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 addition of either okadaic acid or calyculin A desensitizes human platelets to thrombin. One objective of this study was to determine which step(s) leading to secretion reactions may be affected by these protein phosphatase inhibitors. In a dose-dependent manner, okadaic acid or calyculin A inhibits phosphatidylinositol metabolism and Ca(2+)-transients. In all cases, calyculin A was approximately 10-fold more potent than okadaic acid, and it had maximal effects at a concentration of 1 microM. Although thrombin-induced rises in [Ca2+]i were diminished, an increase in the phosphorylation state of myosin light chains (MLC) was still observed. Changes in this phosphorylation were diminished, however, following the addition of thrombin to calyculin A-treated platelets that were loaded with dimethyl-BAPTA. These data demonstrate that calyculin A and okadaic acid lower agonist-induced Ca(2+)-transients, which in turn prevents responses such as secretion reactions. Calyculin A/okadaic acid-induced phosphorylation events were not diminished in BAPTA-loaded platelets, suggesting that these phosphorylations are Ca(2+)-insensitive. Thus, a second objective of this study was to identify the protein kinase(s) that was(were) responsible for the calyculin A-induced phosphorylations. In a platelet lysate system, calyculin A caused an increase in the incorporation of [32P]phosphate into p50. This phosphorylation event was identical to that observed in the intact platelet and was not mimicked by cAMP, cGMP, Ca2+, or a Ca2+/phospholipid/diacylglycerol mixture. Kinase activity was removed after the lysate was incubated with p13suc1-Sepharose. This suggests that a p13suc1-sensitive protein kinase, e.g., a cell cycle-dependent protein kinase, is responsible for the calyculin A-sensitive phosphorylation events.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibitors of protein phosphatase type 1 and 2A attenuate phosphatidylinositol metabolism and Ca(2+)-transients in human platelets. Role of a cdc2-related protein kinase. 132 63

Stimulation of B and T cells via the antigen receptor, by phorbol ester or by phorbol ester and ionomycin, leads to nuclear translocation of the inducible transcription factor NF-kappa B, comprising the p50 and p65 rel-related polypeptides. In this report we show that c-rel is a component of the antigen receptor-induced kappa B binding proteins in both B and T cells. Whereas NF-kappa B can be induced by phorbol ester alone, optimal induction of c-rel requires stimulation by both phorbol ester and ionomycin, the dual signal that is necessary for proliferation of untransformed lymphocytes. Furthermore, c-rel induction is blocked by the immunosuppressive drug FK506 that is known to inhibit B and T cell activation. c-rel-dependent transactivation of the interleukin-2 receptor alpha chain (IL-2R alpha) promoter is augmented by coexpression of calcineurin, suggesting the involvement of a calcineurin-dependent intracellular pathway. Our results identify c-rel as a target of immunosuppressive agents and illustrate the similarity of activation pathways in both B and T cells.
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PMID:FK506 inhibits antigen receptor-mediated induction of c-rel in B and T lymphoid cells. 753 76

Activation of NF-kappa B by various cellular stimuli involves the phosphorylation and subsequent degradation of its inhibitor, I kappa B alpha, although the underlying mechanism remains unclear. In the present study, the role of serine/threonine phosphatases in the regulation of I kappa B alpha phosphorylation was investigated. Our studies demonstrate that incubation of human T cells with low concentrations (approximately 1-5 nM) of calyculin A or okadaic acid, potent inhibitors of protein phosphatase type 1 (PP-1) and type 2A (PP-2A), induces the phosphorylation of I kappa B alpha even in the absence of any cellular stimulus. This action of the phosphatase inhibitors, which is associated with the activation of the RelA.p50 NF-kappa B heterodimer, is not affected by agents that block the induction of I kappa B alpha phosphorylation by tumor necrosis factor alpha (TNF-alpha). Furthermore, the phosphorylated I kappa B alpha from calyculin A-treated cells, but not that from TNF-alpha-stimulated cells, is sensitive to PP-2A in vitro, suggesting the existence of fundamental differences in the phosphorylation of I kappa B alpha induced by the two different NF-kappa B inducers. However, induction of I kappa B alpha phosphorylation by both TNF-alpha and the phosphatase inhibitors is associated with the subsequent degradation of I kappa B alpha. We further demonstrate that TNF-alpha- and calyculin A-induced I kappa B alpha degradation exhibits similar but not identical sensitivities to a proteasome inhibitor. Together, these results suggest that phosphorylation of I kappa B alpha, mediated through both the TNF-alpha-inducible and the PP-2A-opposing kinases, may serve to target I kappa B alpha for proteasome-mediated degradation.
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PMID:Activation of NF-kappa B by phosphatase inhibitors involves the phosphorylation of I kappa B alpha at phosphatase 2A-sensitive sites. 762 57

Two cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12-myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)-expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+-signaling pathways downstream of T cell activation.
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PMID:Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-kappa B/AP-1 in T cells. 813 80

Previous studies have suggested that gangliosides have an important role in cell signaling and recognition. However, their specific function in these processes has not been clearly defined. A mAb, R24, that reacts specifically with a cell surface ganglioside (GD3) has been demonstrated to stimulate proliferation of T cells derived from human peripheral blood. In this study, we have investigated the mechanisms by which the R24 mAb affects T cell functions. We have observed that the R24 mAb stimulates GD3+ T cell proliferation, cytotoxicity, and surface marker expression of IL-2R alpha-chain, IL-2R beta-chain, HLA-DR, CD11a, and CD11c. Additionally, IFN-gamma activity but not IL-1, IL-2, or IL-4 activity was present in culture supernatants 72 h after R24 stimulation. In some donors, increased IL-6 and TNF-alpha activity also was detected after R24 treatment. Furthermore, R24 treatment resulted in translocation of c-rel, but little or no NF kappa B p50 or p65, from the cytoplasm to the nucleus and an increase of NF kappa B binding complexes containing c-rel and p50. This treatment also caused increased tyrosine phosphorylation of specific protein substrates. R24-stimulated increases in proliferation, cytotoxicity, and cell surface protein expression could be blocked by cyclosporin and staurosporin, indicating that cyclophilin/calcineurin and protein kinase C may be involved in the R24 signaling pathway. Additionally, herbimycin A, a tyrosine kinase inhibitor, blocked the R24-stimulated increase in proliferation but not cytotoxicity at concentrations consistent with specificity for tyrosine kinases. These results suggest that multiple biochemical pathways are involved in the activation of human T cells by R24.
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PMID:Increased proliferation, cytotoxicity, and gene expression after stimulation of human peripheral blood T lymphocytes through a surface ganglioside (GD3) 828 32

The phosphotyrosyl phosphatase (PTPase) specificity of phosphotyrosyl-phosphatase-activator-(PTPA)-stimulated protein phosphatase (PP)2A(D) (rabbit muscle) and a bona fide PTP-1B (Xenopus laevis oocytes) were examined in vitro using phosphotyrosine-containing peptides, derived from the phosphorylation sites of p34cdc2, p50/HS1 protein, Abl, c-Src and c-Fgr, as well as the intact phosphoprotein p50/HS1 and the Src-related tyrosine kinases, Lyn and c-Fgr. The local specificity determinants were found to be different for both PTPases. The length of the phosphopeptides is more important for PP2A(D) than for PTP-1B, C-terminal acidic residues adjacent to the phosphotyrosine are detrimental for the PTPase activity of PP2A(D), but they do not affect the PTP-1B activity. Acidic residues at the --2 and --3 position relative to Tyr(P) primarily dictate dephosphorylation by PTP-1B. The higher-order structure of the protein substrates also differentially influences both enzymes: the phospho-octapeptide KDDEYpNPA, which reproduces the autophosphorylation site in c-Fgr (Tyr400), is only dephosphorylated by PP2A(D) if embedded in the intact protein, whereas the opposite is true for PTP-1B. Both the intact p50/HS1 phosphoprotein and the derived phosphopeptide are substrates only for PTP-1B and not for PP2A(D). Lyn and c-Fgr phosphorylated by C-terminal Src kinase (CSK) at their down-regulatory site are resistant to the action of both PTPases while the [Phe6]Src-(514-533) phosphopeptide, representing the highly similar site affected by CSK in c-Src, is readily dephosphorylated by both PTPases, although to a different extent. In vitro dephosphorylation of the c-Fgr Tyr400 site by PP2A(D) is correlated with a decreased tyrosine kinase activity towards exogenous substrates. Under experimental conditions in which both Tyr400 (autophosphorylation site) and Tyr511 (down-regulatory site) of c-Fgr are phosphorylated, PP2A(D) can reverse both phosphorylations.
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PMID:A comparative study of the phosphotyrosyl phosphatase specificity of protein phosphatase type 2A and phosphotyrosyl phosphatase type 1B using phosphopeptides and the phosphoproteins p50/HS1, c-Fgr and Lyn. 861 28

Interferon-gamma (IFN-gamma) is a pleiotropic lymphokine whose production is restricted to activated T cells and NK cells. Along with other cytokines, IFN-gamma gene expression is inhibited by the immunosuppressant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-gamma gene that binds the NF-kappaB protein c-Rel and that shows partial DNA sequence homology with the cyclosporin A-sensitive NFAT binding site and the 3'-half of the NF-kappaB consensus site. Sequence analysis of the IFN-gamma promoter revealed the presence of two additional C3-related elements (C3-1P and C3-3P). In addition, an NF-kappaB site (IFN-gamma kappaB) was identified within the promoter region. Based on this observation, we have analyzed the potential role of NF-kappaB and NFAT family members in regulating IFN-gamma transcription. Electrophoretic mobility shift assay analysis demonstrated that after T cell activation, the p50 and p65 NF-kappaB subunits bind specifically to the newly identified IFN-gamma kappaB and C3-related sites. In addition, we identified the NFAT proteins as a component of the inducible complexes that bind to the C3-3P site. Site-directed mutagenesis and transfection studies demonstrate that calcineurin-inducible transcriptional factors enhance the transcriptional activity of the IFN-gamma promoter through the cyclosporin-sensitive C3-3P site, whereas NF-kappaB proteins functionally interact with the C3-related sites. In addition, when located downstream to the beta-galactosidase gene driven by the IFN-gamma promoter, the intronic C3 site worked in concert with both the IFN-gamma kappaB and the C3-3P site to enhance gene transcription. These results demonstrate that the coordinate activities of NFAT and NF-kappaB proteins are involved in the molecular mechanisms controlling IFN-gamma gene transcription.
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PMID:Interaction of NF-kappaB and NFAT with the interferon-gamma promoter. 937 32

Nuclear factor kappaB (NF-kappaB) and the Rel family of proteins are pleiotropic transcription factors that play central roles in the immune and inflammatory responses, as well as apoptosis. Here, we identified a serine/threonine protein phosphatase X (PPX; also called protein phosphatase 4 (PP4)) that specifically associated with c-Rel, NF-kappaB p50, and RelA. The amino acid sequences of human and mouse PPX are 100% identical, and the PPX gene was mapped to human chromosome 16 p11.2. Overexpression of PPX, but not catalytically inactive PPX mutants, stimulated the DNA-binding activity of c-Rel and activated NF-kappaB-mediated transcription. These results suggest that PPX is a novel activator of c-Rel/NF-kappaB.
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PMID:Protein phosphatase X interacts with c-Rel and stimulates c-Rel/nuclear factor kappaB activity. 983 38

The maturation and activation of newly synthesized molecules of the heme-regulated inhibitor of protein synthesis (HRI) in reticulocytes require their functional interaction with Hsp90. In this report, we demonstrate that protein phosphatase 5 (PP5), a previously documented component of the Hsp90 chaperone machine, is physically associated with HRI maturation intermediates. The interaction of PP5 with HRI is mediated through Hsp90, as mutants of PP5 that do not bind Hsp90 do not interact with HRI. PP5 was also present in Hsp90 heterocomplexes with another Hsp90 cohort, p50(cdc37), and expression of newly synthesized HRI enhanced the amount of p50(cdc37) associated with Hsp90/PP5-HRI heterocomplexes. The functional significance of the interaction of PP5 with Hsp90-HRI heterocomplexes was examined by characterizing the effects of compounds that impact PP5 activity in vitro. The protein phosphatase inhibitors okadaic acid and nodularin enhanced the kinase activity of HRI when applied during HRI maturation/activation, while the PP5 activators arachidonic and linoleic acid repressed HRI activity when applied during HRI maturation/activation. However, application of these compounds after HRI's "transformation" to an Hsp90-independent form did not similarly impact HRI's kinase activity. Furthermore, the Hsp90 inhibitor geldanamycin negated the effects of phosphatase inhibitors on HRI maturation/activation. The finding that PP5 downregulates an Hsp90-dependent process supports models for regulated Hsp90 function and describes a novel potential substrate for PP5 function in vivo.
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PMID:Evidence that protein phosphatase 5 functions to negatively modulate the maturation of the Hsp90-dependent heme-regulated eIF2alpha kinase. 1202 81

The murine chemokine CXCL1/KC is known as a chemoattractant for neutrophil infiltration and as a promoter of tumor growth. To determine its relevance in tumorigenesis, we first asked whether okadaic acid (OKA), a natural tumor promoter and a potent protein phosphatase 1 and 2A inhibitor, stimulates KC expression and if it does, through what pathway, in a promotable mouse epidermal-like JB6 cell line commonly used for studying molecules related to tumor promotion. We found that OKA stimulated the de novo synthesis of KC mRNA and protein in a dose- and time-dependent manner. To determine the mechanism by which OKA stimulated the expression of KC at the transcriptional level, transient transfection assays using serially deleted sections of KC promoter fused to luciferase reporter gene were performed. These studies showed that transactivation of KC promoter by OKA specifically involved the region between -104 and -59 containing the two nuclear factor-kappaB (NF-kappaB) response elements (kappaB1 and kappaB2). Further analyses using the mutated NF-kappaB response elements kappaB1 and kappaB2 indicated that both regions were required for optimum transactivation of KC by OKA with the former NF-kappaB response element playing a more significant role in regulating KC expression. Gel-shift and supershift analyses demonstrated the involvement of three NF-kappaB subunits, p65, p50 and c-Rel, with p65 as the major subunit in the NF-kappaB dimer complex. Additionally, immunohistochemistry and western blot analyses confirmed the presence of p65 in the nucleus with its transactivation domain phosphorylated at serine 536. In summary, this is the first report to show that the tumor promoter OKA can stimulate the de novo synthesis and secretion of KC, and that this stimulation is mediated through the NF-kappaB pathway in JB6 cells.
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PMID:Production of chemokine CXCL1/KC by okadaic acid through the nuclear factor-kappaB pathway. 1600 Apr 1


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