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 ATP.Mg-dependent type-1 protein phosphatase activating factor (FA) was identified as a protein 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. More importantly, more than 90% of the phosphates in 32P-synapsin I phosphorylated by FA could be removed by the activated ATP.Mg-dependent type-1 protein phosphatase and the synapsin I phosphatase activity was found to be strictly FA-dependent. Functional study further revealed that as a synapsin I kinase, factor FA could phosphorylate synapsin I and thereby inhibits crosslinking of synapsin I with tubulin, while as a synapsin I phosphatase activator, FA could promote the crosslinking copolymerization of synapsin I with tubulin. Taken together, the results provide initial evidence that a cyclic modulation of the crosslinking copolymerization of synapsin I with brain microtubules can be controlled by factor FA, representing an efficient cyclic cascade control mechanism for the regulation of axonal transport process during neurotransmission.
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PMID:Cyclic inhibition-potentiation of the crosslinking of synapsin I with brain microtubules by protein kinase FA (an activator of ATP.Mg-dependent protein phosphatase). 131 41

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

Characteristics of the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from the cytosol and in the postsynaptic densities (PSD) of rat brain were investigated. Several proteins were surveyed for their abilities to serve as a substrate for non-autophosphorylated and autophosphorylated CaM kinase IIs from the cytosol and PSD. The tested substrates were separated into two groups. Autophosphorylation of the kinase slightly decreased or did not change its activities towards substrates of the first group: myosin light chain of chicken gizzard, synapsin I, tau factor and microtubule-associated protein 2. In contrast, autophosphorylation of the enzyme increased its activities towards substrates of the second group: syntide-2, histone H1, calcineurin and myelin basic protein. The Ca2+/calmodulin-independent kinase activity increased by autophosphorylation with any of substrates tested. Similar results were obtained with the cytosolic and PSD CaM kinase II. Trifluoperazine and mastoparan, calmodulin binding antagonists, inhibited the activity of the non-autophosphorylated CaM kinase II, but had no effect or only a slight inhibitory effect on the activity of the autophosphorylated CaM kinase II, indicating that the autophosphorylated kinase has no requirement for calmodulin for Ca(2+)-dependent activity and/or a higher affinity for calmodulin The results suggest that the autophosphorylation of CaM kinase II is a subtle mechanism for regulating the interaction between the enzyme and substrate.
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PMID:Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II: effects on interaction between enzyme and substrate. 164 40

Protein phosphorylation is involved in the regulation of a wide variety of physiological processes in the nervous system. Studies in which purified protein kinases or kinase inhibitors have been microinjected into defined cells while a specific response is monitored have demonstrated that protein phosphorylation is both necessary and sufficient to mediate responses of excitable cells to extracellular signals. The precise molecular mechanisms involved in neuronal signal transduction processes can be further elucidated by identification and characterization of the substrate proteins for the various protein kinases. The roles of three such substrate proteins in signal transduction are described in this article: 1) synapsin I, whose phosphorylation increases neurotransmitter release and thereby modulates synaptic transmission presynaptically; 2) the nicotinic acetylcholine receptor, whose phosphorylation increases its rate of desensitization and thereby modulates synaptic transmission postsynaptically; and 3) DARPP-32, whose phosphorylation converts it to a protein phosphatase inhibitor and which thereby may mediate interactions between dopamine and other neurotransmitter systems. The characterization of the large number of additional phosphoproteins that have been found in the nervous system should elucidate many additional molecular mechanisms involved in signal transduction in neurons.
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PMID:Role of protein phosphorylation in neuronal signal transduction. 249 6

A Mn2+/phospholipid-dependent protein phosphatase has been identified and characterized from brain membranes. The phosphatase contains three subunits with molecular weights of 64,000, 54,000, and 35,000 in a 1:1:1 molar ratio. On gel filtration, the enzyme has an apparent molecular weight of approximately 180,000. The phosphatase was active on many substrates, including p-nitrophenyl phosphate, phosphotyrosine, phosphothreonine, phosphorylase a, myelin basic protein, histones, type 1 phosphatase inhibitor-2, microtubule tau protein, and synapsin I. To dephosphorylate phosphoproteins, the phosphatase was dependent on such acidic phospholipids as phosphatidylinositol and phosphatidylserine but not on neutral phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The phospholipid-mediated activation of the phosphatase was time and dose dependent and could be reversed by Triton X-100 or gel filtration. Kinetic study further indicates that phospholipid was able to increase the Vmax of the phosphatase but had no effect on the Km value for substrates, suggesting a direct interaction of phospholipids with the phosphatase. Conversely, in order to dephosphorylate phosphoamino acids such as phosphotyrosine and phosphothreonine, this phosphatase was entirely dependent on Mn2+. Phospholipids had no effect on the dephosphorylation of phosphoamino acids, whereas Mn2+ had no effect on the dephosphorylation of phosphoproteins. It is concluded that this Mn2+/phospholipid-dependent membrane phosphatase has two distinct activation mechanisms. The enzyme requires Mn2+ to dephosphorylate micromolecules, whereas acidic phospholipids are needed to dephosphorylate macromolecules. This suggests that Mn2+ and phospholipids may play a role in regulating the substrate specificity of this multisubstrate membrane phosphatase.
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PMID:Purification and characterization of a Mn2+/phospholipid-dependent protein phosphatase from pig brain membranes. 255 48

This article summarizes some of our knowledge concerning intracellular protein phosphorylation pathways in nerve cells. It also summarizes, very briefly, recent direct experimental evidence involving intracellular injection of protein kinases, protein kinase inhibitors, and substrates, indicating that protein phosphorylation mediates the actions of a variety of neurotransmitters on their target cells. Finally, it summarizes in somewhat greater detail the results of studies of three different types of substrate proteins that appear to regulate different types of biological responses in nerve cells: synapsin I, a substrate protein present in virtually all nerve terminals, which appears to regulate neurotransmitter release from those nerve terminals; the acetylcholine receptor, the phosphorylation of which regulates its rate of desensitization in the presence of acetylcholine; and DARPP-32, the phosphorylation of which converts it into a very potent phosphoprotein phosphatase inhibitor that may be involved in the regulation by the neuromodulator dopamine of the effects of the neurotransmitter glutamate. The identification and characterization of additional neuronal phosphoproteins can be expected to lead to the clarification of numerous additional molecular mechanisms by which signal transduction is carried out in nerve cells.
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PMID:Neuronal phosphoproteins. Mediators of signal transduction. 290 93

We have characterized protein phosphorylation in vitro in subcellular fractions from Drosophila melanogaster heads. Optimal conditions for the incorporation of 32P into proteins, and its dependence on ATP, divalent cations, and cyclic nucleotides have been determined, as well as the effect of inhibitors of ATPase, protein phosphatase, and protein kinase on protein phosphorylation. Among these inhibitors, Zn2+ was found to affect the incorporation of 32P into specific bands and p-hydroxymercuribenzoate was found to be most suited for freezing the activity of both kinases and phosphatases. Cyclic AMP-dependent protein kinase (cAMP-dPK) activity was present in both supernatant (S2) and particulate (P2) fractions, with the majority (60-85%, depending on the homogenization medium) being associated with S2, as determined by phosphorylation of exogenous synapsin I. cAMP-dPK catalyzed the phosphorylation of at least 18 endogenous polypeptides in S2 and at least 10 endogenous polypeptides in P2. These proteins could be classified on the basis of the extent of stimulation of phosphorylation by cyclic nucleotides, dependence on cyclic nucleotide concentration, and rate of phosphorylation. A phosphoprotein of 51 kilodaltons (pp51) was a major component of the S2 and P2 fractions and displayed properties expected from the regulatory subunit of the cAMP-dPK, R-II. A phosphoprotein doublet of approximately 37 kilodaltons (pp37) was stimulated to the largest extent by cAMP in the P2 and S2 fractions. The phosphorylation of several proteins in both fractions was significantly lowered by the mammalian Walsh inhibitor of cAMP-dPK, whereas in some cases the stimulation of phosphorylation of the same proteins by exogeneous cAMP was relatively small. Phosphoproteins from two learning mutants known to be deficient in cAMP metabolism, dnc and rut, were analyzed for their extent of phosphorylation in the presence of a stable cAMP analogue; no significant differences from normal were detected, suggesting that the genetic defect in cAMP metabolism is not accompanied by constituent abnormalities in phosphorylated substrates in the adult fly, and that the physiological defects in these mutants result from aberrations in the interaction of the cAMP cascade with normal substrates. The majority of Ca2+/calmodulin kinase activity (80-90%, depending on the homogenization procedure) was associated with S2, as revealed by phosphorylation of exogenous synapsin I. Two endogenous substrates for this kinase in P2 had molecular masses of approximately 45 and 87 kilodaltons. At least 11 substrates for the Ca2+/calmodulin-dependent kinase were detected in S2.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:In vitro protein phosphorylation in head preparations from normal and mutant Drosophila melanogaster. 304 Sep 7

Physical association of calcineurin with phosphatidylserine (PS) or phosphatidylglycerol (PG) was observed by molecular exclusion chromatography; the enzyme did not associate with phosphatidylethanolamine or phosphatidylcholine. The interactions with PS and PG were enhanced by Ca2+ which implicates a regulatory role for the Ca2+-binding subunit in this process. Addition of PG or PS to standard calcineurin assays elicited profound changes in enzymatic activity; phosphatidylcholine and phosphatidylethanolamine were without effect. Up to 23-fold stimulation of the calmodulin-independent activity was observed with phosphorylated histone H1 or synapsin I as the substrates. In contrast, the activity toward p-nitrophenyl phosphate and tyrosine phosphate was found to be inhibited. A characterization and comparison of the two opposite responses showed that: the phospholipids had insignificant effects on the Km for substrates, the phospholipid specificity for activation and inhibition was nearly indistinguishable, half-maximal activation and inhibition were obtained at similar concentrations of PG (K0.5 = 0.21 and 0.14 mg/ml, respectively), and calmodulin enhanced the responses to PG (K0.5 = 0.064 and 0.033 mg/ml for activation and inhibition, respectively) to similar extents. Together, these observations demonstrate that the two substrate-dependent responses of calcineurin are due to the association of the phosphatase with phospholipids and not a result of substrate-phospholipid interactions. This suggests that Ca2+- and calmodulin-stimulated interactions of calcineurin with acidic phospholipids may play a role in regulating the substrate specificity of this multifunctional phosphatase.
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PMID:Calcium- and calmodulin-sensitive interactions of calcineurin with phospholipids. 361 Oct 55

Calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase found in several tissues, is highly concentrated in mammalian brain. In an attempt to identify endogenous brain substrates for calcineurin, kinetic analyses of the dephosphorylation of several well-characterized phosphoproteins purified from brain were performed. The proteins studied were: G-substrate, a substrate for cyclic GMP-dependent protein kinase; DARPP-32, a substrate for cyclic AMP-dependent protein kinase; Protein K.-F., a substrate for a cyclic nucleotide- and Ca2+-independent protein kinase; and synapsin I, a substrate for cyclic AMP-dependent (site I) and a Ca2+/calmodulin-dependent protein kinase (site II). Calcineurin dephosphorylated each of these proteins in a Ca2+/calmodulin-dependent manner. Similar Km values were obtained for each substrate: G-substrate, 3.8 microM; DARPP-32, 1.6 microM; Protein K.-F., approximately 3 microM (S0.5); synapsin I (site I), 7.0 microM; synapsin I (site II), 4.4 microM. However, significant differences were obtained for the maximal rates of dephosphorylation. The kcat values were: G-substrate, 0.41 s-1; DARPP-32, 0.20 s-1; Protein K.-F., 0.7 s-1; synapsin I (site I), 0.053 s-1; synapsin I (site II), 0.040 s-1. Comparisons of the catalytic efficiency (kcat/Km) for each substrate indicated that DARPP-32, G-substrate, and Protein K.-F. are all potential substrates for calcineurin in vivo.
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PMID:Mammalian brain phosphoproteins as substrates for calcineurin. 633 98

The sequence of molecular events linking depolarisation-dependent calcium influx to calcium-stimulated protein phosphorylation is unknown. In this study the effect of the neuroleptic drug fluphenazine on depolarisation-dependent protein phosphorylation was investigated using an intact postmitochondrial pellet isolated from rat cerebral cortex. Fluphenazine, in a dose-dependent manner, completely inhibited the increases in protein phosphorylation observed previously. The concentration of fluphenazine required for 50% inhibition varied for different phosphoproteins but for synapsin I was 123 microM. Other neuroleptics produced effects similar to fluphenazine with their order of potency being thioridazine greater than haloperidol greater than trifluoperazine greater than fluphenazine greater than chlorpromazine. Fluphenazine also increased the phosphorylation of proteins in nondepolarised controls at concentrations of 20 and 60 microM. The inhibition of depolarisation-dependent phosphorylation was apparently not due to a loss of synaptosomal integrity or viability, a decrease in calcium uptake, a change in substrate availability, or to a change in protein phosphatase activity. The data are most consistent with an inhibition of protein kinase activity by blockade of calmodulin or phospholipid activation.
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PMID:Depolarisation-dependent protein phosphorylation in rat cortical synaptosomes is inhibited by fluphenazine at a step after calcium entry. 674 28


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