okadaic acid). These results suggest that the anti-platelet effects of phosphatase inhibitors are due to the inhibition of Ca2+ influx and that protein phosphatase 1 plays a key role in the regulation of receptor operated Ca2+ channel of human platelets.
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PMID:The possible involvement of protein phosphatase 1 in thrombin-induced Ca2+ influx of human platelets. 838 95

Calyculin A (CLA) and okadaic acid (OA), specific and potent inhibitors of protein phosphatase 1/2A, inhibit platelet aggregation. However, their inhibitory mechanisms remain unknown. We investigated the effects of CLA on the exposure of fibrinogen receptor in thrombin-stimulated platelets, using flow cytometry with a monoclonal antibody against the fibrinogen receptor of activated glycoprotein(Gp)IIb/IIIa complex (PAC-1). CLA inhibited the exposure of fibrinogen receptor in a dose related manner when added either before or 3 min after thrombin stimulation. In contrast, CLA had no significant effect when the expression of GpIIb/IIIa complex was examined in resting platelets, using a monoclonal antibody recognizing non-activated GpIIb/IIIa complex (NNKY1-32). These results suggest that protein phosphatase 1/2A may be directly involved in the exposure of platelet fibrinogen receptor.
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PMID:Calyculin A inhibits the exposure of fibrinogen receptor in thrombin-stimulated platelets. 839 22

Calcineurin, a protein phosphatase found in eukaryotic cells, presents a challenging problem in heterologous protein expression because it is both heterodimeric and posttranslationally modified. In this paper, we describe the cloning of both subunits (catalytic A and regulatory B) of calcineurin from a human cDNA library and their expression at high levels in Escherichia coli. The calcineurin A subunit is expressed as an insoluble glutathione S-transferase fusion protein, while the calcineurin B subunit is soluble upon direct expression. Catalytically active holoenzyme is derived from the separately expressed subunits using a three-step refolding protocol. First, the fusion protein is solubilized, then it is cleaved at the fusion junction with thrombin, and, finally, a catalytically competent calcineurin A:calcineurin B:calmodulin complex is reconstituted by cofolding the separately purified components. In addition, we show that a similar refolding protocol can be applied to a C-terminally truncated form of calcineurin A, which lacks an autoinhibitory and calmodulin-binding domain.
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PMID:Reconstitution of active human calcineurin from recombinant subunits expressed in bacteria. 853 59

Effects of the protein phosphatase inhibitors, tautomycin and calyculin A on protein phosphorylation and cytoskeleton of human platelets. It has been discovered recently that many cytotoxic compounds isolated from a variety of sources are potent phosphatase inhibitors. Two of these, tautomycin (TM) and calyculin-A (CL-A) were applied to human platelets to investigate the role of protein phosphorylation on cytoskeletal structure and function. Exposure to 10 microM TM or 0.1 microM CL-A induced marked morphological changes. The granules were centralized and surrounded by actin filaments, but there was no evidence of granule release. Myosin became more centralized, was occluded from the granulomere, but was not confined to the microfilament ring. These changes occurred without an increase in cytosolic Ca2+ concentrations, as determined by measurements with fura-2. TM and CL-A induced an overall increase in protein phosphorylation. Phosphorylation of the 20,000 dalton light chain of myosin increased markedly and multiple phosphorylation sites were indicated. Cytoskeletons were prepared from control, thrombin- and TM-treated platelets, the latter prepared in the absence of external calcium. The major difference in protein composition was the increased content of myosin associated with the cytoskeleton from TM-treated platelets where the dominant phosphoprotein was the 20,000 dalton light chain. These results suggest that myosin phosphorylation drives the initial shape changes, and via a contractile process results in the formation of the microfilament ring and centralization of granules.
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PMID:Effects of the protein phosphatase inhibitors, tautomycin and calyculin-A, on protein phosphorylation and cytoskeleton of human platelets. 858 89

The preproendothelin-1 (preproET-1) gene is induced by thrombin after phosphorylation of nonreceptor protein tyrosine kinase pathways. This study investigated the contribution of Ca2+/calmodulin-dependent intracellular signaling cascades to this pathway and measured ET-1 mRNA levels by Northern blot analysis in human endothelial cells. Increased intracellular Ca2+ levels in response to Ca2+ ionophore or Ca2+ ATPase inhibitors tert-butylhydroquinone and thapsigargin mimicked thrombin actions on ET-1 mRNA induction. Thrombin-mediated activation of ET-1 mRNA was reduced by specific calmodulin antagonists W7 or calmidazolium and after inhibition of CaM kinase II by KN-62. Inhibition of calcium/calmodulin-dependent phosphatase calcineurin by cyclosporin A, however, stimulated ET-1 mRNA in human endothelial cells. Phosphotyrosine immunoblot assays show that calcium/calmodulin-dependent signaling pathways precede thrombin-induced tyrosine phosphorylation, and that the calcium/calmodulin-dependent phosphatase calcineurin also exerts its effects via activation of protein tyrosine kinases. These observations demonstrate that thrombin stimulates the preproET-1 gene in human endothelial cells through calcium-dependent activation of CaM kinase and protein tyrosine kinases, and that calcineurin may also participate in regulation of the prepro ET-1 gene.
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PMID:Thrombin-mediated ET-1 gene regulation involves CaM kinases and calcineurin in human endothelial cells. 858 30


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