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 catalytic subunits of protein phosphatase-1 and 2A were covalently modified in their reactive sulfhydryl groups with N-(3-Pyrene) maleimide resulting in fluorescent labeling of the proteins to an extent of 0.85 and 0.9 mole dye/mole enzyme, respectively. The reaction of the sulfhydryl group led to the partial inactivation of both phosphatase-1 and 2A. Inhibitor-1 and inhibitor-2 increased markedly the fluorescence intensity of the dye-phosphatase-1 conjugate implying that the labeled enzyme retained its ability to bind these proteins. In contrast, inhibitor-1 or inhibitor-2 had no influence on the fluorescence of the dye-phosphatase-2A conjugate. No change in either the fluorescence intensity or polarization of labeled phosphatase-1 and 2A was observed in the presence of thiophosphorylase a, suggesting a lack of interaction of these enzyme forms with the substrate after modification of the reactive sulfhydryl group.
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PMID:Interaction of the catalytic subunits of protein phosphatase-1 and 2A with inhibitor-1 and 2: a fluorescent study with sulfhydryl-specific pyrene maleimide. 216 68

The catalytic subunit of protein phosphatase 1 (PP1), a key enzyme in the regulation of many cellular functions, has been expressed in insect cells using a baculovirus vector containing PP1 alpha cDNA. The expressed protein had the same apparent molecular mass as PP1 from rabbit skeletal muscle and comprised up to 25% of the total cellular protein. About 5% of expressed PP1 alpha was present as a soluble active species, representing a 15-fold increase over the endogenous activity. Insoluble protein, comprising about 95% of the expressed PP1 was dissolved in 6 M guanidinium chloride and could be fully reactivated by extensive and rapid dilution with buffers containing Mn2+. By a number of criteria (specific activity towards phosphorylase, interaction with inhibitor-1, inhibitor-2 and okadaic acid), this reactivated species was indistinguishable from authentic PP1, and could be concentrated and purified to homogeneity by a single chromatography on DEAE-Sepharose. This procedure yielded about 10 mg active PP1/1 culture, which will facilitate future structural analyses of native and mutant protein phosphatases.
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PMID:Renaturation of protein phosphatase 1 expressed at high levels in insect cells using a baculovirus vector. 216 39

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

Protein phosphatase inhibitor-1 was purified from bovine adipose tissue. The protein had an apparent molecular mass of 32 kDa by SDS/PAGE and a Stokes' radius of 3.4 nm. It was phosphorylated by cAMP-dependent protein kinase on a threonyl residue; this phosphorylation was necessary for inhibition of protein phosphatase-1. Bovine adipose tissue inhibitor-1 was compared directly with rabbit skeletal muscle inhibitor-1 and with a 32000-Mr, dopamine- and cAMP-regulated phosphoprotein from bovine brain (DARPP-32), also an inhibitor of protein phosphatase-1. By the following biochemical and immunochemical criteria, bovine adipose tissue inhibitor-1 was found to be very similar and possibly identical to DARPP-32 and was clearly distinct from skeletal muscle inhibitor-1: molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine residues; Staphylococcus-aureus-V8-protease-generated peptide patterns analyzed by SDS/PAGE; tryptic phosphopeptide maps analysed by two-dimensional thin-layer electrophoresis/chromatography; elution on reverse-phase HPLC; chymotryptic peptide maps as analysed by reverse-phase HPLC; amino acid composition; antibody recognition by immunoprecipitation and immunoblotting; effect of cyanogen bromide cleavage on protein phosphatase inhibitor activity. Based on these results we conclude that bovine brain and adipose tissue contain an identical phosphoprotein inhibitor of protein phosphatase-1 (DARPP-32), which is distinct from that of skeletal muscle (inhibitor-1).
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PMID:Inhibitors of protein phosphatase-1. Inhibitor-1 of bovine adipose tissue and a dopamine- and cAMP-regulated phosphoprotein of bovine brain are identical. 254

Previous studies demonstrated that activation of T lymphocytes by phorbol ester or mitogenic lectin leads to phosphorylation of Ser 126 of the CD3 antigen gamma chain, whereas treatment with ionomycin results in phosphorylation of both Ser 123 and 126 [Davies, A. A. et al. (1987) J. Biol. Chem. 262, 10918-10921]. In the present study, the dephosphorylation of Ser 123 and Ser 126 of the gamma chain was investigated. Phorbol-ester-induced phosphorylation of the gamma-chain Ser 126 in vivo was reversed following removal of phorbol ester. Dephosphorylation of both Ser 123 and 126 was also observed in vitro using the microsome fraction of T lymphocytes. In order to identify the phosphatases acting at these two sites, the immunoprecipitated gamma chain was used as substrate either following treatment with protein kinase C in vitro, in which case phosphorylation occurs mainly at Ser 123, or following in vivo phosphorylation of Ser 126. Purified oligomeric forms of the polycation-stimulated phosphatases were more effective in dephosphorylating both phosphorylated forms of the gamma chain compared with equivalent amounts of ATP,Mg2+-dependent phosphatases or calcineurin. By using phosphopeptide analogues of the CD3 gamma chain containing Ser 123 or Ser 126 as substrates (A3 and A6), it was shown that polycation-stimulated phosphatases selectively dephosphorylated Ser 123 compared to Ser 126. In order to determine which phosphatases dephosphorylate the gamma chain in microsomes, A3 and A6 were used as substrates for characterising phosphatases in microsomes from human T leukaemia Jurkat 6 cells. Three phosphopeptide phosphatases (250-400 kDa) co-eluted through five purification steps with three forms of polycation-stimulated phosphorylase phosphatase. The partially purified A3/A6 phosphopeptide phosphatases were insensitive to Ca2+, calmodulin and inhibitor-1, and dephosphorylated A3 preferentially compared with A6. A latent form of microsomal ATP,Mg2+-dependent phosphorylase phosphatase was stimulated 10-fold by trypsinisation, but did not dephosphorylate phosphopeptides A3 and A6. The results show that high-Mr forms of polycation-stimulated phosphatases are the only enzymes in human T leukaemia cell microsomes which dephosphorylate gamma chain phosphopeptides. The data point to an important role for polycation-stimulated phosphatases in regulating the phosphorylation state, and so function(s), of the CD3 antigen.
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PMID:Dephosphorylation of the human T lymphocyte CD3 antigen. 254 Sep 70

Inhibitor-1 is a potent and specific inhibitor of protein phosphatase 1. Phosphorylation by cAMP-dependent protein kinase is required for expression of its inhibitor activity. In the present study, we have used immobilized inhibitor-1 preparations to study the mechanism underlying protein phosphatase 1 inhibition. Protein phosphatase 1 bound to phosphorylated inhibitor-1 covalently coupled to Sepharose or Affi-Gel beads but did not bind to immobilized preparations of dephosphorylated inhibitor-1 or bovine serum albumin. Phosphorylated inhibitor-1 coupled to Sepharose or Affi-Gel beads retained its ability to inhibit protein phosphatase 1, although the apparent IC50 was decreased about 500-fold. The extent of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was comparable to the degree of protein phosphatase inhibition when the inhibitor protein was present at a concentration near the IC50. The efficiency of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was dependent on the inhibitor concentration on the matrix. Taken together these data indicate that the inhibition of protein phosphatase 1 by phosphorylated inhibitor-1 is a consequence of the binding of the inhibitor protein to one or more sites on protein phosphatase 1.
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PMID:Immobilized inhibitor-1 binds and inhibits protein phosphatase 1. 254 51

Four major serine/threonine-specific protein phosphatase catalytic subunits are present in the cytoplasm of animal cells. Three of these enzymes, PP-1, PP-2A, and PP-2B, are members of the same gene family, while PP-2C appears to be distinct. PP-1, PP-2A, and PP-2B are complexed to other subunits in vivo, whereas PP-2C has only been isolated as a monomeric protein. PP-1, PP-2A, and PP-2C have broad and overlapping specificities in vitro, and account for virtually all measurable activity in tissue extracts toward a variety of phosphoproteins that regulate metabolism, muscle contractility, and other processes. Their precise functions in vivo are unknown, although important clues to the physiological roles of PP-1 and PP-2A are provided by the effects of okadaic acid and by the subcellular localization of PP-1. The active forms of PP-1 are largely particulate, and their association with subcellular structures is mediated by "targetting subunits" that direct PP-1 to particular locations, enhance its activity toward certain substrates, and confer important regulatory properties upon it. This concept is best established for the glycogen-bound enzymes in skeletal muscle and liver (PP-1G) and the myofibrillar form (PP-1M) in skeletal muscle. The activities of PP-1 and PP-2B are controlled by the second messengers cyclic AMP and calcium. The activity of PP-2B is dependent on calcium and calmodulin, while PP-1 is controlled in a variety of ways that depend on the form of the enzyme and the tissue. PP-1 can be inhibited by cyclic AMP in a variety of cells through the A-kinase-catalyzed phosphorylation of inhibitor-1 and its isoforms. Phosphorylation of the glycogen-binding subunit of PP-1G by A-kinase promotes translocation of the catalytic subunit from glycogen particles to cytosol in skeletal muscle, inhibiting the dephosphorylation of glycogen-metabolizing enzymes. Allosteric inhibition of hepatic PP-1G by phosphorylase a occurs in response to signals that elevate cyclic AMP or calcium, and prevents the activation of glycogen synthase in liver. PP-1 can also be activated indirectly by calcium through the ability of PP-2B to dephosphorylate inhibitor-1. This control mechanism may operate in dopaminoceptive neurones of the brain and other cells. The inactive cytosolic form of PP-1 (PP-1I) can be activated in vitro through the glycogen synthase kinase-3-catalyzed phosphorylation of its inhibitory subunit (inhibitor-2), but the physiological significance is unclear.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The structure and regulation of protein phosphatases. 254 56

The activation of the ATP,Mg-dependent protein phosphatase [Fc.M] has been shown to involve a transient phosphorylation of the modulator subunit (M) and consequent isomerization of the catalytic subunit (Fc) into its active conformation (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S. -D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870). The modulator subunit constitutes the inactivating force for the enzyme, but the slow intramolecular inactivation of the phosphatase can be prevented or blocked by the addition of either the phosphorylated inhibitor-1 or Mg2+ ions. Autodephosphorylation of the modulator subunit is not prevented by the phosphoinhibitor-1, suggesting that the ATP,Mg-dependent phosphatase binds the phosphomodulator subunit in a very specific manner, different from the way it binds exogenous phosphoprotein substrates. Alternatively, the autodephosphorylation of the modulator subunit is catalyzed at a separate active site on the enzyme, which is not influenced by the binding of phosphoinhibitor-1. The phosphoinhibitor-1 does not prevent the activation of the enzyme by kinase FA when added at concentrations that totally inhibit the potential phosphorylase phosphatase activity. These results, together with other already published information, suggest separate autonomic controls of the ATP,Mg-dependent phosphatase activity by inhibitor-1 and the modulator protein through the presence of specific regulatory subunits on the enzyme.
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PMID:The ATP,Mg-dependent protein phosphatase. Regulation by inhibitor-1 or modulator protein and stabilizing role of Mg2+ ions. 282 Sep 94

Four classes of protein phosphatases are presumed to play an important role in dephosphorylating the major proteins involved in the control of general metabolism. Based on the enzyme-directed regulation of activity they have been classified as ATP,Mg-dependent-, polycation-stimulated-, Mg2+-dependent protein phosphatases and calcineurin. We have recently purified from rabbit skeletal muscle four distinct PCS protein phosphatases, classified according to the apparent molecular weight of the native enzymes in gel filtration at an early stage of the purification as: PCSH (390 kDa), PCSM (250 kDa) and PCSL (200 kDa) phosphatases. The PCSH phosphatase could be resolved into a 3(65:55 35 kDa)-subunit PCSH1 phosphatase and a 2(65:35 kDa)-subunit PCSH2 enzyme probably derived from the PCSH1 phosphatase, both characterized as specific deinhibitor phosphatases. PCSM phosphatase, a 3(72:65 35 kDa)-subunit enzyme, shows a high degree of stimulation with a low concentration optimum of polycations and is sensitive to a Ca2+-dependent protease, which brings about a five- to ten-fold increase in inhibitor-1 phosphatase activity. PCSL phosphatase is characterized by a 2(65:35 kDa)-subunit structure, a low intrinsic deinhibitor phosphatase activity and a low degree of stimulation of phosphorylase phosphatase activity requiring high concentrations of polycations. At low concentrations of polycations the stimulation of phosphorylase phosphatase activity of the PCS enzymes is enzyme-directed, since it occurs at concentrations far below the substrate concentration. The degree of stimulation is also typical for each type of enzyme (PCSM greater than PCSH1 greater than PCSH2 greater than PCSL greater than PCSC) and dependent on the polycation used; at the optimum concentration the most effective polycations (polylysine, protamine, histone H1) stimulate the phosphorylase phosphatase activity to about the same extent. Polycation concentrations above the optimum are less effective on phosphorylase phosphatase activity and can even become inhibitory to the basal activity. Whether this effect is enzyme- or substrate-directed (or both) is not known. The stimulation by polycations could be completely lost following preincubation of the PCS phosphatase with polycations. This deactivation is time-, temperature- and concentration-dependent. However the polycations did not affect the basal phosphorylase phosphatase activity. In addition to phosphorylase a and inhibitor-1, casein, myosin light chains and phosphorylase b kinase (alpha-subunit) are choice substrates for these enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The polycation-stimulated protein phosphatases: regulation and specificity. 282 47

Inhibitor-1 following phosphorylation by protein kinase A inhibits phosphoprotein phosphatase-1. We have found that in the rat heart inhibitor-1 is present only in the cytosolic fraction and that its phosphorylation in ventricular slices was increased by isoproterenol but not by isoproterenol and propranolol together. Cardiac microsomal phosphoprotein phosphatase activity, with added phosphorylase a as the substrate, was inhibited 33% by phosphorylated inhibitor-1. Phosphorylated inhibitor-1 decreased the dephosphorylation by exogenous phosphoprotein phosphatase-1 of phospholamban present in the sarcoplasmic reticulum membranes. These results suggest an interaction of cytoplasmic inhibitor-1 with either cytoplasmic or membrane-bound phosphoprotein phosphatase-1 with a subsequent effect on the level of phosphorylated phospholamban and the probable involvement of this interaction in the cardiac response to beta-adrenergic hormones.
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PMID:A regulation of the level of phosphorylated phospholamban by inhibitor-1 in rat heart preparations in vitro. 283 40


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