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)

Tissue non-specific alkaline phosphatase is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic, protein phosphatase, and phosphotransferase activities. This enzyme is distributed virtually in all mammalian tissues, particularly during embryonic development. Its expression is stage-specific and can be demonstrated in the developing embryo as early as the 2-cell stage. It has been suggested that tissue non-specific alkaline phosphatase might play a role in tissue formation. In the study reported here, a gene-transfer approach was employed to investigate possible roles for this enzyme by inserting the cDNA for rat tissue non-specific alkaline phosphatase into CHO and LLC-PK1 cells. Permanently transfected cell-lines expressing varying levels of alkaline phosphatase were established. The data showed that functional enzyme was expressed in the transfected cells. Cell spreading and attachment were enhanced in transfected CHO cells expressing high levels of tissue non-specific alkaline phosphatase but not in the LLC-PK1 cells. Further, in CHO cells, proliferation was shown to be inversely proportional to the level of the tissue non-specific alkaline phosphatase expression. Homotypic cell association was demonstrated in both alkaline phosphatase-positive and alkaline phosphatase-negative cells in both CHO and LLC-PK1 cell-lines. Taken together, these findings suggest that in addition to a role in mineralization of bone, tissue non-specific alkaline phosphatase might also play a role in other cell activities, including those related to differentiation, such as cell-cell or cell-substrate interaction and proliferation.
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PMID:Changes in cell adhesion and cell proliferation are associated with expression of tissue non-specific alkaline phosphatase. 750 6

Neurogranin, a peptide capable of binding the calcium-poor form of calmodulin, was tested in vitro for its ability to modulate a typical calmodulin target. The target employed was the calcium/calmodulin-dependent form of nitric oxide synthase, which is produced by several different types of neurons. Neurogranin for the study was purified from perchloric acid-soluble calf brain proteins by a combination of calmodulin-Sepharose affinity chromatography and reverse-phase HPLC. The protocol yielded highly purified neurogranin that was active in assays using purified nitric oxide synthase. The titration of the enzyme activity with neurogranin demonstrated a concentration-dependent effect of the peptide on enzyme activation. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was performed in the presence of different amounts of neurogranin. The effect of neurogranin on the calcium-dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to approximately 1 microM calcium. Treatment of the neurogranin peptide with protein kinase C eliminated its inhibition on nitric oxide synthase activation. Treatment of the protein kinase C-phosphorylated peptide with calcineurin did not restore the ability of neurogranin to inhibit enzyme activity, whereas treatment with alkaline phosphatase did restore this ability. These results suggest that neurogranin may serve as a member of a unique class of endogenous calmodulin inhibitor that functions to regulate the activation of calmodulin-requiring targets in neurons.
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PMID:The dendritic peptide neurogranin can regulate a calmodulin-dependent target. 752 68

An immobilized hepatocyte preparation was used to show that both vasopressin and glucagon could desensitize the ability of glucagon to increase intracellular cyclic AMP concentrations. This process was not dependent on any influx of extracellular Ca2+ and was not mediated by any rise in the intracellular level of Ca2+. The protein kinase C-selective inhibitors chelerythrine, staurosporine and calphostin C acted as potent inhibitors of the desensitization process but with various degrees of selectivity regarding their ability to inhibit the desensitizing actions of glucagon and vasopressin. The protein phosphatase inhibitor okadaic acid was just as potent as vasopressin and glucagon in causing desensitization. Treatment of hepatocyte membranes with alkaline phosphatase restored to near control levels the ability of glucagon to stimulate adenylate cyclase activity in membranes from both glucagon- and vasopressin-treated (desensitized) hepatocytes. It is suggested that the desensitization of glucagon-stimulated adenylate cyclase activity involves a reversible phosphorylation reaction with the likely target being the glucagon receptor itself.
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PMID:A role for protein kinase C-mediated phosphorylation in eliciting glucagon desensitization in rat hepatocytes. 753 13

It has been demonstrated that dephosphorylation of the ferredoxin component of the mitochondrial 25-hydroxyvitamin D3-1-hydroxylase, as a result of a PTH-cAMP mediated activation, involves a protein phosphatase activity. However, the nature and properties of this phosphatase are uncertain. It has been proved that alkaline phosphatase, a magnesium dependent enzyme, could dephosphorylate in vitro the ferredoxin component of the 25-hydroxyvitamin D3-1-hydroxylase. Moreover, some evidence of mitochondrial localization of some alkaline phosphatases has been published. Although the existence of a levamisole inhibitable alkaline phosphatase activity has been described in renal cells, its role remains to be elucidated. In the present work, the existence of an alkaline phosphatase in mitochondrial membrane preparations from LLC-PK1 cells has been described. This alkaline phosphatase is magnesium dependent and levamisole inhibitable. Preparations of mitochondrial membrane from LLC-PK1 cells also showed 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) and 25-hydroxyvitamin D3-24R-hydroxylase (24-hydroxylase) activities being both enzymes responsive to the 8Br-cAMP mediated regulation. The 8Br-cAMP not only stimulated the 1-hydroxylase and inhibited the 24-hydroxylase activities but also increased the mitochondrial alkaline phosphatase activity. In the same way, the levamisole (specific inhibitor of some alkaline phosphatases) inhibited the mitochondrial alkaline phosphatase and also the 1-hydroxylase activity. In addition, the inhibition of mitochondrial alkaline phosphatase by levamisole avoids the effect of 8Br-cAMP on the 1-hydroxylase and 24-hydroxylase activities. On the other hand, the mitochondrial alkaline phosphatase and the 1-hydroxylase activities showed similar behaviour with respect to the magnesium concentrations in the incubation medium. Taking these results together it could be possible to suggest the implication of the Mg(2+)-dependent mitochondrial alkaline phosphatase activity found in LLC-PK1 cells in the regulation of the 1,25(OH)2D3 and 24,25(OH)2D3 synthesis.
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PMID:Possible involvement of a magnesium dependent mitochondrial alkaline phosphatase in the regulation of the 25-hydroxyvitamin D3-1 alpha-and 25-hydroxyvitamin D3-24R-hydroxylases in LLC-PK1 cells. 754 Apr 7

Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding.
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PMID:Phosphorylation of the transcription factor NFATp inhibits its DNA binding activity in cyclosporin A-treated human B and T cells. 765 45

To determine whether protein phosphatases can affect bone regulation, we examined the effects of okadaic acid (OA) and calyculin A (CA), specific inhibitors of protein phosphatases type 1 and type 2A, on alkaline phosphatase activity of mouse osteoblastic cells. Clone MC3T3-E1 cells were cultured with varying concentrations of OA and CA. OA and CA stimulated alkaline phosphatase (ALP) activity in the cells in dose-dependent fashion with a maximal effect at concentrations of 5 nM and 2 nM, respectively. The properties of OA-induced and native ALP in the cells were the same and they were liver-bone-kidney type. These results show that protein phosphatase inhibitors stimulate bone formation in vitro and that phosphorylation and dephosphorylation of specific proteins in the cells may be involved in bone regulation in vivo as well.
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PMID:Protein phosphatase inhibitors, okadaic acid and calyculin A, induce alkaline phosphatase activity in osteoblastic cells derived from newborn mouse calvaria. 766 40

Cystic fibrosis transmembrane conductance regulator (CFTR) is a regulated Cl- channel; in secretory epithelia, it is located in the apical membrane where it regulates transepithelial Cl- secretion. Previous studies have shown that cAMP-dependent protein kinase (PKA) can phosphorylate and activate CFTR Cl- channels. We asked whether other kinases would phosphorylate CFTR in vitro and activate CFTR Cl- channels in excised, inside-out patches of membrane from NIH 3T3 fibroblasts stably expressing recombinant CFTR. We found that both Ca(2+)-independent and Ca(2+)-dependent isoforms of protein kinase C (PKC) activated the CFTR Cl- channel. Consistent with this finding, PKC also phosphorylated CFTR in vitro. In contrast, the multifunctional Ca2+/calmodulin-dependent protein kinase failed to either activate or to phosphorylate CFTR Cl- channels, suggesting that this enzyme has no direct effect on CFTR. We found that cGMP-dependent protein kinase (cGK) (purified from bovine lung) phosphorylated CFTR in vitro. However, cGMP failed to increase the apical membrane Cl- permeability in human airway epithelia, and addition of cGMP, ATP, and cGK failed to activate CFTR Cl- channels. These results suggest that if cGK phosphorylates CFTR in vivo, it does so at sites not involved in CFTR Cl- channel activation. Because cAMP-dependent activation of CFTR Cl- channels and Cl- secretion in intact cells is reversible, we asked whether specific phosphatases can dephosphorylate and inactivate CFTR Cl- channels. Addition of protein phosphatase 2A (PP2A) decreased PKA-activated current by 67% within 10 min. The phosphatase inhibitor calyculin-A blocked the effect of PP2A. In contrast, neither protein phosphatases 1, 2B, nor two preparations of alkaline phosphatase inactivated PKA-phosphorylated CFTR Cl- channels. The effects of protein phosphatases on CFTR function were paralleled by their ability to dephosphorylate CFTR in vitro. Our data indicate that CFTR Cl- channels can be phosphorylated and activated by PKA as well as by Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC and can be dephosphorylated and thus inactivated by PP2A.
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PMID:Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases. 767 14

Acid extracts of thapsigargin-activated Jurkat cells have been shown to have intracellular activity in inducing a dose-dependent rapid chloride current upon microinjection in Xenopus laevis oocytes. The extracts act by elevation of calcium through calcium entry. The factor(s) responsible for this activity have been termed calcium influx factor (CIF) and have been found to be small, relatively polar molecules (< 1000 daltons) whose activity is abolished by alkaline phosphatase treatment and potentiated by co-injection of okadaic acid (a protein phosphatase inhibitor). CIF is produced in a time-dependent manner following thapsigargin treatment of Jurkat cells, being first elevated above basal levels by 2 min. Intracellular CIF activity is completely absent from NG115-401L neuronal cells, which lack capacitative entry. On this basis, it appears that Jurkat cells, activated by stimuli that deplete internal calcium stores, produce one or more CIF activities acting intracellularly, and Xenopus oocytes may be a powerful tool to purify and characterize CIFs.
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PMID:Evaluation of calcium influx factors from stimulated Jurkat T-lymphocytes by microinjection into Xenopus oocytes. 789 75

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary zone electrophoresis (CZE) were evaluated for monitoring protein phosphatase and kinase reactions in vitro. Varying concentrations of peptide C (YIHLEKKYVRRDSG), peptide S (YLIEDNEYTARQGA) and kemptide (LARRSALG) mixed with their corresponding phosphorylated peptides, pC, pS and pkemptide, were analyzed. Comparison between the two techniques indicated that MALDI MS was less quantitative than CZE, showing a bias towards detection of the unphosphorylated peptide S and kemptide. In terms of sensitivity, the MALDI MS and CZE techniques are comparable. Protein kinase A phosphorylation of kemptide was monitored with both MALDI MS and CZE, whereas alkaline phosphatase dephosphorylation of pC could only be monitored with MALDI MS. The absence of inhibition with phosphatase or kinase buffers is a significant advantage of MALDI MS. In contrast to CZE, the MALDI spectra allow identification of the species analyzed by virtue of their mass. The results obtained emphasize the advantage of monitoring enzymatic reactions in buffer solutions using MALDI MS compared with CZE.
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PMID:Monitoring protein kinase and phosphatase reactions with matrix-assisted laser desorption/ionization mass spectrometry and capillary zone electrophoresis: comparison of the detection efficiency of peptide-phosphopeptide mixtures. 791 94

We previously reported that fetal calf serum-induced alkaline phosphatase activity is suppressed due to the activation of protein kinase C in osteoblast-like MC3T3-E1 cells (Miwa et al. (1991) Bone Miner. 14, 15-25; Kotoyori et al. (1994) Horm. Metab. Res. 26, 116-118). In the present study, we examined the effect of okadaic acid, a potent and specific inhibitor of protein phosphatase type 1 and 2A, on fetal calf serum-induced alkaline phosphatase activity in MC3T3-E1 cells. The pretreatment with okadaic acid enhanced the fetal calf serum-induced alkaline phosphatase activity in a dose-dependent manner in the range between 0.1 and 5 nM. 1-Norokadaone, a less potent analogue of okadaic acid, had little effect on the fetal calf serum-induced alkaline phosphatase activity. Okadaic acid partially reversed the suppression of fetal calf serum-induced alkaline phosphatase activity by 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C activator. The effect of okadaic acid was dose-dependent in the range between 0.1 and 5 nM. The patterns of the dose-dependency of both okadaic acid effects on fetal calf serum-induced alkaline phosphatase activity and on the suppression by 12-O-tetradecanoylphorbol-13-acetate were similar. These results strongly suggest that protein phosphatase type 1 and/or 2A act as a regulator of alkaline phosphatase activity at a point downstream from protein kinase C in osteoblast-like cells.
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PMID:Okadaic acid reverses the inhibitory effect of protein kinase C on alkaline phosphatase activity in osteoblast-like cells. 795 88


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