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 cardiac calcium release channel (CRC) of sarcoplasmic reticulum vesicles was incorporated into planar lipid membranes to evaluate modulation of channel activity by phosphorylation and dephosphorylation. For this purpose a microsyringe application directly to the membrane was used to achieve sequential and multiple treatments of channels with highly purified kinases and phosphatases. Cyclic application of protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase II (CalPK) and potato acid phosphatase or protein phosphatase 1 revealed a channel block by Mg2+ (-mM), that is referable to dephosphorylated states of the channel, and that the Mg2+ block could be removed by phosphorylation of the CRC by either PKA or CalPK. By contrast, activation of endogenous CalPK (end CalPK) led to channel closure which could be reversed by dephosphorylation using potato acid phosphatase or protein phosphatase 1. Calmodulin by itself (which activates end CalPK in the presence of MgATP) blocks the channel in the dephosphorylated state, which can be overcome by treatment with CalPK but not PKA. Our findings reveal important insights regarding channel regulation of the ryanodine receptor: 1) the calcium release channel must be phosphorylated to be in the active state at conditions approximating physiological Mg2+ concentrations (-mM); and 2) there are multiple sites of phosphorylation on the calcium release channel with different functional consequences, which may be relevant to the regulation of E-C coupling. Phosphorylation of the CRC may be involved in recruitment of active channels, and/or it may be directly involved in each Ca2+ contraction cycle of the heart. For example, Ca2+ release may require phosphorylation of the CRC by protein kinases at sites which overcome the block by Mg2+. Inactivation may involve CRC block by calmodulin and/or phosphorylation by endogenous CalPK at the junctional face membrane.
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PMID:Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from cardiac muscle. 783 35

Alkaline phosphatase (ALP) hydrolyzed phosvitin and amino acid phosphates demonstrating nonisotropy at different pH. Orthovanadate, a protein phosphatase inhibitor, more specifically inhibited the serine and tyrosine phosphatase activities of ALP than that of threonine phosphatase at concentrations > 0.1 mM or 0.01 mM, respectively. Calyculin A and okadaic acid at increased concentrations increased ALP amino acid phosphatase activity. Bisphosphonates, such as disodium-1-hydroxy-1-aminopropylidine-1,1-diphosphonate (APD) and ethane-1-hydroxy-1,1-diphosphonate (HEBP), at increased concentrations, inhibited ALP amino acid phosphatase activity. These results suggest that ALP may function as a protein phosphatase. In terms of protein kinase inhibitors, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, N-(6-aminoheyxl)-5-chloro-1-naphthalenesulfomide hydrochloride and 4',5,7-trihydroxyisoflavone had little effect on ALP amino acid phosphatase activity. Staurosporine slightly enhanced ALP serine and threonine phosphatase activities at a concentration of 0.1 mM. These results suggest that protein phosphatase activity does not depend on the protein kinase activity of ALP, since duality between the former and the latter is not supported. ALP may function less as a protein kinase than as a protein phosphatase. The coupling mechanism of phosphate dynamics may be regulated indirectly.
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PMID:Amino acid phosphatase activity of alkaline phosphatase. A possible role of protein phosphatase. 785 10

Low-M(r) phosphotyrosine protein phosphatase (PTPase), previously known as low-M(r) acid phosphatase, catalyzes the in-vitro hydrolysis of tyrosine phosphorylated proteins, low-M(r) aryl phosphates and natural and synthetic acyl phosphates. Its activity on Ser/Thr-phosphorylated proteins and on most alkyl phosphates is very poor. In this study the mechanism of benzoyl-phosphate hydrolysis was studied by means of non-mutated and mutated PTPase fusion proteins. The mechanism of benzoyl-phosphate hydrolysis catalyzed by the enzyme was compared to the known mechanism of p-nitrophenyl-phosphate hydrolysis. The results demonstrated that both hydrolytic processes proceed through common enzyme-catalyzed mechanisms. Nevertheless, the performed phosphoenzyme-trapping experiments enable us to identify Cys12 as the active-site residue that performs the nucleophilic attack at the phosphorus atom of the substrate to produce a phosphoenzyme covalent intermediate. In addition, while the role of Cys17 in the substrate binding was confirmed, its participation a second time in the step that involves the Cys12 dephosphorylation was suggested by the results of phosphoenzyme-trapping experiments. The participation of Arg18 in the substrate-binding site was demonstrated by site-directed mutagenesis that produced the conservative Lys18 and the non-conservative Met18 mutants. Both these mutants were almost inactive and not able to bind the substrate and a competitive inhibitor. Furthermore, phosphoenzyme-trapping experiments clearly excluded that Cys62 and Cys145 (that were indicated by another laboratory to be involved in the active site of the enzyme as powerful nucleophilic agents) are the residues directly involved in the formation of the phosphoenzyme covalent intermediate.
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PMID:The role of Cys12, Cys17 and Arg18 in the catalytic mechanism of low-M(r) cytosolic phosphotyrosine protein phosphatase. 831 76

The substrate specificity of the cyanobacterial dual-specificity protein phosphatase, IphP, was explored using a variety of potential substrates. The enzyme displayed phosphomonoesterase activity toward a broad range of peptide, protein, and low molecular weight organophosphate compounds. It displayed little or no hydrolase activity toward phosphodiesters, phosphoramides, carboxyl esters, or sulfoesters. However, it did display measurable pyrophosphatase activity, especially toward ADP and ATP. Among the low molecular weight phosphomonoesters, the presence of an aromatic ring either as part of the leaving group alcohol or immediately adjacent thereto, as in 5'-AMP, was a strong positive determinant for hydrolysis. Among peptide and protein substrates, a rough, but imperfect, correlation between charge character and hydrolysis was noted in which proteins and phosphorylation sites of an acidic nature seemed favored. Heparin affected IphP activity in a substrate-dependent manner. Toward small organophosphates, heparin had no significant effect, but it was inhibitory toward most protein and peptide substrates. However, toward phosphoseryl casein and MAP kinase, it enhanced activity as much as 10-fold. This enhancement was attributed to the ability of heparin to bind to these substrate proteins, as well as IphP, and recruit them to the same microenvironment.
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PMID:Substrate specificity of IphP, a cyanobacterial dual-specificity protein phosphatase with MAP kinase phosphatase activity. 865 37

Pho85, a protein kinase with significant homology to the cyclin-dependent kinase, Cdc28, has been shown to function in repression of transcription of acid phosphatase (APase, encoded by PHO5) in high phosphate (Pi) medium, as well as in regulation of the cell cycle at G1/S. We described several unique phenotypes associated with the deletion of the PHO85 gene including growth defects on a variety of carbon sources and hyperaccumulation of glycogen in rich medium high in Pi. Hyperaccumulation of glycogen in the pho85 strains is independent of other APase regulatory molecules and is not signaled through Snfl kinase. However, constitutive activation of cAPK suppresses the hyperaccumulation of glycogen in a pho85 mutant. Mutation of the type-1 protein phosphatase encoded by GLC7 only partially suppresses the glycogen phenotype of the pho85 mutant. Additionally, strains containing a deletion of the PHO85 gene show an increase in expression of GSY2. This work provides evidence that Pho85 has functions in addition to transcriptional regulation of APase and cell-cycle progression including the regulation of glycogen levels in the cell and may provide a link between the nutritional state of the cell and these growth related responses.
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PMID:Deletion of the gene encoding the cyclin-dependent protein kinase Pho85 alters glycogen metabolism in Saccharomyces cerevisiae. 872 62

The FeZn derivative of purple acid phosphatase from porcine uterus (FeZnUf) and its phosphate complex (FeZnUf.PO4) have been characterized by X-ray absorption spectroscopy at both the iron and zinc K-edges, to gain insight into the nature of the FeZn active site as well as the phosphate binding mode. Pre-edge data show that both FeZnUf and FeZnUf.PO4 have a 6-coordinate iron site. The iron site has an average Fe-O/N bond length of 2.01-2.02 A, which can be resolved into subshells of 1.92 and 2.11 A for FeZnUf.PO4. On the other hand, the zinc site has a shell of scatterers at 2.02-2.03 A plus one scatterer at ca. 2.4 A. These metal-ligand bond lengths are consistent with the nature of the ligands deduced from spectroscopic studies or identified in the crystal structure of the related kidney bean purple acid phosphatase (KBPAP). The outer-sphere analysis indicates an Fe-Zn separation of approximately 3.3 A in both FeZnUf and FeZnUf.PO4, consistent with the presence of an M2(mu-OR)2 diamond core as found in the crystal structures of KBPAP, calcineurin, and protein phosphatase 1. The Fe-P and Zn-P bond distances in FeZnUf.PO4 are determined to be 3.23 and 3.18 A, respectively, indicating that phosphate binds to the dinuclear center in a bidentate mode; such a mode has been observed in oxoanion complexes of KBPAP, calcineurin, and alkaline phosphatase, as well as in a number of synthetic FeFe and FeZn complexes. The implications of these structural results on the mechanism of phosphatase action are discussed.
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PMID:X-ray absorption spectroscopic studies of the FeZn derivative of uteroferrin. 890 92

Crystal structures of the Ser/Thr phosphatase calcineurin (protein phosphatase 2B) have recently been solved by X-ray crystallography, both in the free-protein state, and complexed with the immunophilin/immunosuppressant FKBP12/FK506. Core elements of the calcineurin phosphatase have been found to be similar to the corresponding elements of Ser/Thr phosphatase 1 and purple acid phosphatase. The structures provide a basis for understanding calcineurin inhibition by a ternary complex of immunophilin and immunosuppressant proteins.
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PMID:Calcineurin-immunosuppressor complexes. 899 77

The Ah receptor binds aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with high affinity. After binding aryl hydrocarbons, the receptor releases the 90-kDa heat shock protein and forms a dimer with the Arnt protein capable of binding at xenobiotic-responsive elements (XREs) and stimulating the transcription of genes involved in the metabolism of aryl hydrocarbons. The activity of the Ah receptor/ Arnt dimer can be decreased by treatments causing the down-regulation of protein kinase C and decreasing the nuclear accumulation of the receptor. Incubation with acid phosphatase or with alkaline phosphatase has been reported to block XRE binding. Thus the literature suggests that phosphorylation regulates Ah receptor activity by affecting DNA binding and/or nuclear transport. A reporter plasmid containing two XREs was used to investigate the effects of phosphatase inhibitors on TCDD-dependent transcription by the Hepa-1 mouse liver cell line. The inhibitors calyculin A and okadaic acid caused two- to threefold increases in TCDD-dependent transcription at concentrations capable of selectively inhibiting protein phosphatase 1 and protein phosphatase 2A. The inhibitor cyclosporin A doubled TCDD-dependent transcription at a concentration capable of selectively inhibiting protein phosphatase 2B. All three of the phosphatase inhibitors increased TCDD-dependent transcription without affecting transcription in the absence of TCDD. Nuclear extracts were prepared from cells treated with concentrations of okadaic acid or cyclosporin A which substantially stimulated TCDD-dependent transcription. Neither of the inhibitors significantly increased the level of TCDD-dependent XRE binding in the extracts. GAL4-Arnt fusion proteins were used to further investigate whether the phosphatase inhibitors affected a step other than DNA binding. Okadaic acid treatment specifically increased the ability of a GAL4 fusion protein containing the Arnt PAS and transactivation domains to stimulate transcription. These results suggest that serine/threonine-specific protein phosphatases can act at a level subsequent to XRE binding to inhibit the ability of the Ah receptor/Arnt dimer to stimulate transcription.
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PMID:Inhibitors of serine/threonine-specific protein phosphatases stimulate transcription by the Ah receptor/Arnt dimer by affecting a step subsequent to XRE binding. 912 79

Acyl analogs of platelet-activating factor (PAF) (1-acyl-2-acetyl-sn-glycero-3-phosphocholine, acylacetyl -GPC) are the predominant products synthesized during thrombin or ionophore A23187-mediated activation of endothelial cells. However, the biosynthetic pathway responsible for the production of acylacetyl-GPC is not well understood. In the present investigation, we have demonstrated that the acyl analogs of PAF are also the major products from calf pulmonary artery endothelial cells in response to a time-dependent stimulation of ATP (10(-3) M), bradykinin (10(-8) M), or ionophore A23187 (2 microM). In addition, we have found that the CoA-independent PAF:acyllyso-GPC transacetylase recently identified by us is concurrently and transiently induced with maximal 4-fold enhancement at 5 min and returned to near basal level by 10 min treatment of endothelial cells with ATP. Acid phosphatase reduces the increased PAF:acyllyso-GPC transacetylase activity from the homogenates of ATP-activated endothelial cells. Reduced PAF:acyllyso-GPC transacetylase activity can be restored by incubating the acid phosphatase-treated homogenates with ATP (5 mM) and Mg2+ (10 mM). Furthermore, okadaic acid, a protein phosphatase 1 and 2A inhibitor, incubated with endothelial cells in a dose-dependent manner (1-100 nM) for 10-min potentiates and sustained the stimulation of PAF:acyllyso-GPC transacetylase activity by ATP. On the other hand, genistein, tyrphostin-25 (inhibitors of tyrosine-specific protein kinase), and calphostin C (an inhibitor of protein kinase C) block the activation of PAF:acyllyso-GPC transacetylase by ATP. These results are consistent with the notion that ATP regulates the transacetylase activity by reversible activation and inactivation via the phosphorylation and dephosphorylation cycle. ATP also augments the activities of alkyllyso-GPC/acyllyso-GPC:acetyl-CoA acetyltransferase. However, the activation of the acetyltransferases precedes that of the transacetylase with peak activation occurring at 1-2 min of the ATP treatment. In addition, sodium vanadate, also an inhibitor of protein phosphatase, stimulates the increase in the incorporation of [3H]acetate into acyl[3H]acetyl-GPC of the ATP-treated endothelial cells. Collectively, our data show that both acetyltransferases and transacetylase participate in and contribute to the biosynthesis of acyl analogs of PAF in a coordinate fashion in endothelial cells.
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PMID:The role of platelet-activating factor-dependent transacetylase in the biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine by stimulated endothelial cells. 921 86

An aromatic carboxylate, anthracene-9-carboxylic acid (9-AC), is known as a Cl- channel blocker. However, variable 9-AC effects have hitherto been reported on the cardiac cAMP-activated Cl- conductance, when applied extracellularly. We have reexamined the 9-AC effect on the Cl-conductance activated by isoproterenol or forskolin in guinea pig ventricular myocytes under whole-cell patch-clamp conditions. The inward current was blocked by 9-AC at > or = 0.5 mmol/L, but in contrast, the outward current was enhanced at much lower concentrations (ED50, approximately 13 mumol/L). 9-AC applied by the intracellular perfusion technique increased both the inward and outward currents. In the presence of intracellular 9-AC, deactivation of the conductance after washout of isoproterenol or forskolin was largely prevented. 9-AC produced an enhancing effect, even after inhibiting the deactivation process by okadaic acid (OA), whereas it failed to produce additional-effects in the presence of orthovanadate. Intracellular application of 9-AC together with OA virtually abolished the current deactivation. The 9-AC effects on the Cl-conductance were not dependent on intracellular Ca2+ or pH. Putative inhibitors of alkaline (bromotetramisole) and acid phosphatases (tartrate) were without effect. 9-AC failed to inhibit the activities of purified protein phosphatase (PP)-1, -2A, and -2C. In the extract of guinea pig ventricle, 9-AC (> or = 10 mumol/L for full action) significantly inhibited a fraction of endogenous phosphatase activity that was sensitive to orthovanadate but not to OA, bromotetramisole, and tartrate. It is concluded that 9-AC blocks cardiac cAMP-activated (cystic fibrosis transmembrane conductance regulator) Cl- conductance from the extracellular side but enhances the conductance from the intracellular side by inhibiting an orthovanadate-sensitive phosphatase distinct from PP-1, -2A, -2B, or -2C and alkaline or acid phosphatase.
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PMID:Phosphatase-mediated enhancement of cardiac cAMP-activated Cl-conductance by a Cl- channel blocker, anthracene-9-carboxylate. 924 83


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