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)

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.
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PMID:In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase. 200 28

Activity of crude histidine decarboxylases (HisDC) from the hypothalamus and the lungs, was markedly reduced by incubating with ATP.Mg, cAMP and cAMP-dependent protein kinase A, whereas activity of the crude glandular stomach enzyme changed only slightly under equal condition. The omission of one of these components failed to reduce HisDC activity by as much as the complete system. Addition of bovine heart (type II) or rat cerebellum protein kinase A (types I and II) inhibitor to the assay prevented enzyme inactivation; moreover, protein kinase A inhibitors permitted moderate activation under phosphorylating and control conditions. Cytosolic hypothalamus HisDC activity was elevated 2-2.2-fold by incubating the cytosol for 15 min in the presence of MnCl2, a known stimulator of phosphoprotein phosphatase; this was prevented when 20 mM NaF, a common inhibitor of phosphoprotein phosphatase, was added to the cytosol. The apparent Km of ATP.Mg-treated hypothalamus HisDC for histidine was elevated 5-10-fold compared to controls, whereas the Vmax was approximately the same. Under this condition, the Km was calculated as high as 0.5-2.2 mM (depending on phosphorylating conditions), while controls had a Km of 0.1-0.3 mM (depending on the initial phosphorylating states). Addition of rabbit muscle (type I), bovine heart (type II) or rat cerebellum (types I and II) inhibitor of protein kinase A, to the phosphorylating mixture, abolished the difference in Km between control and ATP.Mg-treated HisDC. Moreover, rat cerebellum protein kinase A inhibitors increased Vmax to above the control level; while 20 mM NaF (inhibitor of phosphoprotein phosphatase) decreased Vmax to approximately one half of that of the controls. These data indicate that HisDC activity in the hypothalamus and the lungs, but not in the stomach, is affected in oppositely by protein kinase A and phosphoprotein phosphatases.
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PMID:Possible regulation of hypothalamus and lung histidine decarboxylase activity by cAMP-dependent protein kinase. 201 19

We have investigated the role of protein phosphorylation in the control of exocytosis in sea urchin eggs by treating eggs with a thio-analogue of ATP. ATP gamma S (adenosine 5'-O-3-thiotriphosphate) is a compound which can be used as a phosphoryl donor by protein kinases, leading to irreversible protein thiophosphorylation (Gratecos, D., and E.H. Fischer. 1974. Biochem. Biophys. Res. Commun. 58:960-967). Microinjection of ATP gamma S inhibits cortical granule exocytosis, but has no effect on the sperm-egg signal transduction mechanisms which normally cause exocytosis by generating an increase in [Ca2+]i. ATP gamma S requires cytosolic factors for its inhibition of cortical granule exocytosis: it does not affect exocytosis when applied directly to the isolated exocytotic apparatus. Our data suggest that ATP gamma S irreversibly inhibits exocytosis via thiophosphorylation of proteins associated with the egg cortex. We have identified two thiophosphorylated proteins (33 and 27 kD) that are associated with the isolated exocytotic apparatus. They may mediate the inhibition of exocytosis by ATP gamma S. In addition, we show that okadaic acid, an inhibitor of phosphoprotein phosphatases, prevents cortical granule exocytosis at fertilization without affecting calcium mobilization. Like ATP gamma S, okadaic acid has no effect on exocytosis in vitro. Our results suggest that an inhibitory phosphoprotein can obstruct calcium-stimulated exocytosis in sea urchin eggs; on the other hand, they do not readily support the idea that a protein phosphatase is an essential component of the mechanism controlling exocytosis.
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PMID:Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs. 202 49

The Na/K/2Cl cotransport system in the avian erythrocyte can be activated by agents that raise intracellular cAMP suggesting the involvement of cAMP-dependent protein kinase (cAMP-PK) in its regulation. Another group of stimuli including fluoride and hypertonicity stimulate cotransport via cAMP-independent means. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase inhibitors of 8 (p-Cl-phenylthio)-cAMP (cpt-cAMP), fluoride and hypertonic activation of cotransport in duck red cells, and [3H]bumetanide binding to isolated membranes. Preincubation of cells with the kinase inhibitors K-252a (Ki approximately 1.6 microM) and H-9 (Ki approximately 100 microM) blocked cpt-cAMP activation of bumetanide-sensitive 86Rb influx and bumetanide binding. These inhibitors also led to a rapid deactivation of cotransport and decrease in bumetanide binding when added to cells maximally stimulated by cpt-cAMP. K-252a and H-9 inhibited cotransport activation by cAMP-independent stimuli, but 10-fold higher concentrations were required, implying the involvement of a cAMP-independent phosphorylation process in the mechanism of action of these agents. Removal of stimuli that elevate cAMP leads to a rapid reversal of cotransport indicating the presence of active protein phosphatases in these cells. The protein phosphatase inhibitor okadaic acid (OA, EC50: 630 nM) stimulated both Na/K/2Cl cotransport and bumetanide binding to membranes. As with fluoride and hypertonic stimulation, the OA effect was inhibited only at relatively high concentrations of K-252a. Phosphorylation of the membrane skeletal protein goblin (Mr 230,000) at specific cAMP-dependent sites was used as an in situ marker for the state of activation of cAMP-PK. Goblin phosphorylation at these sites was increased by norepinephrine and cpt-cAMP and rapidly reversed by K-252a and H-9, confirming that both inhibitors do block cAMP-PK activity. While OA markedly increased overall phosphorylation of many erythrocyte membrane proteins, including goblin, it did not affect goblin phosphorylation at specific cAMP-dependent sites. These results implicate a cAMP-independent protein kinase in the mediation of the OA effect on cotransport and bumetanide binding. The bumetanide-binding component of the avian erythrocyte cotransporter, an Mr approximately 150,000 protein that can be photolabeled with the bumetanide analog [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)-benzoic acid was found to be a phosphoprotein. These results strongly support the hypothesis that phosphorylation and dephosphorylation, possibly of the Na/K/2Cl cotransporter itself, regulates the activity of
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PMID:The regulation of Na/K/2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephosphorylation. Effects of kinase inhibitors and okadaic acid. 214 26

The cloning and characterization of cDNAs for the catalytic subunit of calcineurin (CN) from murine and human brain libraries were carried out using nonisotopic methods. A murine cDNA clone encoding a protein of 521 amino acids (Mr approximately 58,650) was isolated; overlapping clones established a 3'-untranslated region of 554 base pairs preceding the poly(A) tail. Homologous cDNAs from human brain showed greater than 92% nucleotide sequence identity in both coding and non-coding regions with greater than 99% conservation of amino acid sequence. A second class of cDNAs lacking a specific 30-base pair region following the calmodulin-binding domain was found in four murine and human libraries. Oligonucleotide probes for both cDNA isoforms hybridized to mRNA from several brain regions indicating the existence of transcripts in vivo. The nucleotide sequences of the two forms were identical except for the inserted sequence, and Southern blot analysis of mouse and rat DNA was consistent with their having originated from the same gene; these data suggest that alternative splicing may give rise to molecular isoforms of the catalytic subunit in brain. Northern blots showed a predominant mRNA for CN in most tissues of approximately 4.0 kilobases (kb) with lower amounts of a 3.6-kb species. Brain showed 10 times more of these mRNAs than skeletal muscle while other tissues had less than or equal to 5% that in brain. In testis, multiple mRNAs were observed, with the major forms being approximately 2.8 and 1.6 kb; the total amount of CN message was about 15% that in brain. The presence of mRNA isoforms of the catalytic subunit may provide for isoenzymes of this phosphatase having distinct phosphoprotein substrate specificities or regulatory properties. The structural relatedness of CN to other mammalian serine/threonine protein phosphatases was highest over a region of approximately 240 amino acids near the amino terminus of this subunit, with greater similarity to protein phosphatase 2A than protein phosphatase 1. The conservation of many regions found in lambda phage phosphatase (Cohen, P.T.W., and Cohen, P. (1989) Biochem. J. 260, 931-934) indicates a common origin for the catalytic domain of this enzyme.
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PMID:Cloning and characterization of molecular isoforms of the catalytic subunit of calcineurin using nonisotopic methods. 216 44

Cultures of cerebellar granule neurons have been utilized to examine morphological and biochemical consequences of methyl mercury (MeHg). Exposure to MeHg for 24 h was found to exert toxic effects at concentrations below 1 microM characterized by neuron degeneration and neuritic varicosities. Dose-response and time course profiles for cell death were established using the 51Cr release assay, which revealed that 1 microM MeHg produced 15% cell death at 24 h, progressing to 50% at 48 h. Labeling of cultures with [32P]orthophosphate following 24-h exposure to 1 microM MeHg disclosed abnormalities in both protein and lipid phosphorylation. After 24-h exposure to 5 microM MeHg, phospholabeling of protein and lipid increased 174 and 128%, respectively, compared with controls. This stimulation of phosphorylation appeared to be neuron specific since cultures enriched in cerebellar glial cells and devoid of granule neurons displayed dose-dependent inhibition of total phosphorylation. Measurement of 32P labeling of ATP using a cyclic AMP-dependent protein kinase assay in conjunction with the firefly luciferase assay for ATP indicated no significant change in either total ATP levels or [32P]ATP specific activity at 1 or 4 h as a function of [MeHg]. Studies measuring 32P-phosphoprotein turnover indicated that MeHg had no effect on intracellular protein phosphatase activity. We conclude that one of the manifestations associated with in vitro cerebellar granule cell neurotoxicity is an abnormality in protein phosphorylation that is independent of [32P]ATP specific activity and protein phosphatase activity.
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PMID:Methyl mercury stimulates protein 32P phospholabeling in cerebellar granule cell culture. 216 77

Synthetic peptides based on the threonine phosphorylation site and proposed inhibitory site of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were prepared and analyzed as substrates for cAMP-dependent protein kinase and protein phosphatases-1c, -2Ac (the catalytic subunits of protein phosphatase-1 and 2A, respectively) and -2B, and as inhibitors of protein phosphatase-1c. Studies of the kinetics of phosphorylation of the peptides by cAMP-dependent protein kinase indicated an important role in facilitating phosphorylation for the region COOH-terminal to the phosphorylatable threonyl residue. Studies of the dephosphorylation of the phosphopeptides demonstrated that they were effectively dephosphorylated by protein phosphatase-2A and -2B and poorly dephosphorylated by protein phosphatase-1. The active inhibitory region of phospho-DARPP-32 was analyzed by determining the effects of synthetic phosphopeptides on the activity of protein phosphatase-1c. Phospho-D32-(8-48) and phospho-D32-(8-38) inhibited protein phosphatase-1c with IC50 values of 2 x 10(-8) and 4 x 10(-8) M, respectively, compared with an IC50 of 8 x 10(-9) M for intact phospho-DARPP-32. Phospho-D32-(9-38) was equipotent with phospho-D32-(8-38); however, further NH2-terminal deletions resulted in marked reductions in IC50 values. An analog of an active DARPP-32 phosphopeptide containing a phosphoseryl residue in place of the phosphothreonyl residue also exhibited a much reduced IC50. These data identify the essential inhibitory region of phospho-DARPP-32 as residues 9-38, which contains the phosphorylation site (Thr34). This region exhibits extensive amino acid sequence identity with phosphatase inhibitor-1, a distinct inhibitor of protein phosphatase-1. Kinetic studies of the inhibition of protein phosphatase-1c by phospho-D32-(9-38), a potent inhibitor, as well as by phospho-D32-(10-38), a weak inhibitor, indicated a mixed competitive/noncompetitive mechanism of inhibition, as has been previously found for both intact phospho-DARPP-32 and intact phospho-inhibitor-1. These findings support the hypothesis that a 30-amino acid domain in the NH2-terminal region of phospho-DARPP-32 is sufficient for the inhibition of protein phosphatase-1.
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PMID:Synthetic peptide analogs of DARPP-32 (Mr 32,000 dopamine- and cAMP-regulated phosphoprotein), an inhibitor of protein phosphatase-1. Phosphorylation, dephosphorylation, and inhibitory activity. 217 4

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca2+-binding subunits of the Ca2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase (Mr 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.
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PMID:Immunological characterization of phosphoprotein phosphatases. 241 61

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

During ATP hydrolysis the K+-translocating Kdp-ATPase from Escherichia coli forms a phosphorylated intermediate as part of the catalytic cycle. The influence of effectors (K+, Na+, Mg2+, ATP, ADP) and inhibitors (vanadate, N-ethylmaleimide, bafilomycin A1) on the phosphointermediate level and on the ATPase activity was analyzed in purified wild-type enzyme (apparent Km = 10 microM) and a KdpA mutant ATPase exhibiting a lower affinity for K+ (Km = 6 mM). Based on these data we propose a minimum reaction scheme consisting of (i) a Mg2+-dependent protein kinase, (ii) a Mg2+-dependent and K+-stimulated phosphoprotein phosphatase, and (iii) a K+-independent basal phosphoprotein phosphatase. The findings of a K+-uncoupled basal activity, inhibition by high K+ concentrations, lower ATP saturation values for the phosphorylation than for the overall ATPase reaction, and presumed reversibility of the phosphoprotein formation by excess ADP indicated similarities in fundamental principles of the reaction cycle between the Kdp-ATPase and eukaryotic E1E2-ATPases. The phosphoprotein was tentatively characterized as an acylphosphate on the basis of its alkali-lability and its sensitivity to hydroxylamine. The KdpB polypeptide was identified as the phosphorylated subunit after electrophoretic separation at pH 2.4, 4 degrees C of cytoplasmic membranes or of purified ATPase labeled with [gamma-32P]ATP.
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PMID:Characterization of the phosphorylated intermediate of the K+-translocating Kdp-ATPase from Escherichia coli. 252 40


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