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)

The rubella virus (RV)-encoded protein NSP90, which contains the retinoblastoma protein (Rb)-binding motif LXCXE, interacts with Rb and RV replication is reduced in cells lacking Rb. Whether the LXCXE motif of RV NSP90 itself is essential for Rb binding and virus replication is not known. Therefore, in the present study, the functional role of this motif was investigated by site-directed mutagenesis in a plasmid from which infectious RV RNA can be produced. Three critical mutations in the motif, two substitutions at the conserved cysteine residue (C --> G and C --> R) and a deletion of the entire motif, were created. A cell-free translated NSP90 C terminus polypeptide containing the deletion did not bind to Rb and a polypeptide carrying the C --> R substitution had barely detectable binding affinity for Rb. Rb binding by the C --> G mutant was reduced significantly compared to that of wild-type protein. Correlating with the binding results, mutant viruses containing the LXRXE and LXGXE motifs had a reduction in replication to < 0.5% and 47% of the wild-type, respectively, while deletion of the motif was found to be lethal. By the first serial passage, replication of the LXRXE-carrying virus had increased from < 0.5% to 2% of the wild-type. Sequencing of the genome of this virus revealed a nucleotide change that altered the motif from LXRXE to LXSXE, which is a known Rb-binding motif in two protein phosphatase subunits. Thus, our results clearly demonstrate that the LXCXE motif is required for efficient RV replication.
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PMID:Mutations in the retinoblastoma protein-binding LXCXE motif of rubella virus putative replicase affect virus replication. 1007 91

Reactive oxygen species (ROS) have been implicated as second messengers that activate protein kinase cascades, although the means by which ROS regulate signal transduction remains unclear. In the present study, we show that interleukin 1beta (IL1beta), H2O2, and sorbitol-induced hyperosmolarity mediate a 5- to 10-fold increase in phosphorylation (activation) of the p38 protein kinase in rat primary glial cells as measured by analyses of Western blots using an antibody directed against the dually phosphorylated (active) p38. Additionally, IL1beta was found to elicit H2O2 synthesis in these cells. Concurrent with p38 phosphorylation, all three stimulation paradigms caused an inhibition of protein phosphatase activity. Phenyl-tert-butyl nitrone (PBN), a nitrone-based free radical trap and N-acetyl-cysteine (NAC), a thiol reducing agent, were examined for their effects on the phosphorylation of p38 as well as phosphatase activity. Pretreatment of cells with either PBN or NAC at 1.0 mM suppressed IL1beta H2O2, and sorbitol-mediated activation of p38 and significantly increased phosphatase activity. These data suggest that ROS, particularly H2O2, are used as second messenger substances that activate p38 in part via the transient inactivation of regulatory protein phosphatases.
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PMID:Redox-sensitive protein phosphatase activity regulates the phosphorylation state of p38 protein kinase in primary astrocyte culture. 1022 Jan 13

The intracellular parasite Theileria parva transforms bovine T-lymphocytes, inducing uncontrolled proliferation. Upon infection, cells cease to require antigenic stimulation and exogenous growth factors to proliferate. Earlier studies have shown that pathways triggered via stimulation of the T-cell receptor are silent in transformed cells. This is reflected by a lack of phosphorylation of key signalling molecules and the fact that proliferation is not inhibited by immunosuppressants such as cyclosporin and ascomycin that target calcineurin. This suggests that the parasite bypasses the normal T-cells activation pathways to induce proliferation. Among the MAP-kinase pathways, ERK and p38 are silent, and only Jun N-terminal kinase is activated. This appears to suffice to induce constitutive activation of the transcription factor AP-1. More recently, it could be shown that the presence of the parasite in the host cell cytoplasm also induces constitutive activation of NF-kappaB, a transcription factor involved in proliferation and protection against apoptosis. Activation is effectuated by parasite-induced degradation of IkappaBs, the cytoplasmic inhibitors which sequester NF-kappaB in the cytoplasm. NF-kappaB activation is resistant to the antioxidant N-acetyl cysteine and a range of other reagents, suggesting that activation might occur in an unorthodox manner. Studies using inhibitors and dominant negative mutants demonstrate that the parasite activates a NF-kappaB-dependent anti-apoptotic mechanism that protects the transformed cell form spontaneous apoptosis and is essential for maintaining the transformed state of the parasitised cell.
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PMID:Interference by the intracellular parasite Theileria parva with T-cell signal transduction pathways induces transformation and protection against apoptosis. 1061 98

Thiol-disulfides cause a time- and a concentration-dependent inactivation of the low-M(r) phosphotyrosine protein phosphatase (PTP). We demonstrated that six of eight enzyme cysteines have similar reactivity against 5,5'-dithiobis(nitrobenzoic acid): Their thiolation is accompanied by enzyme inactivation. The inactivation of the enzyme by glutathione disulfide also is accompanied by the thiolation of six cysteine residues. Inorganic phosphate, a competitive enzyme inhibitor, protects the enzyme from inactivation, indicating that the inactivation results from thiolation of the essential active-site cysteine of the enzyme. The inactivation is reversed by dithiothreitol. Although all PTPs have three-dimensional active-site structures very similar to each other and also have identical reaction mechanisms, the thiol group contained in the active site of low-M(r) PTP seems to have lower reactivity than that of other PTPs in the protein thiolation reaction.
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PMID:Thiolation of low-Mr phosphotyrosine protein phosphatase by thiol-disulfides. 1063 66

We have reported that treatment with okadaic acid, a potent protein phosphatase inhibitor, has the ability to enhance the synthesis of the 78-kDa glucose-regulated protein (GRP78). This article reports our investigation of another protein phosphatase inhibitor, calyculin A, demonstrating the signaling pathways elicited by the protein phosphatase inhibitors that lead to the induction of grp78. Our data showed that the induction process is abolished by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38(MAPK)). Phosphorylation-activation of p38(MAPK) in the treated cells was indicated by its own phosphorylation, as shown by double Western blotting analyses and directly confirmed by the in vitro kinase assay using MAPK-activated protein kinase-2, a well-known downstream effector of p38(MAPK), as a substrate. The involvement of p38(MAPK) in this process is further substantiated by using transient transfection assays with a plasmid, pGRP78-Luc, which contains a 0.72-kbp stretch of the grp78 promoter. By exploiting the same transfection assay, we demonstrated that the up-regulation of the grp78 promoter by the protein phosphatase inhibitors is suppressed in the presence of the cytoplasmic calcium chelator bis(aminophenoxy)ethane N,N'-tetraacetic acid, the mitochondria calcium uniporter inhibitor ruthenium red as well as the antioxidants N-acetyl cysteine and pyrrolidinedithiocarbamate. Taken together, our results lead us to conclude that treatment with the protein phosphatase inhibitors would activate the signaling pathways involving p38(MAPK) and mitochondrial calcium-mediated oxidative stress and that these pathways must act in concert in order to confer the induction of grp78 by okadaic acid and calyculin A.
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PMID:Activation of p38 mitogen-activated protein kinase and mitochondrial Ca(2+)-mediated oxidative stress are essential for the enhanced expression of grp78 induced by the protein phosphatase inhibitors okadaic acid and calyculin A. 1065 78

HePTP is a tyrosine specific protein phosphatase that is strongly expressed in activated T-cells. It was recently demonstrated that in transfected T-cells HePTP impairs TCR-mediated activation of the MAP-kinase family members ERK2 and p38 and it was suggested that both ERK and p38 MAP-kinases are substrates of HePTP. The HePTP gene has been mapped to human chromosome 1q32.1. Abnormalities in this region are frequently found in various hematopoietic malignancies. HePTP is highly expressed in acute myeloid leukemia and its expression in fibroblasts resulted in transformation. To address a possible involvement of HePTP in hematopoietic malignancies we sought to identify HePTP substrate(s) in leukemic cells. Using substrate trapping mutants we have identified the MAP-kinase ERK2 as a specific target of HePTP in the myelogenous leukemia cell line K562. Tyrosine phosphorylated ERK2, but not ERK1, p38, or JNK1, efficiently bound to catalytically inactive HePTP mutants in which the active site cysteine (HePTP-C/S) or the conserved aspartic acid residue (HePTP-D/A) had been exchanged for serine and alanine, respectively. Moreover, the interaction of ERK2 with HePTP trapping mutants was dependent on ERK2 tyrosine phosphorylation, indicating that HePTP is specifically targeted to activated ERK2. Using a deletion mutant of HePTP (HePTP-dLD), in which 14 amino acid residues within the N-terminus are missing, we show that regions outside the catalytic domain are also required for the interaction. Furthermore, overexpression of HePTP in K562 cells and fibroblasts interfered with PMA or growth factor induced MAP-kinase activation and HePTP efficiently dephosphorylated active ERK2 on the tyrosine residue in the activation loop in vitro. Together, these data identify ERK2 as a specific and direct target of HePTP and are consistent with a model in which HePTP negatively regulates ERK2 activity as part of a feedback mechanism. Oncogene (2000) 19, 858 - 869.
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PMID:The MAP-kinase ERK2 is a specific substrate of the protein tyrosine phosphatase HePTP. 1070 94

Previous research has indicated that oxidants, antioxidants and the intracellular redox state regulate the activities of a variety of protein tyrosine kinases, protein tyrosine phosphatases, phospholipases and transcription factors. In order to explore the redox regulation of the serine/threonine phosphatase calcineurin, we have investigated the effects of a variety of oxidants and antioxidants on calcineurin phosphatase activity in vitro. The oxidants hydrogen peroxide, superoxide and glutathione disulfide inhibited the phosphatase activity of calcineurin in a dose-dependent manner. Incubation of purified calcineurin with the antioxidants ascorbate, ascorbate 2-phosphate, alpha-lipoic acid, N-acetyl-L-cysteine and glutathione increased phosphatase activity relative to untreated controls. In contrast, several other commonly used antioxidants, including butylated hydroxytoluene, butylated hydroxyanisole, TEMPOL (4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl), Trolox (6-hydroxy-2,5,7, 8-tetramethyl-chroman-2-carboxylic acid) and dihydrolipoic acid decreased the activity of purified calcineurin, possibly through prooxidative mechanisms. Although the antioxidant pyrrolidine dithiocarbamate increased the activity of purified calcineurin, it significantly inhibited the activity of calcineurin present in crude fibroblast lysates. These results support and extend the hypothesis that redox factors modulate the phosphatase activity of calcineurin and suggest that further in vivo studies are warranted.
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PMID:Modulation of the phosphatase activity of calcineurin by oxidants and antioxidants in vitro. 1075 56

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to the receptor leads to receptor activation and the induction of intracellular signals that result in apoptotic cell death. In the present study, the expression of mRNA and proteins of Fas receptor and Fas ligand were examined in human submandibular gland ductal (HSG) cells treated with okadaic acid by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Six hundred and eighty-two bp of the PCR product of Fas receptor mRNA was detected in HSG cells and a protein with an estimated molecular weight of 58,000 was expressed in HSG cells. Treatment of HSG cells with an agonistic anti-Fas monoclonal antibody resulted in death of HSG cells, indicating that the functional Fas receptor protein is expressed in HSG cells. Fas receptor protein expression stimulated by okadaic acid was elevated in a dose- and time-dependent manner, with maximal expression at 20 nM and 48 h treatment. Fas ligand mRNA was also detected constitutively in HSG cells by RT-PCR. Okadaic acid stimulated the expression of Fas ligand protein in HSG cells in a time-dependent manner, while the expression of the ligand was low in untreated HSG cells. The molecular weight of Fas ligand was estimated as 68,000. An antagonistic anti-Fas ligand monoclonal antibody prevented okadaic acid-induced death in HSG cells in a dose-dependent fashion as determined by WST-1 assay. The results indicate that the expression of Fas receptor and ligand is regulated by protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in HSG cells. The results also suggest that the Fas receptor-ligand system might regulate apoptosis in HSG cells.
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PMID:Upregulation of the expression of Fas antigen and Fas ligand in a human submandibular gland ductal cell line by okadaic acid. 1086 77

The K(+)-Cl(-) cotransporters (KCCs) are members of the cation-chloride cotransporter gene family and fall into two phylogenetic subgroups: KCC2 paired with KCC4 and KCC1 paired with KCC3. We report a functional comparison in Xenopus oocytes of KCC1 and KCC4, widely expressed representatives of these two subgroups. KCC1 and KCC4 exhibit differential sensitivity to transport inhibitors, such that KCC4 is much less sensitive to bumetanide and furosemide. The efficacy of these anion inhibitors is critically dependent on the concentration of extracellular K(+), with much higher inhibition in 50 mm K(+) versus 2 mm K(+). KCC4 is also uniquely sensitive to 10 mm barium and to 2 mm trichlormethiazide. Kinetic characterization reveals divergent affinities for K(+) (K(m) values of approximately 25.5 and 17.5 mm for KCC1 and KCC4, respectively), probably due to variation within the second transmembrane segment. Although the two isoforms have equivalent affinities for Cl(-), they differ in the anion selectivity of K(+) transport (Cl(-) > SCN(-) = Br(-) > PO(4)(-3) > I(-) for KCC1 and Cl(-) > Br(-) > PO(4)(-3) = I(-) > SCN(-) for KCC4). Both KCCs express minimal K(+)-Cl(-) cotransport under isotonic conditions, with significant activation by cell swelling under hypotonic conditions. The cysteine-alkylating agent N-ethylmaleimide activates K(+)-Cl(-) cotransport in isotonic conditions but abrogates hypotonic activation, an unexpected dissociation of N-ethylmaleimide sensitivity and volume sensitivity. Although KCC4 is consistently more volume-sensitive, the hypotonic activation of both isoforms is critically dependent on protein phosphatase 1. Overall, the functional comparison of these cloned K(+)-Cl(-) cotransporters reveals important functional, pharmacological, and kinetic differences with both physiological and mechanistic implications.
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PMID:Functional comparison of the K+-Cl- cotransporters KCC1 and KCC4. 1091 27

Immunoassays are increasingly used to investigate the production, properties and fates of the cyanobacterial hepatotoxic microcystins in vitro and in vivo. Responses of an ELISA immunoassay to microcystins have been determined using the authentic toxin antigen, microcystin-LR, and conjugation products between the toxin and glutathione, cysteine-glycine and cysteine. The antibodies against microcystin-LR crossreacted with the toxin conjugation products with similar affinities (96-112%) to that of microcystin-LR, when assayed at a concentration of 1 microg l(-1). Toxicity assessment of the conjugates, in comparison to microcystin-LR, indicated a reduction according to mouse bioassay. In vitro protein phosphatase inhibition assay indicated that the conjugates possessed approximately 3-9-fold lower toxicity than microcystin-LR.
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PMID:Immuno-crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin, microcystin-LR. 1093 Jul 30


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