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)

We investigated the effects of the protein phosphatase inhibitors okadaic acid and microcystin-LR upon transport of newly synthesized proteins through the exocytic pathway. Treatment of CHO cells with 1 microM okadaic acid rapidly inhibited movement of a marker protein (vesicular stomatitis virus G protein) from the endoplasmic reticulum to the Golgi compartment. Both okadaic acid and microcystin-LR also inhibited transport in an in vitro assay reconstituting movement to the Golgi compartment, at concentrations equivalent to those required to inhibit phosphorylase phosphatase activity. Inhibition both in vivo and in vitro could be antagonized by protein kinase inhibitors, suggesting that protein phosphorylation was directly responsible for this effect. An early stage in the transport reaction associated with vesicle formation or targeting was inhibited by protein phosphorylation, which could be reversed by fractions enriched in protein phosphatase 2A. Protein kinase antagonists did not inhibit transport between sequential compartments of the exocytic pathway in vitro, suggesting that protein phosphorylation is not itself required for vesicular transport. During mitosis, vesicular transport is inhibited simultaneous to the activation of maturation-promoting factor. It is proposed that the inhibition caused by okadaic acid and microcystin-LR involves a similar mechanism to that responsible for the mitotic arrest of vesicular transport.
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PMID:Evidence for the regulation of exocytic transport by protein phosphorylation. 131 11

A combination of immunocytochemical and electron microscopic methods was used to study the effects of okadaic acid, a specific inhibitor of protein phosphatase types 1 and 2A, on the Golgi complex and the microtubule system of interphase CHO cells. At a concentration of 0.25 microM and within 2-3 h of exposure, okadaic acid caused a reversible disorganization of the Golgi complex, observed as a disintegration of the stacks of cisternae and formation of clusters of tubules and vesicles dispersed in the cytoplasm. At the same time, staining for mannosidase II was shifted from the Golgi stacks to the endoplasmic reticulum, whereas the clusters of tubules and vesicles for the main part were negative. This change in localization of the enzyme was not blocked by cycloheximide and thus not dependent on ongoing protein synthesis. The changes in the morphology of the Golgi complex were coordinated in time with a remodelling of the microtubule system, observed as a reduction in the number of microtubules, a tendency of the remaining microtubules to arrange in an aster-like pattern, and an increased sensitivity to low concentrations of the microtubule-disruptive drug nocodazole. After removal of the drug, the microtubule system was rapidly normalized (1-2 h) and subsequently also the Golgi complex (4-8 h). The results suggest that okadaic acid induces a redistribution of the Golgi stacks into the endoplasmic reticulum, leaving the trans-most elements behind as tubules and vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Disorganization of the Golgi complex and the cytoplasmic microtubule system in CHO cells exposed to okadaic acid. 133 78

The glucose regulated proteins (GRPs) are major structural components of the endoplasmic reticulum (ER) and are involved in the import, folding, and processing of ER proteins. Expression of the glucose regulated proteins (GRP78 and GRP94) is greatly increased after cells are exposed to stress agents (including A23187 and tunicamycin) which inhibit ER function. Here, we demonstrate that three novel inhibitors of ER function, thapsigargin (which inhibits the ER Ca(2+)-ATPase), brefeldin A (an inhibitor of vesicle transport between the ER and Golgi) and AIF4-, (which inhibits trimeric G-proteins), can increase the expression of both GRP78 and 94. The common characteristic shared by activators of GRP expression is that they disrupt some function of the ER. The increased levels of GRPs may be a response to the accumulation of aberrant proteins in the ER or they may be increased in response to structural/functional damage to the ER. The increased accumulation of GRP78 mRNA after exposure of cells to either thapsigargin, brefeldin A, AIF4-, A23187, or tunicamycin can be blocked by pre-incubation in cycloheximide. In contrast, accumulation of GRPs after exposure to hypoxia was independent of cycloheximide. In addition, the protein kinase inhibitor genistein blocked the thapsigargin induced accumulation of GRP78 mRNA, whereas the protein phosphatase inhibitor okadaic acid caused increased accumulation of GRP78 mRNA. The data indicates that there are at least 2 mechanisms for induced expression of GRPs, one of which involves a phosphorylation step and requires new protein synthesis (e.g., thapsigargin, A23187) and one which is independent of both these steps (hypoxia).
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PMID:Brefeldin A, thapsigargin, and AIF4- stimulate the accumulation of GRP78 mRNA in a cycloheximide dependent manner, whilst induction by hypoxia is independent of protein synthesis. 150 13

Studies on sphingomyelin metabolism in rat hepatocytes were facilitated by the use of choline-deficient cells which allowed for the rapid labeling of phosphatidylcholine and as a result sphingomyelin. Pulse and pulse-chase studies with [methyl-3H]choline and [methyl-3H]methionine demonstrated that both compounds were effectively used for sphingomyelin biosynthesis and that newly made and pre-existing phosphatidylcholine could be used for sphingomyelin biosynthesis. When hepatocytes were incubated with brefeldin A, there was a 2.4-fold stimulation of the conversion of phosphatidylcholine into sphingomyelin. Since brefeldin A causes collapse of the cis/medial Golgi into the endoplasmic reticulum the stimulation of sphingomyelin biosynthesis could be due to more rapid access of the labeled phosphatidylcholine in the endoplasmic reticulum to sphingomyelin synthase in the collapsed Golgi. Forskolin inhibited the brefeldin A-induced stimulation of sphingomyelin biosynthesis. To investigate whether or not phosphorylation reactions regulate sphingomyelin metabolism, hepatocytes were incubated with okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A. Rather than stimulating sphingomyelin biosynthesis, okadaic acid enhanced the catabolism of sphingomyelin. In contrast, a cyclic AMP analogue and forskolin had no effect on sphingomyelin biosynthesis or catabolism. Surprisingly, other pulse-chase studies demonstrated that okadaic acid stimulated the catabolism of only newly made sphingomyelin. The brefeldin A and okadaic acid effects were independent of lysosomal involvement. Subcellular fractionation studies revealed that brefeldin A and okadaic acid effects were generalized in all sphingomyelin containing membranes. The brefeldin A studies suggest that the rate of transfer of phosphatidylcholine from the endoplasmic reticulum to the Golgi might be limiting for sphingomyelin biosynthesis. The okadaic acid studies indicate that the catabolism of sphingomyelin by a sphingomyelinase is regulated by an unidentified protein kinase and by either protein phosphatase 1 and/or 2A activity in hepatocytes.
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PMID:Stimulation of sphingomyelin biosynthesis by brefeldin A and sphingomyelin breakdown by okadaic acid treatment of rat hepatocytes. 161 52

Several rat liver HMG-CoA-reductase (HMG-CoA-Rd) phosphatase activities have been shown to be associated with the endoplasmic reticulum. These activities were not due to glycogen contamination, as judged not only from different patterns of solubilization of the microsomal membranes and the glycogen pellet but also by differential centrifugation behavior under standard conditions and in a sucrose gradient. We present evidence that at least three forms of protein phosphatase are associated with microsomal membranes: a polycation-stimulated type 2A phosphatase, a type 2C phosphatase, and a non-2A, non-2B, non-2C phosphatase. This last HMG-CoA-Rd phosphatase activity corresponding to an 85 kDa protein was partially purified by several chromatographic procedures. The IC50 value for the inhibition of the HMG-CoA-Rd phosphatase by I-2 was 10-fold higher than for the inhibition of the purified type 1 catalytic subunit from rabbit skeletal muscle. The microsomal HMG-CoA-Rd phosphatase activity was slightly affected by the protein inhibitor that inhibits type 2A activity when HMG-CoA reductase is the substrate. The HMG-CoA-Rd phosphatase activity is spontaneously active and it is not reactivated in the presence of Mg2+ or polycations. The holoenzyme does not contain the inhibitor-2 and it is not reactivated by incubation with ATP and glycogen synthase kinase-3. Proteolytic treatment of the enzyme yielded a polypeptide fragment of low Mr (37 kDa) with reduced activity. A model of holoenzymatic HMG-CoA-Rd phosphatase and its relation to the microsomal membranes is presented.
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PMID:Isolation and partial characterization of a protein with HMG-CoA reductase phosphatase activity associated with rat liver microsomal membranes. 175 9

Phosphatidylcholine is apparently essential for mammalian life, since there are no known inherited diseases in the biosynthesis of this lipid. One of its critical roles appears to be in the structure of the eucaryotic membranes. Why phosphatidylcholine is required and why other phospholipids will not substitute are unknown. The major pathway for the biosynthesis of phosphatidylcholine occurs via the CDP-choline pathway. Choline kinase, the initial enzyme in the sequence, has been purified to homogeneity from kidney and liver and also catalyzes the phosphorylation of ethanolamine. Most evidence suggests that the next enzyme in the pathway, CTP:phosphocholine cytidylyltransferase, catalyzes the rate-limiting and regulated step in phosphatidylcholine biosynthesis. This enzyme has also been completely purified from liver. Cytidylyltransferase appears to exist in the cytosol as an inactive reservoir of enzyme and as a membrane-bound form (largely associated with the endoplasmic reticulum), which is activated by the phospholipid environment. There is evidence that the activity of this enzyme and the rate of phosphatidylcholine biosynthesis are regulated by the reversible translocation of the cytidylyltransferase between membranes and cytosol. Three major mechanisms appear to govern the distribution and cellular activity of this enzyme. (i) The enzyme is phosphorylated by cAMP-dependent protein kinase, which results in release of the enzyme into the cytosol. Reactivation of cytidylyltransferase by binding to membranes can occur by the action of protein phosphatase 1 or 2A. (ii) Fatty acids added to cells in culture or in vitro causes the enzyme to bind to membranes, where it is activated. Removal of the fatty acids dissociates the enzyme from the membrane. (iii) Perhaps most importantly, the concentration of phosphatidylcholine in the endoplasmic reticulum feedback regulates the distribution of cytidylyltransferase. A decrease in the level of phosphatidylcholine causes the enzyme to be activated by binding to the membrane, whereas an increase in phosphatidylcholine mediates the release of enzyme into the cytosol. The third enzyme in the CDP-choline pathway, CDP-choline:1,2-diacylglycerol choline-phosphotransferase, has been cloned from yeast but never purified from any source. In liver an alternative pathway for phosphatidylcholine biosynthesis is the methylation of phosphatidylethanolamine by phosphatidylethanolamine N-methyltransferase. This enzyme is membrane bound and has been purified to homogeneity. It catalyzes all three methylation reactions involved in the conversion of phosphatidylethanolamine to phosphatidylcholine.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Boehringer Mannheim Award lecture. Phosphatidylcholine metabolism: masochistic enzymology, metabolic regulation, and lipoprotein assembly. 226 10

The smooth endoplasmic reticulum (ER) and cytosol fractions of liver homogenates exhibit phosphoprotein phosphatase activity towards glycogen synthase D and phosphorylase a. The following observations suggest that liver contains multiple forms of these phosphatases. Synthase phosphatase activity in either fraction was more readily inactivated by heating than phosphorylase phosphatase activity. Both synthase phosphatase and phosphorylase phosphatase activities in smooth ER were non-competitively inhibited by Mg2+, but were activated by this ion in the cytosol. Synthase phosphatase activities in cytosol and smooth ER were stimulated by a number of sugar phosphates, particularly glucose-1-phosphate, galactose-6-phosphate and fructose-6-phosphate. Erythrose-4-phosphate stimulated synthase phosphatase activity in the cytosol, but inhibited the microsomal enzyme. Phosphorylase phosphatase activities in either fraction were inhibited by most sugar phosphates. Adenosine mono-, di- and tri-phosphates inhibited phosphatase activities in both fractions. Low concentrations of AMP and ADP inhibited phosphorylase phosphatase activities to a greater extent than synthase phosphatase activities. Chromatography of the smooth ER fraction on DEAE-cellulose resulted in the separation of synthase phosphatase from phosphorylase phosphatase, as soluble proteins. The elution profile for the microsomal phosphatase was different from that for the cytosol enzymes. It is concluded that: both synthase phosphatase and phosphorylase phosphatase in liver have at least two isoenzyme forms; synthase phosphatase and phosphorylase phosphatase are separate enzymes; the different behaviour of microsomal and cytosol phosphatases towards divalent cations and sugar phosphates provides a potential mechanism for the differential regulation of these activities in liver.
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PMID:Multiple forms of synthase D phosphatase and phosphorylase a phosphatase in liver and regulatory effects of metabolites on their activities. 298 42

The expressed catalytic activity of liver microsomal HMG CoA reductase, the limiting enzyme in cholesterol synthesis, is reversibly diminished by phosphorylation in vitro. In intact hepatocytes the expressed activity of HMG CoA reductase is enhanced by incubation of cells with insulin, and diminished by treatment with glucagon or with mevalonate. In the latter situations the level of total reductase activity falls following initial inactivation (phosphorylation) of the enzyme. This observation suggested that the phosphorylated form of HMG CoA reductase is more sensitive to proteolysis. HMG CoA reductase is a 97,000 dalton (97 K) integral protein of the endoplasmic reticulum with a cytosolic domain that includes the catalytic site and serine residues that may be reversibly phosphorylated. In vitro the Ca2+-activated proteolytic enzyme, calpain, generates two catalytically-active fragments: a membrane bound 62 K and a soluble 53 K form of the enzyme which are quantified by specific immunoblot procedures. Cleavage of the native 97 K HMG CoA reductase is enhanced by pretreatment (inactivation) of microsomes with ATP (Mg2+) and liver reductase kinase compared to microsomes pretreated with protein phosphatase. This is reflected in a loss of 97 K reductase and an increase in the soluble 53 K form of the enzyme. Degradation of HMG CoA reductase in hepatocytes is partially blocked by lysosomotropic agents and insulin. A steady state model for the turnover of proteins subject to reversible phosphorylation has been developed which recognizes fractional degradative rate constants for the phosphorylated and dephosphorylated species.
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PMID:Phosphorylation state of HMG CoA reductase affects its catalytic activity and degradation. 302 50

Enzyme cytochemical and immunocytochemical techniques at the light and electron microscope levels were used to study the distribution of potential markers of chemical transformation in rodent bladders. In rat tumours induced by in vivo treatment with methylnitrosourea, alkaline phosphatase localization was normal on the external surface of the plasma membranes of some cells but abnormal in others where reaction product was seen only on intracellular membranes. 5'-Nucleotidase localization was abnormal in all cells, being seen on endoplasmic reticulum and nuclear membranes only, while in normal bladders only ectoenzyme localization was seen. Heterogeneity of alkaline phosphatase amd 5'-nucleotidase localization was seen on the plasma membranes of these tumours after 15 days in organ culture. Some cells produced enzyme and others did not; in other cells only parts of the membrane reacted heavily, while other regions were negative. In transformed cell cultures and tumours of mouse bladder derived by in vitro treatment of explants with dimethylbenz (a) anthracene, a bimodal pattern of alkaline phosphatase localization was seen. Cells had either normal ectoenzyme reaction product or abnormal intracellular membrane reaction product. 5'-Nucleotidase and ADPase were lost after transformation while cAMP-phosphodiesterase was retained as an ectoenzyme. Mg.ATPase and a cAMP-independent, calcium-insensitive 'protein phosphatase' were induced in transformed cell cultures. An epithelial antigen was detected in the cytoplasm of both normal and transformed cells associated with reticular cytoplasmic ground substance, plasma membrane vesicles and cytoskeletal elements.
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PMID:Cytochemical markers of bladder carcinogenesis. 627 42

Three stages of development of hepatic glycogen metabolism in the rat were studied. These included the last stage of gestation, in which large scale synthesis and accumulation of glycogen takes place, the perinatal period of glycogenolysis, and the suckling period up to and including weaning. The role of insulin in the regulation of the key rate-limiting enzymes of glycogen synthesis (glycogen synthase) and glycogen breakdown (glycogen phosphorylase) was studied as was the role of the key phosphoprotein phosphatase enzymes that regulate activation of synthase (synthase phosphatase) and inactivation of phosphorylase (phosphorylase phosphatase). Glycogen accumulates in significant quantities on days 20-21 of gestation in the rat (term, 22 days). Associated with this increased rate and amount of glycogen accumulation is an increase in glycogen synthase a and synthase phosphatase and phosphorylase phosphatase activities associated with the endoplasmic reticulum (ER). Concomitantly, fetal insulin levels are elevated as is the insulin to glucagon molar ratio and the synthase a/phosphorylase a ratio. At birth, these hepatic glycogen stores are rapidly degraded, and synthase a levels are diminished, as are ER-associated synthase phosphatase and phosphorylase phosphatase activities. Phosphorylase a levels are markedly elevated at this time as well. Insulin levels are decreased, as is the insulin to glucagon molar ratio. Gradually over a period of 4 weeks after birth, glycogen levels increase in the liver, accompanied by increased ER-associated phosphatase activities and an increased insulin to glucagon molar ratio. The data support a role for increased ambient insulin concentrations in regulation of the periods of active glycogen synthesis and accumulation in pre- and postnatal rat liver. A possible site of action of insulin is the ER and associated phosphoprotein phosphatase activities.
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PMID:Regulation of hepatic glycogen metabolism in pre- and postnatal rats. 640 92


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