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)

Cholinergic interneurons have been implicated in striatally mediated associative learning. In classical conditioning paradigms, conditioned stimuli trigger a transient suppression of neuronal activity that is dependent upon an intact dopaminergic innervation. Our hypothesis was that this suppression reflected dopaminergic enhancement of sensory-linked GABAergic input. As a test, the impact of dopamine on interneuronal GABA(A) receptor function was studied by combined patch-clamp recording and single-cell reverse transcription PCR. Activation of D5 dopamine receptors reversibly enhanced a Zn2+-sensitive component of GABA(A) currents. Although dependent upon protein kinase A (PKA) activation, the modulation was blocked by protein phosphatase 1 (PP1) inhibition, suggesting it was dependent upon dephosphorylation. These results establish a novel mechanism by which intrastriatally released dopamine mediates changes in GABAergic signaling that could underlie the initial stages of associative learning.
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PMID:D5 dopamine receptors enhance Zn2+-sensitive GABA(A) currents in striatal cholinergic interneurons through a PKA/PP1 cascade. 939 May 24

Sulfhydryl reagents, such as dithiothreitol (DTT), affected the activity of Ser/Thr phosphoprotein phosphatases. Addition of DTT to the assay buffer increased the affinity of lambda Ser/Thr phosphoprotein phosphatase (lambda-PPase) for its Mn2+ cofactor. On the other hand, the enzyme was found to be inactivated simply by dilution in Tris buffer. The inactivation could be completely prevented by the presence of DTT or Mn2+ in the buffer. Further studies showed that oxidation or reduction of cysteine residues in lambda-PPase may not be the cause of the change in the enzyme activity. Without exception, mutation of all cysteine residues in lambda-PPase to serine did not convert the enzyme into a thiol-insensitive mutant. By careful examination of the effects of different sulfhydryl reagents, metal ion cofactors and substrates on lambda-PPase, it was found that the role of sulfhydryl reagents was the chelation of small amounts of inhibitory metal ions, which were present in plastic laboratory ware, such as disposable cuvets and tubes, with prevention of the enzyme from inactivation. One of the main contaminants found in plastic cuvets was Zn2+, which is a potent inhibitor of lambda-PPase. The inhibition of lambda-PPase by Zn2+ was characterized. Pre-treatment of the enzyme (1-4 nM) with 1 microM of ZnCl2 almost completely inhibited the enzymatic activity in response to 2 mM Mn2+. However, no significant inhibition was found when the enzyme was added to the assay mixture containing 1 microM Zn2+ and 2 mM Mn2+ . This confirms the sensitivity of the holoenzyme to inhibitory metal ions in vitro. The kinetic analysis indicated that the inhibitory metal ion might compete with Mn2+ to bind to the active site of lambda-PPase. This was further supported by the mutation of metal cofactor binding amino acid residues of the enzyme. Mutants which have less affinity for Mn2+ are also less sensitive to Zn2+. Our results suggest that inhibitory metal ions may induce a different structural conformation for lambda-PPase.
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PMID:Effects of sulfhydryl regents on the activity of lambda Ser/Thr phosphoprotein phosphatase and inhibition of the enzyme by zinc ion. 954 6

Four putative yeast transcription factors (Hal6-9p) have been identified which upon overexpression in multicopy plasmids increase sodium and lithium tolerance. This effect is mediated, at least in part, by increased expression of the Enalp Na+/Li+ extrusion pump. Hal6p and Hal7p are bZIP proteins and their gene disruptions affected neither salt tolerance nor ENA1 expression. Hal8p and Hal9p are putative zinc fingers and their gene disruptions decreased both salt tolerance and ENA1 expression. Therefore, Hal8p and Hal9p, but not Hal6p and Hal7p, qualify as transcriptional activators of ENA1 under physiological conditions. Hal8p seems to mediate the calcineurin-dependent part of ENA1 expression.
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PMID:Yeast putative transcription factors involved in salt tolerance. 955 73

Freshly isolated protein phosphatase 2A (PP2A) was highly active as to the dephosphorylation of protein substrates, but lost most of its spontaneous activity on prolonged storage, and was converted to a latent form requiring Mn2+ or Co2+ ions for activity. In this report, we show that the latent form of PP2A can be activated by the Fe2+/ascorbate system. Activation of the phosphatase required both Fe2+ ions and ascorbate, and the level of activation was dependent on the concentrations of both Fe2+ ions and ascorbate. Both the holoenzyme and catalytic subunit of phosphatase 2A could be activated by the Fe2+/ascorbate system, indicating that direct modulation of the catalytic subunit of the phosphatase by the Fe2+/ascorbate system may cause this activation. Several common divalent metal ions, including Ca2+, Mg2+, Cu2+, Zn2+, and Ni2+ ions, cannot cooperate with ascorbate to activate the phosphatase. Dithiothreitol, a SH-containing reducing agent, could replace ascorbate in the Fe2+/ascorbate system to activate the phosphatase, whereas H2O2, a strong oxidizer, significantly diminished the phosphatase activation by the Fe2+/ascorbate system. The results indicate that iron ions stabilized in the +2 state by reducing agents can activate the phosphatase. Overall, the present study provides initial biochemical evidence suggesting that Fe2+ could be a biologically important metal ion cofactor responsible for PP2A activation.
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PMID:Activation of protein phosphatase 2A by the Fe2+/ascorbate system. 964 67

SV40 small tumor antigen (small-t) was used as a model to identify structural elements involved in the interactions between regulatory proteins and protein phosphatase 2A (PP2A). Using mutant proteins and synthetic peptides, we identified a small domain within small-t that is a major site for interaction with the dimeric form of PP2A. A series of small-t truncation mutants identified a region surrounding the first of two conserved cysteine clusters that was critical for interaction with PP2A. These mutants also identified additional regions of small-t that contribute to high affinity interaction. Deletion of residues 110-119, which encompass the first cysteine cluster, resulted in a protein that failed to bind to PP2A. Synthetic peptides that contained residues 105-122 of small-t blocked binding of small-t to PP2A. These peptides also inhibited the phosphatase activity of PP2A in a manner analogous to full-length small-t. The active small-t peptides adopt a beta-strand structure that was essential for high affinity interaction with the PP2A dimer. Based on circular dichroism measurements, the same cysteine cluster-containing peptides that bind to PP2A also interact with zinc. Interaction with zinc required the conserved cysteines but was not required for interaction with PP2A.
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PMID:Identification of structural elements involved in the interaction of simian virus 40 small tumor antigen with protein phosphatase 2A. 985 76

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.
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PMID:The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis. 1006 78

A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme.
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PMID:Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one. 1021 76

In free-living Amoeba proteus (strain B), acid phosphatase (AcP) was examined by disc-electrophoresis in polyacrylamide gel. The tartrate-sensitive amebian AcP was greatly inhibited by dithiothreitol and Cu2+, and only partly inhibited by sodium orthovanadate, ammonium molybdate, EDTA, disodium salt and Mg2+, Ca2+, Zn2+ and Mn2+. On the contrary, it appeared to be resistant to sulfhydryl reagents--4(hydroxymercury) benzoic acid, sodium salt and N-ethylmaleimide. Unlike the tartrate-sensitive enzyme, the tartrate-resistant AcP was greatly inhibited by EDTA and partly inhibited by dithiothreitol, Mg2+ and Cu2+ (Mn2+ > Cu2+), being activated by orthovanadate, molybdate, sulfhydryl reagents, Mg2+, Ca2+ and Zn2+. Both tartrate-sensitive and tartrate-resistant AcPs lack apparently free SH-groups necessary for their catalytic activities. Using 2-naphthyl phosphate as a substrate at pH 4.5, six AcP electromorphs were revealed in cytosol and sediment, four of these being most frequently localized in the former, and two in the latter. Two other AcP electromorphs were confined to the sediment only. Depending on the quantity of sedimented amoebae making a homogenate (0.5 or 2.0 cm3), that was added to Percoll solution, the lysosomal AcP fraction in polyacrylamide gel was represented by one or two tartrate-sensitive electromorphs. Therefore, tartrate-resistant AcP in A. proteus may be a lysosomal enzyme, while tartrate-resistant AcP may correspond to serine/threonine protein phosphatase.
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PMID:[Tartrate-sensitive and tartrate-resistant acid phosphatases in Amoeba proteus]. 1095 68

Characterization of ceramide-effector(s), which includes protein phosphatase 2A (PP2A) is an important prelude to understanding the molecular basis of sphingolipid-mediated biological effects such as cell growth, differentiation and apoptosis. Recently, the existence of a metal-dependent form of PP2A has been reported (Cai et al., 1995). In this study, we investigated the effects of metal ions and chelators on ceramide-activated PP2A (CAPP). Our study demonstrates that at 0.5 mM concentration, Mg2+ appears to have no significant effect on either basal or ceramide-stimulated phosphatase activities, whereas Ca2+ stimulated the basal phosphatase activity, but was inhibitory towards CAPP. Moreover, the divalent cations Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ and Zn2+ were tested and all were found to be inhibitory towards both CAPP and basal phosphatase activities. By contrast, Cs+ and Li+ had almost no effect on CAPP, although both stimulated basal phosphatase activity. The effects of EDTA and EGTA were tested and it was observed that EDTA decreased CAPP activity in a dose-dependent fashion, but had no effect upon basal phosphatase activity. These results suggest that CAPP is a metal-dependent protein, but, because Ca2+ inhibitied CAPP and EGTA was much less potent than EDTA in inhibiting CAPP, Ca2+ is unlikely to be its metal cofactor.
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PMID:Effects of cations on ceramide-activated protein phosphatase 2A. 1179 86

Zinc is essential for cell proliferation and differentiation, especially for the regulation of DNA synthesis and mitosis. On the molecular level, it is a structural constituent of a great number of proteins, including enzymes of cellular signaling pathways and transcription factors. Zinc homeostasis in eukaryotic cells is controlled on the levels of uptake, intracellular sequestration in zinc storing vesicles ('zincosomes'), nucleocytoplasmic distribution and elimination. These processes involve the major zinc binding protein metallothionein as a tool for the regulation of the cellular zinc level and the nuclear translocation of zinc in the course of the cell cycle and differentiation. In addition, there is also increasing evidence for a direct signaling function for zinc on all levels of signal transduction. Zinc can modulate cellular signal recognition, second messenger metabolism, protein kinase and protein phosphatase activities, and it may stimulate or inhibit activities of transcription factors, depending on the experimental systems studied. Zinc has been shown to modify specifically the metabolism of cGMP, the activities of protein kinase C and mitogen activated protein kinases, and the activity of transcription factor MTF-1 which controls the transcription of the genes for metallothionein and the zinc transporter ZnT-1. As a conclusion of these observations new hypotheses regarding regulatory functions of zinc ions in cellular signaling pathways are proposed.
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PMID:Functions of zinc in signaling, proliferation and differentiation of mammalian cells. 1183 63


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