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)

Oxidative inactivation of protein tyrosine phosphatases and calcineurin is a well established mechanism; however, little information with regard to the effect of oxidants on PP1 and PP2A activity is available. Herein, we show that PP1 activity is inhibited by H(2)O(2) treatment in differentiated PC12 cells both in vitro and in vivo experiments. Thiol-antioxidant N-acetyl-cysteine (NAC) and reduced glutathione (GSH), when added in vitro to lysates from H(2)O(2)-treated cells, reversed PP1 inhibition. H(2)O(2) treatment increased eIF2 alpha phosphorylated levels (eIF2 alpha P) in a time- and dose-dependent fashion and promoted protein synthesis inhibition. Interestingly, NAC pretreatment protected cells from H(2)O(2)-induced PP1 inactivation and, consequently, it abolished increased H(2)O(2)-induced eIF2 alpha phosphorylation and protein synthesis inhibition. In addition, PP1 inhibitor tautomycin prevented both NAC-induced PP1 reactivation and eIF2 alpha P dephosphorylation in H(2)O(2)-treated cells. Taken together, our findings support a role for PP1 in eIF2 alpha phosphorylation and oxidative stress-triggered translation down regulation.
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PMID:Reversible inhibition of the protein phosphatase 1 by hydrogen peroxide. Potential regulation of eIF2 alpha phosphorylation in differentiated PC12 cells. 1294 1

14-3-3-interacting proteins were isolated from extracts of proliferating HeLa cells using 14-3-3 affinity chromatography, eluting with a phosphopeptide that competes with targets for 14-3-3 binding. The isolated proteins did not bind to 14-3-3 proteins (14-3-3s) after dephosphorylation with protein phosphatase 2A (PP2A), indicating that binding to 14-3-3s requires their phosphorylation. The binding proteins identified by tryptic mass fingerprinting and Western blotting include many enzymes involved in generating precursors such as purines (AMP, GMP and ATP), FAD, NADPH, cysteine and S-adenosylmethionine, which are needed for cell growth, regulators of cell proliferation, including enzymes of DNA replication, proteins of anti-oxidative metabolism, regulators of actin dynamics and cellular trafficking, and proteins whose deregulation has been implicated in cancers, diabetes, Parkinsonism and other neurological diseases. Several proteins bound to 14-3-3-Sepharose in extracts of proliferating cells, but not in non-proliferating, serum-starved cells, including a novel microtubule-interacting protein ELP95 (EMAP-like protein of 95 kDa) and a small HVA22/Yop1p-related protein. In contrast, the interactions of 14-3-3s with the N-methyl-D-aspartate receptor 2A subunit and NuMA (nuclear mitotic apparatus protein) were not regulated by serum. Overall, our findings suggest that 14-3-3s may be central to integrating the regulation of biosynthetic metabolism, cell proliferation, survival, and other processes in human cells.
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PMID:14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking. 1506 4

The present study examined in subcellular fractions from rat brain the nature and sensitivity to hydrogen peroxide of constitutively expressed mitogen-activated protein kinase (MAPK) phosphatase activity. MAPK phosphatase activity was defined as the activity directed towards a dual-phosphorylated (pT/pY) peptide corresponding to the activation domain of the extracellular-regulated kinase (ERK) subtype of the MAPKs. The use of phosphatase inhibitors and biochemical analyses demonstrate that the MAPK phosphatase activity, which was highest in the microsomal membrane and soluble fractions, was attributable mainly, if not entirely, to protein phosphatase 2A (PP2A). Moreover, hydrogen peroxide (in the absence and presence of reduced glutathione) and glutathione disulfide inhibited the MAPK phosphatase activity by a dithiothreitol-reversible mechanism. These results provide direct support for mounting evidence that PP2A is a major regulator of MAPK phosphorylation in brain and suggest that inhibition of PP2A activity via reversible oxidation of a cysteine thiol(s) may underlie at least in part the activation of MAPKs occurring in response to hydrogen peroxide and oxidative stress.
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PMID:Identification and H2O2 sensitivity of the major constitutive MAPK phosphatase from rat brain. 1497 38

Many small G proteins require post-translational modification to allow functional association to the cell membrane. This process often involves the enzymic addition of hydrophobic prenyl groups to a conserved cysteine residue near the C-terminus of the protein. The enzymes that catalyse these reactions include protein farnesyltransferase and protein geranylgeranyltransferases. The human fungal pathogen Cryptococcus neoformans requires functional Ras and Rho proteins in order to undergo normal growth and differentiation. Since farnesylation and geranylgeranylation are likely required for the proper function of these small G proteins, we hypothesized that inhibition of these prenylation events would alter the growth and cellular morphogenesis of this fungus. We cloned the RAM1 gene encoding the single protein-farnesyltransferase beta-chain homologue in C. neoformans. Using a gene-disruption strategy in a diploid C. neoformans strain, we demonstrated that this gene encodes an essential function, in contrast to the case in Saccharomyces cerevisiae, in which the homologous RAM1 gene is not essential for growth. Pharmacological inhibition of farnesyltransferase activity resulted in dose-dependent cytostasis of C. neoformans, as well as prevention of hyphal differentiation. Simultaneous inhibition of farnesylation and calcineurin signalling results in a synthetic effect on growth. Protein farnesylation is required for the growth and cellular differentiation of C. neoformans and may provide novel targets for antifungal therapy.
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PMID:The RAM1 gene encoding a protein-farnesyltransferase beta-subunit homologue is essential in Cryptococcus neoformans. 1518 78

In Arabidopsis, there is a family of receptor-like protein kinases (RLKs) containing novel cysteine-rich repeats in their extracellular domains. Genes encoding many of these cysteine-rich RLKs (CRKs) are induced by pathogen infection, suggesting a possible role in plant defense responses. We have previously generated Arabidopsis plants expressing four pathogen-regulated CRK genes (CRK5, 6, 10 and 11) under control of a steroid-inducible promoter and found that induced expression of CRK5, but not the other three CRK genes, triggered hypersensitive response-like cell death in transgenic plants. In the present study, we have analyzed the structural relationship of the CRK family and identified three CRKs (CRK4, 19 and 20) that are structurally closely related to CRK5. Genes encoding these three CRKs are all induced by salicylic acid and pathogen infection. Furthermore, induced expression of CRK4, 19 and 20 all activates rapid cell death in transgenic plants. Thus, the activity of inducing rapid cell death is shared by these structurally closely related CRKs. We have also performed yeast two-hybrid screens and identified proteins that interact with the kinase domains of CRKs. One of the identified CRK-interacting proteins is the kinase-associated type 2C protein phospohatase known to interact with a number of other RLKs through its kinase-interacting FHA domain. Other CRK-interacting proteins include a second protein with a FHA domain and another type 2C protein phosphatase. Interactions of CRKs with these three proteins in vivo were demonstrated through co-immunoprecipitation. These CRK-interacting proteins may play roles in the regulation and signaling of CRKs.
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PMID:Activation of hypersensitive cell death by pathogen-induced receptor-like protein kinases from Arabidopsis. 1560 43

Novel dual-specificity protein phosphatases (DSPs), which catalyse the removal of phosphate from both phosphotyrosine and phosphoserine/phosphothreonine substrates, have recently been identified in two viruses within the family Circoviridae. Viral protein 2 (VP2) of chicken anemia virus (CAV) and ORF2 of TT virus have been shown to possess DSP activity in vitro. CAV VP2 is unusual in possessing two vicinal cysteines within the protein phosphatase signature motif. The first cysteine residue (C95) within the motif has been identified by mutagenesis as the essential catalytic cysteine. In this study, it was shown that virus mutated at this residue displayed a marked inhibition of growth, with titres reduced 10(4)-fold, and reduced cytopathogenic effect in cell culture, indicating that viral DSP activity may be significant during infection. As with virus mutated at the first cysteine residue, mutation of the second cysteine (C97) within the motif resulted in a marked reduction in viral growth and attenuation of cytopathogenicity in infected cell cultures. However, mutagenesis of this second cysteine only reduced phosphotyrosine phosphatase activity to 70 % of that of wild-type VP2, but increased phosphoserine/phosphothreonine phosphatase activity by as much as 700 %. The differential effect of the C97S mutation on VP2 activity does not appear to have parallels in other DSPs and suggests a unique role for the second cysteine in the function of these viral proteins, particularly in vivo.
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PMID:Mutation of chicken anemia virus VP2 differentially affects serine/threonine and tyrosine protein phosphatase activities. 1572 22

Intracellular signaling pathways and their relationship to malignant progression have become a major focus of cancer biology. The dual-specificity phosphatase (DSP) family is a more recently identified family of intracellular signaling modulators. We have identified a novel protein phosphatase with a well-conserved DSP catalytic domain containing the DSP catalytic motif, xHCxxGxSRS, and mitogen-activated protein kinase phosphatase (MKP) motif, AYLM. Because of these unique characteristics, the protein was named mitogen-activated protein kinase phosphatase-8 (MKP-8). This protein is approximately 20kDa in size and mainly localizes to the nuclear compartment of the cell. MKP-8 is expressed in embryonal cancers (retinoblastoma, neuroepithelioma, and neuroblastoma) and has limited expression in normal tissues. MKP-8 displays significant phosphatase activity that is inhibited by a cysteine to serine substitution in the catalytic domain. When co-expressed with activated MAPKs, MKP-8 is able to inhibit p38 kinase phosphorylation and downstream activity.
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PMID:MKP-8, a novel MAPK phosphatase that inhibits p38 kinase. 1579 12

Calcium- and integrin-binding protein 1 (CIB1) is involved in the process of platelet aggregation by binding the cytoplasmic tail of the alpha(IIb) subunit of the platelet-specific integrin alpha(Iib)beta(3). Although poorly understood, it is widely believed that CIB1 acts as a global signaling regulator because it is expressed in many tissues that do not express integrin alpha(Iib)beta(3). We report the structure of human CIB1 to a resolution of 2.3 A, crystallized as a dimer. The dimer interface includes an extensive hydrophobic patch in a crystal form with 80% solvent content. Although the dimer form of CIB1 may not be physiologically relevant, this intersub-unit surface is likely to be linked to alpha(IIb) binding and to the binding of other signaling partner proteins. The C-terminal domain of CIB1 is structurally similar to other EF-hand proteins such as calmodulin and calcineurin B. Despite structural homology to the C-terminal domain, the N-terminal domain of CIB1 lacks calcium-binding sites. The structure of CIB1 revealed a complex with a molecule of glutathione in the reduced state bond to the N-terminal domain of one of the two subunits poised to interact with the free thiol of C35. Glutathione bound in this fashion suggests CIB1 may be redox regulated. Next to the bound GSH, the orientation of residues C35, H31, and S48 is suggestive of a cysteine-type protein phosphatase active site. The potential enzymatic activity of CIB1 is discussed and suggests a mechanism by which it regulates a wide variety of proteins in cells in addition to platelets.
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PMID:The crystal structure of calcium- and integrin-binding protein 1: insights into redox regulated functions. 1584 Aug 29

Transcriptional signaling from the Ca(2+)-calmodulin-activated phosphatase calcineurin to its substrate NFAT (nuclear factor of activated T cells, also termed NFATc) is critically dependent on a protein-protein docking interaction between calcineurin and the PXIXIT motif in NFAT. Several inhibitors of NFAT-calcineurin association (INCA compounds) prevent binding of NFAT or the peptide ligand PVIVIT to calcineurin. Here we show that the binding site on calcineurin for INCA1, INCA2, and INCA6 is centered on cysteine 266 of calcineurin Aalpha and does not coincide with the core PXIXIT-binding site. Although ample evidence indicates that INCA1 and INCA2 react covalently with cysteine 266, covalent derivatization alone is not sufficient for maximal inhibition of the calcineurin-PVIVIT interaction, because the maleimide INCA12 reacts with the same site and produces only very modest inhibition. Thus, inhibition arises through an allosteric change affecting the PXIXIT docking site, which may be assisted by covalent binding but depends on other specific features of the ligand. The spatial arrangement of the binding sites for PVIVIT and INCA makes it probable that the change in conformation involves the beta11-beta12 loop of calcineurin. The finding that an allosteric site controls NFAT binding opens new alternatives for inhibition of calcineurin-NFAT signaling.
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PMID:Inhibition of the calcineurin-NFAT interaction by small organic molecules reflects binding at an allosteric site. 1614 11

Intracellular reduction and oxidation pathways regulate protein functionality through both reversible and irreversible mechanisms. The Cdc25 phosphatases, which control cell cycle progression, are potential subjects of oxidative regulation. Many of the more potent Cdc25 phosphatase inhibitors reported to date are quinones, which are capable of redox cycling. Therefore, we used the previously characterized quinolinedione Cdc25 inhibitor DA3003-1 [NSC 663284 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] and a newly synthesized congener JUN1111 [7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] to test the hypothesis that quinone inhibitors of Cdc25 regulate phosphatase activity through redox mechanisms. Like DA3003-1, JUN1111 selectively inhibited Cdc25 phosphatases in vitro in an irreversible, time-dependent manner and arrested cells in the G1 and G2/M phases of the cell cycle. It is noteworthy that both DA3003-1 and JUN1111 directly inhibited Cdc25B activity in cells. Depletion of glutathione increased cellular sensitivity to DA3003-1 and JUN1111, and in vitro Cdc25B inhibition by these compounds was sensitive to pH, catalase, and reductants (dithiothreitol and glutathione), consistent with oxidative inactivation. In addition, both DA3003-1 and JUN1111 rapidly generated intracellular reactive oxygen species. Analysis of Cdc25B by mass spectrometry revealed sulfonic acid formation on the catalytic cysteine of Cdc25B after in vitro treatment with DA3003-1. These results indicate that irreversible oxidation of the catalytic cysteine of Cdc25B is indeed a mechanism by which these quinolinediones inactivate this protein phosphatase.
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PMID:Redox regulation of Cdc25B by cell-active quinolinediones. 1615 9


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