Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In response to hypoxia, sickle red blood cells (SS RBC) and leukocytes exhibit increased adherence to the vascular endothelium, while diapedesis of leukocytes through the blood vessel increases. However, the cellular signaling pathway(s) caused by hypoxia is poorly understood. We utilized CoCl2 as a mimetic molecule for hypoxia to study cellular signaling pathways. We found that in human umbilical vein endothelial cells (HUVEC), CoCl2 at 2 mM concentration induced the surface expression of a subset of CAMs (VCAM-1) and activation of transcription factor NF-kappaB in the nuclear extracts of HUVEC. Furthermore, CoCl2 also caused time-dependent tyrosine phosphorylation of mitogen-activated protein (MAP) kinase isoform ERK2 without significantly affecting ERK1, indicating ERK2 is the preferred substrate for upstream kinase of the MAPK pathway. Inhibitors of MAP kinase (PD98059) or platelet-activating factor (PAF)- receptor antagonist (CV3988) inhibited the CoCl2-induced NF-kappaB activation and VCAM-1 expression. Augmented expression of VCAM-1 led to increased SS RBC adhesion, inhibitable by a VCAM-1 antibody. Additionally, CoCl2 caused a two- to threefold increase in the rate of transendothelial migration of monocyte-like HL-60 cells and a twentyfold increase in phosphorylation of platelet endothelial cell adhesion molecules (PECAM-1). The transendothelial migration of monocytes was inhibited by an antibody to PECAM-1. Both phosphorylation of PECAM-1 and transendothelial migration of monocytes in response to CoCl2 were inhibited by protein kinase inhibitor (GF109203X) and augmented by protein phosphatase inhibitor (Calyculin A). Our data suggests that CoCl2-induced cellular signals directing increased expression of VCAM-1 in HUVEC involve downstream activation of MAP kinase and NF-kappaB, while the phosphorylation of PECAM-1 occurs as a result of activation of PKC. We conclude that PAF-receptor antagonist inhibits the CoCl2- or hypoxia-induced increase in the adhesion of SS RBC, PECAM-1 phosphorylation, and the concomitant transendothelial migration of monocytes.
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PMID:Cobalt chloride-induced signaling in endothelium leading to the augmented adherence of sickle red blood cells and transendothelial migration of monocyte-like HL-60 cells is blocked by PAF-receptor antagonist. 1008 34

The influence of the phosphorylation dephosphorylation states on the gamma-aminobutyric acid (GABA) transporter activity of synaptic plasma membranes (SPM) was studied by using either specific phosphatase inhibitors or activators. Calyculin A and okadaic acid (phosphatase 1 and phosphatase 2A inhibitors) inhibited the GABA uptake by isolated SPM vesicles, whereas cyclosporin A (phosphatase 2B inhibitor) had a stimulatory effect (approximately 10%) which was higher (approximately 38%) when all these drugs were present in the reaction medium. On the other hand, intravesicular Ca2+, up to about 10 microM, inhibited the GABA uptake (approximately 50%) in a manner which appeared to be facilitated in the presence of PP1 and PP2A inhibitors and this inhibition was relieved by the calmodulin antagonist W-7. We also observed that isolated SPM vesicles contain both Ca(2+)-independent phosphatase activity that is significantly inhibited by PP1 and PP2A inhibitors, and Ca(2+)-dependent phosphatase activity that is abolished in the presence of the PP2B inhibitor, cyclosporin A. These results indicate that regulation of the SPM GABA transporter is determined by the internally localized Ca-calmodulin-dependent phosphatase activity (calcineurin), and that other phosphorylated sites, sensitive to PP1 and PP2A inhibitors, potentiate either the positive or negative effects exerted by those internal sites when they are in their phosphorylated or dephosphorylated states, respectively.
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PMID:Regulation of the gamma-aminobutyric acid transporter activity by protein phosphatases in synaptic plasma membranes. 1009 70

1. Na+-K+-2Cl- cotransport (NKCC) was studied in turkey red cells using Na+ dependence or bumetanide sensitivity of 86Rb+ influx to monitor activity of the transporter. 2. Deoxygenation was the major physiological stimulus for NKCC activity: oxygen tensions (PO2) over the physiological range modulated the transporter, with a PO2 for half-maximal activation of about 41 mmHg (n = 3). In air, activity of NKCC was also stimulated by shrinkage and isoproteronol (isoprenaline, 5 microgr;M). By contrast, in deoxygenated cells, although the transporter activity was markedly elevated, it was no longer sensitive to volume or beta-adrenergic stimulation. 3. Calyculin A, a protein phosphatase inhibitor, stimulated cotransport with a lag of about 5 min. N-Ethylmaleimide (NEM) inhibited cotransport and also blocked the stimulatory effect of calyculin A if administered before calyculin A. Stimulation by calyculin A and deoxygenation were not additive. Staurosporine (2 microM) inhibited deoxygenated-stimulated K+ influxes, but not those stimulated by calyculin A. NEM added during calyculin A stimulation, i.e. during the 5 min lag, caused transport activity to be clamped at levels intermediate between maximal (calyculin A alone) and control. Cells treated with calyculin A alone or with calyculin A followed by NEM were no longer sensitive to volume, isoproteronol or PO2. 4. The results have characterized the interaction between deoxygenation and other stimuli of NKCC activity. They have also shown that it is possible to manipulate the transporter in a reciprocal way to that shown previously for K+-Cl- cotransport.
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PMID:Regulation of Na+-K+-2Cl- cotransport in turkey red cells: the role of oxygen tension and protein phosphorylation. 1033 92

The feasibility of chemically-induced premature chromosome condensation (PCC) was tested in two human fibroblast and two human malignant melanoma cell lines. To induce PCCs, calyculin A, a potent protein phosphatase inhibitor, was used at a final concentration of 80 nM. Attached cells and cells put into suspension by trypsinization were incubated with calyculin A at 37 degrees C for 1 hour. Calyculin A was able to induce PCCs in all the phases of the cell cycle with the tumour cell lines giving the highest PCC frequency. No systematic differences were observed between attached cells and cells put into suspension by trypsinization. However, a cytotoxic effect that led to the loss of 50% to 80% of the treated cells was observed. The cytotoxic effect was more severe in the fibroblast than in the tumour cell lines. The appearance of deformed, fragile and fragmented nuclei with no particular chromatin condensation would explain to some extent this cytotoxic effect. A better understanding of the mechanism of action of calyculin A would help generalizing its use to study interphase chromosome aberrations.
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PMID:Differential induction of premature chromosome condensation by calyculin A in human fibroblast and tumor cell lines. 1036 32

Calyculin A, a protein phosphatase inhibitor, enhanced phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2-) production and translocation of the cytosolic NADPH oxidase factor, p47phox, to the plasma membrane in guinea pig polymorphonuclear leukocytes (PMNs). When PMNs were treated with t-(5-isoquino-line-sulfonyl)-3-methyl-piperazine (H-7), a protein kinase C (PKC) inhibitor, after exposure to PMA, inhibition of O2- production and of translocation of p47phox to the membrane fraction in PMA-stimulated PMNs were observed. When calyculin A was added to the PMA-stimulated PMNs after the addition of H-7, O2- production was again observed, and translocation of p47phox to the membrane fraction also occurred. The activity of NADPH oxidase, the amount of p47phox and the level of phosphorylation of p47phox in the membrane fraction prepared from PMA-stimulated PMNs, were reduced by the addition of the cytosol fraction from unstimulated PMNs. These reductions were attenuated by calyculin A. These results indicate that the active form of NADPH oxidase in PMNs can be reconstituted after the active complex of the enzyme has disappeared once, and that one of the mechanisms of regulation of this enzyme activity involves the phosphorylation of p47phox in the cyotosol and dephosphorylation of phosphorylated p47phox in the NADPH oxidase complex by protein kinase and protein phosphatase, respectively.
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PMID:Participation of cytosolic protein phosphatase in regulation of NADPH oxidase in polymorphonuclear leukocytes. 1040 25

The role of nutritional factors during CryIVA protoxin expression in Bacillus thuringiensis israelensis (Bti) has been investigated. Inorganic phosphate (Pi) was found to stimulate 135 kD protoxin synthesis by Bti cells. There was a corresponding increase in the cryIVA specific mRNA in the presence of Pi. Inorganic phosphate inhibited HPr kinase but activated HPr phosphatase, the two enzymes responsible for regulating the concentration of phosphorylated HPr in the cell. Addition of protein phosphatase inhibitors NaF and calyculin A during resuspension resulted in the inhibition of toxin synthesis by Bti cells. Calyculin A inhibited HPr phosphatase activity in the in vitro assay also. The concentration of phosphorylated HPr was upregulated when the cells were resuspended in the presence of calyculin A, while the levels of the same were lowered in the presence of Pi, as determined by Western blotting the respective cells. The efficiency of sporulation of Bti was not affected when Pi was added alone or along with the phosphatase inhibitor calyculin A.
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PMID:Inorganic phosphate regulates CryIVA protoxin expression in Bacillus thuringiensis israelensis. 1046 80

During activation of platelets by thrombin phosphorylation of Thr(558) in the C-terminal domain of the membrane-F-actin linking protein moesin increases transiently, and this correlates with protrusion of filopodial structures. Calyculin A enhances phosphorylation of moesin by inhibition of phosphatases. To measure this moesin-specific activity, a nonradioactive enzyme-linked immunosorbent assay method was developed with the synthetic peptide Cys-Lys(555)-Tyr-Lys-Thr(P)-Leu-Arg(560) coupled to bovine serum albumin as the substrate and moesin phosphorylation state-specific polyclonal antibodies for the detection and quantitation of dephosphorylation. Calyculin A-sensitive and -insensitive protein-threonine phosphatase activities were detected in platelet lysates and separated by DEAE-cellulose chromatography. The calyculin A-sensitive enzyme was identified as a type 1 protein phosphatase. The calyculin A-insensitive enzyme activity was purified to homogeneity by phenyl- Sepharose, protamine-, and phosphonic acid peptide-agarose chromatography and characterized biochemically and immunologically as a 53-kDa protein(s) and a type 2C protein phosphatase (PP2C). Phosphorylation of Thr(558) is necessary for F-actin binding of moesin in vitro. The purified enzyme, as well as bacterially made PP2Calpha and PP2Cbeta, efficiently dephosphorylate(s) highly purified platelet phospho-moesin. This reverses the activating effect of phosphorylation, and moesin no longer co-sediments with actin filaments. In vivo, regulation of these phosphatase activities are likely to influence dynamic interactions between the actin cytoskeleton and membrane constituents linked to moesin.
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PMID:Protein phosphatase 2C inactivates F-actin binding of human platelet moesin. 1048 Aug 73

We cloned two novel Trypanosoma cruzi proteins by using degenerate oligonucleotide primers prepared against conserved domains in mammalian serine/threonine protein phosphatases 1, 2A, and 2B. The isolated genes encoded proteins of 323 and 330 amino acids, respectively, that were more homologous to the catalytic subunit of human protein phosphatase 1 than to those of human protein phosphatase 2A or 2B. The proteins encoded by these genes have been tentatively designated TcPP1alpha and TcPP1beta. Northern blot analysis revealed the presence of a major 2.3-kb mRNA transcript hybridizing to each gene in both the epimastigote and metacyclic trypomastigote developmental stages. Southern blot analysis suggests that each protein phosphatase 1 gene is present as a single copy in the T. cruzi genome. The complete coding region for TcPP1beta was expressed in Escherichia coli by using a vector, pTACTAC, with the trp-lac hybrid promoter. The recombinant protein from the TcPP1beta construct displayed phosphatase activity toward phosphorylase a, and this activity was preferentially inhibited by calyculin A (50% inhibitory concentration [IC(50)], approximately 2 nM) over okadaic acid (IC(50), approximately 100 nM). Calyculin A, but not okadaic acid, had profound effects on the in vitro replication and morphology of T. cruzi epimastigotes. Low concentrations of calyculin A (1 to 10 nM) caused growth arrest. Electron microscopic studies of the calyculin A-treated epimastigotes revealed that the organisms underwent duplication of organelles, including the flagellum, kinetoplast, and nucleus, but were incapable of completing cell division. At concentrations higher than 10 nM, or upon prolonged incubation at lower concentrations, the epimastigotes lost their characteristic elongated spindle shape and had a more rounded morphology. Okadaic acid at concentrations up to 1 microM did not result in growth arrest or morphological alterations to T. cruzi epimastigotes. Calyculin A, but not okadaic acid, was also a potent inhibitor of the dephosphorylation of (32)P-labeled phosphorylase a by T. cruzi epimastigotes and metacyclic trypomastigote extracts. These inhibitor studies suggest that in T. cruzi, type 1 protein phosphatases are important for the completion of cell division and for the maintenance of cell shape.
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PMID:Identification of novel serine/threonine protein phosphatases in Trypanosoma cruzi: a potential role in control of cytokinesis and morphology. 1067 47

The effects of marine substances with various cytotoxic mechanisms on the integrity of the human intestinal Caco-2 cell monolayer were examined by measuring the transepithelial electrical resistance (TEER). TEER was rapidly decreased by apical exposure of the monolayers to discodermin A, a membrane pore-forming substance. The decrease in TEER occurred in an earlier stage of incubation than the release of intracellular lactate dehydrogenase (LDH) which is commonly used as a parameter of cell damage or death. Mycalolide B (an actin-depolymerizing substance), calyculin A and okadaic acid (protein phosphatase inhibitors) also rapidly decreased the TEER value, although no cell membrane damage or resultant LDH release by these toxicants were detected. The TEER decrease caused by the toxicants was associated with the increased transepithelial permeability of the cell monolayer. Treatment with these toxicants, except calyculin A, caused morphological changes in the intracellular actin filament, suggesting that these toxicants altered the cytoskeletal structure, by which the tight junction was opened. Calyculin A was likely to loosen the cellular junctions rapidly and induce cell detachment from the monolayer. Although onnamide A, a protein synthesis inhibitor, did not cause any decrease in TEER, at least during a 90-min incubation, TEER sensitively reflects the cytotoxic effects of various types of toxicants with acute toxicity.
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PMID:Assessment of the marine toxins by monitoring the integrity of human intestinal Caco-2 cell monolayers. 1080 72

We examined the possibility that p38 mitogen-activated protein kinase and caspase-3 would be activated for execution of apoptosis and excitotoxicity, the two major types of neuronal death underlying hypoxicischemic and neurodegenerative diseases. Mouse cortical cell cultures underwent widespread neuronal apoptosis 24 h following exposure to 10-30 nM calyculin A, a selective inhibitor of Ser/Thr phosphatase I and IIA. Activity of p38 was increased 2-4 h following exposure to 30 nM calyculin A. Addition of 3-10 microM PD169316, a selective p38 inhibitor, partially attenuated calyculin A neurotoxicity. Activity of caspase-3-like proteases was increased in cortical cell cultures exposed to 30 nM calyculin A for 8-16 h as shown by cleavage of DEVD-p-nitroanilide and phosphorylated tau. Proteolysis of tau was completely blocked by addition of 100 microM N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), a broad-spectrum inhibitor of caspases, but incompletely by 10 microM PD169316. Calyculin A neurotoxicity was partially sensitive to 100 microM z-VAD-fmk. Cotreatment with 10 microM PD169316 and 100 microM z-VAD-fmk showed additive neuroprotection against calyculin A. Neither PD169316 nor z-VAD-fmk showed a beneficial effect against excitotoxic neuronal necrosis induced by exposure to 20 microM NMDA. Thus, caspase-3-like proteases and p38 likely contribute to calyculin A-induced neuronal apoptosis but not NMDA-induced neuronal necrosis.
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PMID:Synergetic activation of p38 mitogen-activated protein kinase and caspase-3-like proteases for execution of calyculin A-induced apoptosis but not N-methyl-d-aspartate-induced necrosis in mouse cortical neurons. 1082 Feb 6


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