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)

Alterations in content of glycogen as well as in activity of phosphorylase and phosphoprotein phosphatase were studied in various rat tissues after intravenous administration of a new hypothalamic hexapeptide Tyr-Gly-Leu-Pro-Gly-NH2. An increase in phosphoprotein phosphatase activity was observed in heart muscle, which correlated with transformation of phosphorylase A to the B form and with accumulation of glycogen. The opposite effect was found in liver tissue and sceletal muscle.
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PMID:[Molecular mechanisms of hypothalamic hexapeptide regulation of the links in glycogen metabolism]. 22 71

Functional expression of recombinant wild-type phosphatase 2A catalytic subunit has been unsuccessful in the past. A nine-amino-acid peptide sequence (YP-YDVPDYA) derived from the influenza hemagglutinin protein was used to modify the NH2 and/or COOH terminus of the phosphatase 2A catalytic subunit. Addition of the nine-amino-acid sequence at the NH2 terminus allowed recombinant phosphatase 2A expression as a predominantly cytosolic phosphatase 2A enzyme. The 12CA5 monoclonal antibody that recognizes the nine-amino-acid hemagglutinin peptide sequence was used to immunoprecipitate the epitope-tagged phosphatase 2A catalytic subunit. Assay of the immunoprecipitated epitope-tagged phosphatase 2A demonstrated an okadaic acid-sensitive dephosphorylation of [32P] histone H1 and [32P]myelin basic protein similar to that measured with the wild-type enzyme. Functional phosphatase activity could be demonstrated for the NH2-terminal modified phosphatase 2A catalytic subunit following transient expression in COS cells or stable expression in Rat1a cells. In contrast, the COOH-terminal-modified phosphatase 2A catalytic subunit was very poorly expressed. The NH2-, COOH-modified subunit, having the nine-amino-acid hemagglutinin peptide sequence encoded at both termini of the polypeptide, was also expressed as a functional phosphatase 2A enzyme. Thus, NH2-terminal modification of the phosphatase 2A catalytic subunit results in a functional plasmid-expressed enzyme. The unique nine-amino-acid epitope-tag sequence also provides a method to easily resolve the recombinant phosphatase 2A from the endogenous wild-type gene product and related phosphatases expressed in cells.
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PMID:NH2-terminal modification of the phosphatase 2A catalytic subunit allows functional expression in mammalian cells. 138 Sep 55

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

DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP.
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PMID:Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase II. 255 37

The physico-chemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were investigated. The enzyme was shown to possess a wide substrate specificity and to catalyze dephosphorylation of phosphocasein, ATP, ADP and p-nitrophenylphosphate (pNPP). The Km values for ATP, ADP and pNPP are 0.44, 0.43 and 1.25 mM, respectively. The molecular weight of the enzyme as determined by gel filtration on Sephadex G-75 and electrophoresis in polyacrylamide gel of different concentrations is approximately 33 000. SDS-polyacrylamide gel electrophoresis revealed two protein bands with Mr 12 000 and 18 000. The enzyme molecule predominantly contains acidic amino acid residues, two free SH-groups and two disulphide bonds. Phosphoprotein phosphatase is a glycoprotein with the carbohydrate content of about 22%, and has an additional absorption maximum at 560 nm. The enzyme is competitively inhibited by ammonium molybdate (Ki = 0.37 microM) and non-competitively by sodium fluoride (Ki = 1.3 mM). Incubation of phosphoprotein phosphatase with 2 mM phenylmethylsulfonylfluoride (PMSF) for 25 hours resulted in a approximately 46% loss of the enzyme activity. Ammonium molybdate, sodium fluoride and PMSF reversibly inhibit the enzyme. Modification of aminoacid SH-groups, NH2-groups and histidine led to a decrease of the enzyme activity. Incubation of phosphoprotein phosphatase with [gamma-33P]ATP resulted in the incorporation of 0.33 mol of 33P per mol of the enzyme. The mechanism of the enzyme-catalyzed hydrolysis of the phosphoester bond is discussed.
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PMID:[Phosphoprotein phosphatases from cell nuclei of the bovine spleen: physico-chemical properties]. 299 59

The complete amino acid sequence of bovine brain DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein, which is a potent and specific inhibitor of the catalytic subunit of protein phosphatase-1, has been determined. The S-14C-carboxymethylated protein was subjected to enzymatic cleavage by endoproteinase Lys-C, endoproteinase Arg-C, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease, and to chemical cleavage by cyanogen bromide. The overlapping sets of peptides were purified by high performance liquid chromatography and subjected to amino acid sequencing by automated Edman degradation to deduce the complete sequence. The protein consists of a single NH2-terminal blocked polypeptide chain of 202 residues, with a calculated molecular mass of 22,591 daltons, excluding the unidentified NH2-terminal blocking group. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or hydrodynamic measurements. The threonine residue that is phosphorylated by cyclic AMP-dependent protein kinase (Hemmings, H. C., Jr., Williams, K. R., Konigsberg, W. H., and Greengard, P. (1984) J. Biol. Chem. 259, 14486-14490), and that must be phosphorylated for the expression of inhibitory activity, is located at position 34. The molecule contains only 1 cysteine residue and 1 tryptophan residue, at positions 72 and 161, respectively. DARPP-32 is very hydrophilic, and contains a stretch of 16 consecutive acidic residues from position 119 to 134. The predicted secondary structure suggests the presence of 47% alpha-helix, 7% beta-sheet, and 46% random coil, with 11 beta-turns. Comparison of the complete amino acid sequence of bovine DARPP-32 with that of rabbit skeletal muscle protein phosphatase inhibitor-1 revealed a significant amount of sequence identity in the NH2-terminal regions of these two proteins. The active region of inhibitor-1 has been localized to an NH2-terminal fragment (Aitken, A., and Cohen, P. (1982) FEBS Lett. 147, 54-58), the part of the molecule that is most similar to DARPP-32. These data suggest that these two protein phosphatase inhibitors may share a common structural basis for their inhibitory activity and may be related by a common ancestral gene.
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PMID:DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein. Primary structure and homology with protein phosphatase inhibitor-1. 351 Oct 54

The Ca2+-dependent regulator protein (CDR), also frequently termed "calmodulin" was determined to influence the dephosphorylation of mixed calf thymus histones or purified histones 1, 2A, or 2B by a partially purified bovine brain phosphoprotein phosphatase. CDR increase the rate of dephosphorylation of mixed histones more than 20-fold. With increasing concentrations of mixed histones as substrate, a proportionate increase of CDR concentration was required to maintain maximal expression of histone phosphatase activity. Mixed histones suppressed the activation by CDR of a bovine brain cyclic nucleotide phosphodiesterase activity, with activation being restored by increased quantities of CDR. Dephosphorylation of casein and phosphorylase alpha by the phosphatase preparation was not affected by CDR. These observations support the interpretation that the effects of CDR on histone dephosphorylation are substrate-directed. The rates of dephosphorylation of histones 1, 2A, and 2B by the phosphatase were 4- to 12-fold more rapid at low (sub-micromolar) concentrations of free Ca2+ than at high (200 microM) Ca2+ in incubations containing CDR, but they were unaffected by Ca2+ in incubations without CDR. The addition of stoichiometric quantities of calmodulin increased the apparent Km of the phosphatase for the various histones 2- to 6-fold, while maximal velocities were 4- to 12-fold higher at low than at high added Ca2+. The inhibitory effect of Ca2+ on histone dephosphorylation was immediately reversible by chelation of Ca2+ with EDTA. Ca2+-dependent inhibition of histone 1 or 2B phosphatase activities was also produced by rabbit skeletal muscle troponin C, but not by rabbit skeletal muscle parvalbumin, by poly(L-aspartate) or poly(L-glutamate). The phosphorylated fragment from the NH2-terminal region of either H2A (generated by treatment with N-bromosuccinimide) or H2B (generated by treatment with cyanogen bromide) was dephosphorylated by the phosphatase, with the rates of dephosphorylation being reduced 3- to 6-fold by Ca2+ in incubations containing CDR.
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PMID:Interaction of calmodulin with histones. Alteration of histone dephosphorylation. 625 89

The synthetic phosphohexapeptides Arg-Arg-Ala-Thr(35P)-Val-Ala and Arg-Arg-Ala-Ser(32P)-Val-Ala, phosphorylated by the cAMP-dependent protein kinase and differing only in the nature of the phosphorylated residue, have been used as substrates of a partially purified rat liver protein phosphatase-T, distinct from the multifunctional protein phosphatase-1. While the phosphothreonyl hexapeptide is readily dephosphorylated (exhibiting a Km = 15 microM), the phosphoseryl one is almost unaffected. Such a behavior is not shared by protein phosphatase-1, calf intestine alkaline phosphatase, and potato acid phosphatase, all of which are more active on the phosphoseryl hexapeptide. The NH2-terminal basic residues critical for cAMP-dependent phosphorylation are not required in the dephosphorylation reaction, as both Arg can be removed without impairing the efficiency of protein phosphatase-T toward the phosphothreonyl peptide. On the other hand, the replacement of 2 Pro for the Ala and Val flanking Thr(32P), to give a new phosphohexapeptide reproducing the phosphorylated site of protein phosphatase inhibitor-1, prevents the protein phosphatase-T activity. Moreover, IgG heavy chain 32P labeled in tyrosine is not affected by protein phosphatase-T, while it is dephosphorylated by alkaline phosphatase. These results would indicate that protein phosphatase(s)-T represent a distinct class of protein phosphatases specifically involved in the dephosphorylation of phosphothreonyl residues fulfilling definite structural requirements.
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PMID:Dephosphorylation of synthetic phosphopeptides by protein phosphatase-T, a phosphothreonyl protein phosphatase. 628 35

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.
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PMID:Agonist and antagonist properties of calmodulin fragments. 632 72

The NH2-terminal blocking group of the Ca2+-binding B-subunit of calcineurin (protein phosphatase-2B) has been identified as myristic acid by fast atom bombardment mass spectrometry and gas chromatography. The sequence, myristyl-Gly-Asn-Glu-Ala-, is very similar to that of the catalytic subunit of cyclic AMP-dependent protein kinase, the only other protein known to contain this fatty acid. This finding, and the elution of all myristyl peptides at 57% acetonitrile on reverse phase HPLC, may facilitate the identification of other proteins with this blocking group.
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PMID:Identification of the NH2-terminal blocking group of calcineurin B as myristic acid. 716 Apr 76


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