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)

Elevated dosage of the GAC1 gene from the yeast Saccharomyces cerevisiae causes hyperaccumulation of glycogen whereas a gene disruption of GAC1 results in reduced glycogen levels. Glycogen synthase is almost entirely in the active, glucose 6-phosphate-independent, form in cells with increased gene dosage of GAC1 whereas the enzyme is mostly in the inactive form in strains lacking GAC1. GAC1 encodes an 88 kDa protein that is similar to the regulatory subunit (RG1) of phosphoprotein phosphatase type 1 (PP-1) from skeletal muscle that targets PP-1 to glycogen particles. Taken together, these results suggest that GAC1 encodes a regulatory subunit of PP-1. As previously shown for glycogen phosphorylase (GPH1), GAC1 RNA accumulates concomitantly with the appearance of glycogen. A strain with a mutation in the regulatory subunit of the cAMP-dependent protein kinase (bcy1) fails to accumulate GPH1 and GAC1 RNA. These results point to coordinate regulation of enzymes involved in glycogen metabolism at the level of RNA accumulation and indicate that at least part of this control is exerted by the RAS-cAMP pathway.
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PMID:GAC1 may encode a regulatory subunit for protein phosphatase type 1 in Saccharomyces cerevisiae. 131 Sep 38

The Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is inhibited by the immunosuppressant drug cyclosporin A in the presence of cyclophilin A or B. Of the two isoforms, cyclophilin B is more potent by a factor of 2-5 when either the phosphoprotein [32P]casein or the [32P]phosphoserine [Ser(32P)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide RII, [Ser(32P)15]RII, is used as substrate. With [Ser(32P15]RII as substrate, the concentrations of the cyclosporin A.cyclophilin A and cyclosporin A.cyclophilin B complexes, which cause 50% inhibition of calcineurin activity, are 120 and 50 nM, respectively. Lowering the concentration of calcineurin 80% with [32P]casein as substrate lowered the apparent inhibition constant for each complex even further; 50% inhibition of calcineurin was observed at 40 nM for cyclosporin A.cyclophilin A, whereas it was less than 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [32P]casein or [Ser(32P)15]RII, the concentration of calcineurin required for measurable phosphatase activity is such that these complexes behave as tight-binding inhibitors of calcineurin, and steady-state kinetics cannot be used to assess inhibition patterns or Ki values. Limited trypsinization of calcineurin produces a fragment that is still inhibited, indicating that the interaction of cyclosporin.cyclophilin with calcineurin does not require either calmodulin or Ca2+.
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PMID:Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B. 131 36

In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.
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PMID:Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle. 132 72

The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factor FA has further been identified as a cAMP and Ca(2+)-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factor FA could phosphorylate synapsin I with a low Km value of about 2 microM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factor FA specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca2+/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factor FA could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factor FA as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.
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PMID:Identification of the ATP.Mg-dependent protein phosphatase activator (FA) as a synapsin I kinase that inhibits cross-linking of synapsin I with brain microtubules. 133 16

We have used a monoclonal antibody (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder epithelial cells, to isolate a chloride channel protein by the use of an immunoaffinity column and FPLC. This protein (M(r) 219,000) has been reconstituted into a planar lipid bilayer, where it behaves as a chloride-selective channel (PCl/PNa = 20.2; PNa/PK = 1) whose unit conductance is 62.4 +/- 4.6 pS. Antibody added to the trans side (there is no effect from the cis side) causes channel open probability to drop to virtually zero, but has no effect on the conductance or the selectivity of single channels. To test the role of phosphorylation in the activity of the native channel, we studied the effects of the protein phosphatase inhibitor okadaic acid (OA) on intact gallbladders, and showed that channels opened by theophylline treatment and closed by antibody are reopened reversibly by OA (0.01-1.0 microM). Addition of the catalytic subunit of protein phosphatase 2A (PP-2A) to the cis side of a bilayer containing reconstituted chloride channels caused closure of the channels after a delay, and subsequent addition of ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA) caused immediate reopening. These data indicate that (a) this chloride channel protein inserts in a directed way into the bilayer such that the cis side is 'intracellular', (b) the purified channel protein is phosphorylated, and (c) gating from the cellular side is controlled by the direct phosphorylation and dephosphorylation of the channel protein.
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PMID:Reconstitution and regulation of an epithelial chloride channel. 133 26

It has been recently proposed that DARPP-32 participates, as third messenger, in the mediation of effects induced by dopamine at the cellular level. DARPP-32 is indeed localized almost exclusively on dopaminoceptive neurons bearing the D1 receptor subtype and it is phosphorylated by cAMP-dependent protein kinase. In its phospho-form, DARPP-32 acts as an inhibitor of protein phosphatase-1. In vivo pharmacological treatment with selective D1 agonists and antagonists induces changes in the phosphorylation state of DARPP-32 that can be correlated to changes in cAMP, mediated in turn by D1 and D2 receptors. These data demonstrate that the measurement of the phosphorylation state of DARPP-32 with the back-phosphorylation assay can represent a useful biochemical tool to gain further insight into the sequence of events elicited by specific dopaminergic drugs in vivo.
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PMID:The phosphorylation state of DARPP-32, a third messenger for dopamine, is regulated by in vivo pharmacological treatments. 136 18

The voltage-dependent Na+ channel of the brain is a good substrate for phosphorylation by the cAMP-dependent protein kinase (protein kinase A, or PKA), but the physiological effects of PKA on Na+ channels are poorly documented. We studied modulation by PKA of voltage-dependent Na+ channels expressed in Xenopus oocytes injected with RNA coding for the alpha-subunit of the channel protein (rat brain type IIA and its variant VA200), using the two electrode voltage-clamp technique. Intracellularly injected cAMP or catalytic subunit of PKA, or extracellularly applied forskolin, inhibited the Na+ current by 20-30%. The effect of cAMP was attenuated by prior injection of PKA inhibitors. Injection of small doses of protein phosphatase 2A increased the Na+ current by 10%, whereas larger doses of protein phosphatase 1 and alkaline phosphatase were without effect. The inhibition by PKA showed little voltage dependence, being only slightly stronger at holding potentials at which the availability of the channels was reduced. The voltage dependence of activation and inactivation processes was not altered by cAMP. Similar effects were exerted by forskolin and cAMP on the Na+ channels expressed after the injection of heterologous (total) RNA from rat brain. Thus, PKA modulates the Na+ channel by a mechanism that does not involve major changes in the voltage dependency of the current and is exerted on the channel-forming alpha-subunit.
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PMID:Protein kinase A reduces voltage-dependent Na+ current in Xenopus oocytes. 138 76

Calmodulin-dependent phosphodiesterase was purified to apparent homogeneity from the total calmodulin-binding fraction of bovine heart in a single step by immunoaffinity chromatography. The isolated enzyme had significantly higher affinity for calmodulin than the bovine brain 60-kDa phosphodiesterase isozyme. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of the purified cardiac calmodulin-dependent phosphodiesterase with the incorporation of 1 mol of phosphate/mol of subunit. The phosphodiesterase phosphorylation rate was increased severalfold by histidine without affecting phosphate incorporation into the enzyme. Phosphorylation of phosphodiesterase lowered its affinity for calmodulin and Ca2+. At constant saturating concentrations of calmodulin (650 nM), the phosphorylated calmodulin-dependent phosphodiesterase required a higher concentration of Ca2+ (20 microM) than the nonphosphorylated phosphodiesterase (0.8 microM) for 50% activity. Phosphorylation could be reversed by the calmodulin-dependent phosphatase (calcineurin), and dephosphorylation was accompanied by an increase in the affinity of phosphodiesterase for calmodulin.
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PMID:Phosphorylation and characterization of bovine heart calmodulin-dependent phosphodiesterase. 164 4

The activating factor of ATP.Mg-dependent protein phosphatase (FA) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates, FA could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with a Km value of 0.4 microM, and tau proteins to 4 moles of phosphates per mole of proteins with a Km value of about 3 microM. When using microtubules as substrates, FA could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca+2/phospholipid-dependent protein kinase, the FA-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced by FA. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed that FA could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca+2/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due to FA. Taken together, the results provide initial evidence that the ATP.Mg-dependent protein phosphatase activating factor (FA) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.
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PMID:Identification and characterization of the ATP.Mg-dependent protein phosphatase activator (FA) as a microtubule protein kinase in the brain. 165 23

The effect of CPT-cAMP and okadaic acid on phosphatidylcholine catabolism in suspension cultures of choline-deficient rat hepatocytes was investigated. Choline-deficient hepatocytes were pulse-labeled for 30 min with [methyl-3H]choline and subsequently chased for up to 60 min with choline in the absence or presence of 0.5 mM CPT-cAMP or 0.5 microM okadaic acid. Radioactivity in phosphatidylcholine and lysophosphatidylcholine were unchanged during the chase. However, the radioactivity incorporated into glycerophosphocholine was significantly increased (P less than 0.05) 59 and 77% after 60 min of chase in hepatocytes incubated with either okadaic acid or CPT-cAMP, respectively. Incubation of choline-deficient hepatocytes with both okadaic acid and CPT-cAMP produced an additive effect on radioactivity incorporated ino glycerophosphocholine. Crude mitochondrial, microsomal, and cytosolic phospholipaselysophospholipase activities, assayed in the presence of exogenously labeled phosphatidylcholine, were unchanged in both CPT-cAMP and okadaic acid treated hepatocytes compared with control. Phospholipase-lysophospholipase activity, assayed with endogenously labeled phosphatidylcholine, was increased 28 and 47% (P less than 0.05) in the crude mitochondrial fraction of hepatocytes treated with either okadaic acid or CPT-cAMP, respectively, compared with the control. Incubation of choline-deficient hepatocytes, labeled with L-[methyl-3H]methionine, with CPT-cAMP or okadaic acid caused a 31 and 20% increase (P less than 0.05) in the radioactivity incorporated into glycerophosphocholine, respectively, compared with the control. We postulate that phosphatidylcholine catabolism in choline-deficient hepatocytes may be regulated by a phosphorylation-dephosphorylation mechanism mediated through cAMP-dependent protein kinase and phosphoprotein phosphatase activities.
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PMID:CPT-cAMP and okadaic acid enhance phosphatidylcholine catabolism in choline-deficient rat hepatocytes. 166 52


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