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 phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of 32Pi from [32P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [32P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions--notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated 1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and 2) by membrane deformation that alters enzyme-spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane-bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell-free systems.
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PMID:Phosphorylation and dephosphorylation of spectrin. 3 38

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.
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PMID:On the mechanism of ATP-induced shape changes in human erythrocyte membranes. II. The role of ATP. 19 4

Similar time courses were obtained for decreases in the rate of calcium transport by cardiac sarcoplasmic reticulum vesicles previously phosphorylated by cAMP-dependent protein kinase and dephosphorylation of the 22,000-dalton phosphoprotein in these membranes. Dephosphorylation of the 22,000-dalton phosphoprotein can be attributed to a phosphoprotein phosphatase in the sarcoplasmic reticular membranes. This membrane-bound phosphoprotein phosphatase may play a role in the reversal of the relaxation-promoting effect of catecholamines on the heart.
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PMID:Phosphoprotein phosphatase-catalyzed dephosphorylation of the 22,000-dalton phosphoprotein of cardiac sarcoplasmic reticulum. 20 87

The ATP.Mg-dependent type-1 protein phosphatase and its activating factor (protein kinase FA) were identified to exist in brain synaptosome. The inactive protein phosphatase was found to exist in the synaptosomal cytosol whereas its activating factor (protein kinase FA) was present in the synaptosomal membrane, indicating that the inactive protein phosphatase and its activating factor FA are localized in two separate subcellular compartments. The membrane-bound FA was found to exist in two forms; approximately 75% of FA is inactive and trypsin-resistant, whereas 25% of FA is active and trypsin-labile. When membranes were incubated with exogenous phospholipase C, the inactive/trypsin-resistant FA could be activated and sequestered to become the active/trypsin-labile FA in a time- and dose-dependent manner. Taken together, the results provide initial evidence that the activation-sequestration of membrane-bound protein kinase FA may represent one mode of control modulating the activity of protein kinase FA and thereby to activate protein phosphatase in brain synaptosome, representing an efficient regulatory mechanism for regulating neurotransmission in the central nervous system.
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PMID:The mechanism of activation of protein kinase FA (the activator of type-1 protein phosphatase) in brain synaptosomes. 131 12

1. In voltage-clamped whole cells dialysed with GTP, extracellular application of ACh elicits an inwardly rectifying K+ current which subsequently decreases to a steady-state level well below the maximally induced current (desensitization). The mechanism of desensitization of the acetylcholine (ACh)-activated K+ channel current was studied in rat neonatal atrial cells at the single-channel level using the patch-clamp technique. 2. In cell-attached patches with ACh in the pipette, a similar pattern of K+ channel current desensitization was present. Single-channel analyses revealed that the initial rapid decrease in channel activity was associated with progressive shortening of the mean open time (tau o) and prolongation of the mean closed time (tau c) of the K+ channel. 3. In excised, inside-out patches with ACh in the pipette, GTP activated K+ channels with a tau o of approximately 1.0 ms. Addition of ATP to the cytosolic surface resulted in progressive increases in tau o (from 1 to 5 ms) and channel activity. These changes are similar but opposite in direction to those observed during the early phase of ACh-induced channel desensitization in cell-attached patches. 4. The effect of ATP on the channel kinetics was abolished in Mg(2+)-free solution AMP-PNP (adenylyl-imidodiphosphate, a non-hydrolysable analogue of ATP), ADP, CTP (cytidine triphosphate), ITP (inosine triphosphate) or UTP (uridine triphosphate) did not alter the channel kinetics, suggesting that the ATP effect on channel gating probably occurs via phosphorylation by a membrane-bound kinase. H-8 (an isoquinolinesulphonamide derivative which inhibits protein kinases A and C) failed to prevent the action of ATP on the channel. 5. The increases in tau o and channel activity produced by ATP could be completely reversed by an elevation of cytosolic [Ca2+] to 3 x 10(-5) M or above. 6. The effect of Ca2+ on the ATP-induced changes in channel kinetics was blocked by sodium vanadate, a general phosphatase inhibitor. Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, did not block the Ca2+ effect. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), trifluoroperazine, and calmidazolium, partially blocked the effect of Ca2+. 7. Alkaline phosphatase (20 units/ml) reversed the ATP-induced increases in tau o and channel activity. These results suggest that the ACh-activated K+ channel can be modulated by phosphorylation and dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Modulation of acetylcholine-activated K+ channel function in rat atrial cells by phosphorylation. 165 50

Okadaic acid, a potent inhibitor of Type 1 and Type 2A protein phosphatases, was used to investigate the mechanism of insulin action on membrane-bound low Km cAMP phosphodiesterase in rat adipocytes. Upon incubation of cells with 1 microM okadaic acid for 20 min, phosphodiesterase was stimulated 3.7- to 3.9-fold. This stimulation was larger than that elicited by insulin (2.5- to 3.0-fold). Although okadaic acid enhanced the effect of insulin, the maximum effects of the two agents were not additive. When cells were pretreated with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), the level of phosphodiesterase stimulation by okadaic acid was rendered smaller, similar to that attained by insulin. In cells that had been treated with 2 mM KCN, okadaic acid (like insulin) failed to stimulate phosphodiesterase, suggesting that ATP was essential. Also, as reported previously, the effect of insulin on phosphodiesterase was reversed upon exposure of hormone-treated cells to KCN. This deactivation of previously-stimulated phosphodiesterase was blocked by okadaic acid, but not by insulin. The above KCN experiments were carried out with cells in which A-kinase activity was minimized by pretreatment with H-7. Okadaic acid mildly stimulated basal glucose transport and, at the same time, strongly inhibited the action of insulin thereon. It is suggested that insulin may stimulate phosphodiesterase by promoting its phosphorylation and that the hormonal effect may be reversed by a protein phosphatase which is sensitive to okadaic acid. The hypothetical protein kinase thought to be involved in the insulin-dependent stimulation of phosphodiesterase appears to be more H-7-resistant than A-kinase.
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PMID:Effects of okadaic acid on insulin-sensitive cAMP phosphodiesterase in rat adipocytes. Evidence that insulin may stimulate the enzyme by phosphorylation. 165 32

The Ca2(+)- and phospholipid-dependent protein kinase, protein kinase-C (PK-C), was studied in fetal rat liver between d 17 and 21 of gestation. Initial studies showed that rat liver, membrane-associated PK-C could be detected as a protein of Mr = 80,000 using a polyclonal rat brain PK-C antiserum. Fetal hepatic membrane-bound PK-C activity rose as gestation progressed with adult levels (21 +/- 3 pmol/min/mg protein) being attained by term. Although fasting for 48 h led to PK-C activation in adult livers, fetal hepatic PK-C was not activated by 48 h of maternal fasting. Membrane-associated protein phosphatase activity that might reverse PK-C action was also studied. PK-C sites in casein (an artificial PK-C substrate) were selectively dephosphorylated by a membrane-associated, poly-cation-stimulated protein phosphatase. This activity, thus classified as protein phosphatase type-2A, was constitutively expressed in fetal liver membranes from 17-21 d gestation. We have previously reported that the other major hepatic protein phosphatase, protein phosphatase type-1, also is constitutively expressed during the later stage of gestation. Taken together with the results of our present study, our data indicate that PK-C-dependent phosphorylation in fetal liver probably increases with advancing gestation.
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PMID:Hepatic protein kinase-C and protein phosphatase type-2A in the fetal rat. 216 16

A systematic study of protein kinase activity and phosphorylation of membrane proteins by ATP was carried out with vesicular fragments of longitudinal tubules (light SR) and junctional terminal cisternae (JTC) derived from skeletal muscle sarcoplasmic reticulum (SR). Following incubation of JTC with ATP, a 170,000-Da glycoprotein, a 97,500-Da protein (glycogen phosphorylase), and a 55,000-60,000-Da doublet (containing calmodulin-dependent protein kinase subunit) underwent phosphorylation. Addition of calmodulin in the presence of Ca2+ (with no added protein kinase) produced a 10-fold increase of phosphorylation involving numerous JTC proteins, including the large (approximately 450,000 Da) ryanodine receptor protein. Calmodulin-dependent phosphorylation of the ryanodine receptor protein was unambiguously demonstrated by Western blot analysis. The specificity of these findings was demonstrated by much lower levels of calmodulin-dependent phosphorylation in light SR as compared to JTC, and by much lower cyclic AMP dependent kinase activity in both JTC and light SR. These observations indicate that the purified JTC contain membrane-bound calmodulin-dependent protein kinase that undergoes autophosphorylation and catalyzes phosphorylation of various membrane proteins. Protein dephosphorylation was very slow in the absence of added phosphatases, but was accelerated by the addition of phosphatase 1 and 2A (catalytic subunit) in the absence of Ca2+, and calcineurin in the presence of Ca2+. Therefore, in the muscle fiber, dephosphorylation of SR proteins relies on cytoplasmic phosphatases. No significant effect of protein phosphorylation was detected on the Ca2(+)-induced Ca2+ release exhibited by isolated JTC vesicles. However, the selective and prominent association of calmodulin-dependent protein kinase and related substrates with junctional membranes, its Ca2+ sensitivity, and its close proximity to the ryanodine and dihydropyridine receptor Ca2+ channels suggest that this phosphorylation system is involved in regulation of functions linked to these structures.
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PMID:Specific association of calmodulin-dependent protein kinase and related substrates with the junctional sarcoplasmic reticulum of skeletal muscle. 216 64

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 phosphorylation and dephosphorylation of the dihydropyridine-sensitive Ca2+ channel was studied in transverse-tubule membranes isolated from rabbit skeletal muscle. Exposure of these membranes to either the cAMP-dependent protein kinase or a Ca2+/calmodulin-dependent protein kinase resulted in a rapid phosphorylation of a protein with properties similar to the major component of the skeletal muscle Ca2+ channel. The molecular mass of the phosphoprotein was 140 or 160 kDa, depending on the electrophoretic conditions. The stoichiometry of the phosphorylation was calculated to be 0.4-1.0 mol of phosphate per mol of protein. Neither the rate nor the extent of phosphorylation was affected by dihydropyridines. Limited proteolytic digestion of the protein that had been phosphorylated by either or both protein kinases yielded a single phosphopeptide of approximately equal to 5.4 kDa. The Ca2+-dependent phosphatase calcineurin dephosphorylated the membrane-bound Ca2+ channel that had been previously phosphorylated by either protein kinase. The results suggest that the major component of the dihydropyridine-sensitive Ca2+ channel from skeletal muscle can be effectively phosphorylated and dephosphorylated in its native state by cAMP- and Ca2+-dependent processes.
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PMID:Phosphorylation and dephosphorylation of dihydropyridine-sensitive voltage-dependent Ca2+ channel in skeletal muscle membranes by cAMP- and Ca2+-dependent processes. 242 10


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