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)

Molecular cloning of glycogen synthase kinase-3 (GSK-3) has demonstrated the existence of a novel form, termed GSK-3 beta, which is highly related to the well characterised GSK-3 alpha protein but derived from a distinct gene. The cDNA cloning also revealed a striking degree of amino acid identity between the two GSK-3 proteins, particularly the beta-form, and the zeste-white3/shaggy (zw3sgg) homeotic gene of Drosophila melanogaster. Abrogation of zw3sgg causes pleiotropic effects on fruitfly development affecting segmental organisation and cell fate determination. In view of the potential importance of GSK-3 beta in mammalian development and the lack of previous characterisation, we have expressed this protein in insect cells using recombinant baculovirus. A rapid purification scheme has been developed yielding essentially pure GSK-3 beta protein in three chromatographic steps. The protein has autonomous protein kinase activity and similar, but not identical, substrate preferences to GSK-3 alpha. Both GSK-3 proteins activate the MgATP-dependent form of protein phosphatase-1 and thus display 'factor A' activity. Since GSK-3 beta exhibits an identical site specificity to GSK-3 alpha with respect to phosphorylation of the proto-oncogene/transcription factors c-jun and c-myc, it is likely that the Drosophila zw3sgg protein kinase has a similar specificity for such transcription factors which may underlie the pleiotropic phenotypes observed when the Drosophila homologue is mutationally inactivated.
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PMID:Baculovirus-mediated expression and characterisation of rat glycogen synthase kinase-3 beta, the mammalian homologue of the Drosophila melanogaster zeste-white 3sgg homeotic gene product. 134 4

The in vivo phosphorylation stoichiometries of 4 serines on the glycogen-binding (G)-subunit of protein phosphatase 1 (PP1) have been determined. In fed rabbits injected with propranolol stoichiometries (mol/mol) were: site 1 (0.67 +/- 0.09), site 2 (0.20 +/- 0.07), site 3a (0.23 +/- 0.01) and site 3b (0). After injection with adrenalin they became: site 1 (0.90 +/- 0.02), site 2 (0.72 +/- 0.01), site 3a (0.23 +/- 0.02) and site 3b (0). These results, together with other data, establish that site 2 phosphorylation by cyclic AMP-dependent protein kinase triggers dissociation of PP1 from the G-subunit in vivo. They also demonstrate that a residue phosphorylated in vitro by glycogen synthase kinase 3 (site 3a) is phosphorylated in vivo.
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PMID:Identification of three in vivo phosphorylation sites on the glycogen-binding subunit of protein phosphatase 1 from rabbit skeletal muscle, and their response to adrenaline. 215 82

Rat fat cells contain three species of spontaneously active inhibitor proteins of protein phosphatase 1, as resolved by SDS-PAGE, with apparent molecular masses of 40 kDa, and 28 kDa respectively. The 33-kDa, thermostable inhibitor was highly purified from bovine adipose tissue and shown to be very similar to inhibitor-2 of skeletal muscle. It was phosphorylated, on threonine only, by glycogen synthase kinase 3. It formed an inactivated complex with protein phosphatase 1, that was reactivated by incubation with ATP-Mg and glycogen synthase kinase 3. By gel filtration it had a Stokes radius of 3.4 nm. Peptide and phosphopeptide maps, generated by Staphylococcus aureus V8 proteinase, trypsin or thermolysin, of the inhibitor and of the skeletal muscle inhibitor-2 were similar. The 40-kDa inhibitor, which was denatured by boiling, represents a novel protein phosphatase inhibitor protein or an undegraded precursor of inhibitor-2. The total activity of inhibitor-2-like material (thermostable and macromolecular) in an adipocyte cytosol extract corresponded to an intracellular concentration of 0.3 microM inhibitor-2.
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PMID:Adipose tissue protein phosphatase inhibitor-2. 282 48

Immunoaffinity purified pp60v-src was found to activate the MgATP-dependent protein phosphatase in the presence of MgATP. Although preliminary evidence suggested that phosphorylation of the inhibitor-2 subunit on tyrosine residues was responsible for the activation, preincubation of the pp60v-src preparation at 41 degrees C resulted in a rapid loss of its protein kinase activities towards both casein and inhibitor-2 while its ability to activate the protein phosphatase complex was relatively insensitive to this treatment. This result demonstrated that pp60v-src was not responsible for activation of the MgATP-dependent protein phosphatase. A protein kinase activity which phosphorylated glycogen synthase on serine residues was detected in the pp60v-src preparation. The protein kinase was active in the presence of inhibitors of phosphorylase kinase, glycogen synthase kinase 5/casein kinase II, and cAMP-dependent protein kinase. It is, therefore, likely that activation of the MgATP-dependent protein phosphatase resulted from the presence of a glycogen synthase kinase 3 like activity in the pp60v-src preparation. Our results illustrate the importance of applying multiple criteria to link the phosphorylation of a protein with an observed change in its activity.
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PMID:Apparent activation of the MgATP-dependent protein phosphatase by pp60v-src. Identification of an activity like that of glycogen synthase kinase 3 in immunoaffinity purified pp60v-src preparations. 301 36

The 'native' Mg-ATP-dependent protein phosphatase was isolated from rabbit skeletal muscle by a procedure that avoided the use of organic solvents or heating at 90-100 degrees C. The purified enzyme was composed of two major proteins (molecular mass 37 kDa and 31 kDa) that were present in a 1:1 molar ratio, and accounted for 70-80% of the material. The 37-kDa component comigrated with the catalytic subunit of protein phosphatase-1, and its identity with this protein was established by peptide mapping, and by its cleavage to the characteristic 34-kDa and 33-kDa fragments following incubation with chymotrypsin. The 31-kDa protein comigrated with inhibitor-2, and its identity with this protein was established by its heat stability, ability to inhibit protein phosphatase-1 at nanomolar concentrations, and its phosphorylation on a threonine residue by glycogen synthase kinase 3. It is therefore concluded that the 'native' Mg-ATP-dependent protein phosphatase is composed of the catalytic subunit of protein phosphatase-1 (37 kDa) and inhibitor-2 (31 kDa) in a 1:1 molar ratio. The 'native' Mg-ATP-dependent protein phosphatase had virtually identical properties to the enzyme reconstituted from inhibitor-2 and the 37-kDa catalytic subunit of protein phosphatase-1. Each preparation had a similar specific activity and was inhibited by identical concentrations of inhibitor-1. Both enzymes could be activated by incubation with glycogen synthase kinase-3 and Mg-ATP, or by Mn2+ and trypsin (or chymotrypsin). However, Mn2+ alone, or proteinase digestion in the absence of Mn2+, failed to activate either preparation. Incubation with glycogen synthase kinase-3 and Mg-ATP did not dissociate the 'native' or 'reconstituted' enzymes, whereas treatment with Mn2+ and trypsin decreased their apparent molecular masses from 70 kDa to 35 kDa. Incubation with chymotrypsin converted the 'native' and 'reconstituted' enzymes to forms that required preincubation with glycogen synthase kinase-3, Mg-ATP and inhibitor-2, in order to exhibit catalytic activity. The Mg-ATP-dependent protein phosphatase reconstituted from the 'nicked' 33-kDa catalytic subunit dissociated upon activation, in contrast to the enzyme reconstituted from the undegraded 37-kDa catalytic subunit. The results suggest that a 3-4-kDa fragment at one end of the polypeptide is involved in strengthening interaction between the undegraded 37-kDa catalytic subunit and the phosphorylated form of inhibitor-2.
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PMID:The protein phosphatases involved in cellular regulation. Comparison of native and reconstituted Mg-ATP-dependent protein phosphatases from rabbit skeletal muscle. 609 83

The MgATP-dependent phosphorylase phosphatase was found to have a broad substrate specificity. Its activity against all phosphoproteins tested was dependent upon preincubation with the activating factor FA and MgATP. The enzyme dephosphorylated and inactivated phosphorylase kinase and inhibitor 1, and dephosphorylated and activated glycogen synthase and acetyl-CoA carboxylase. Glycogen synthase was dephosphorylated at similar rates whether it had been phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase or glycogen synthase kinase 3. The enzyme also catalysed the dephosphorylation of ATP citrate lyase, initiation factor eIF-2, and troponin I. The properties of the MgATP-dependent protein phosphatase from either dog liver or rabbit skeletal muscle showed a remarkable similarity to highly purified preparations of protein phosphatase 1 from rabbit skeletal muscle. The relative activities of the two enzymes against all phosphoproteins tested was very similar. Both enzymes dephosphorylated the beta-subunit of phosphorylase kinase 40-fold faster than the alpha-subunit, and both enzymes were inhibited by identical concentrations of the two proteins termed inhibitor 1 and inhibitor 2, which inhibit protein phosphatase 1 specifically. These results demonstrate that the MgATP-dependent protein phosphatase is a type-1 protein phosphatase, and is distinct from type-2 protein phosphatases which dephosphorylate the alpha-subunit of phosphorylase kinase and are unaffected by inhibitor 1 and inhibitor 2. The possibility that the MgATP-dependent protein phosphatase is an inactive form of protein phosphatase 1 and that both proteins share the same catalytic subunit is discussed.
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PMID:The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities. 626 81

The signal transduction mechanism of protein kinase FA/GSK-3 alpha by tyrosine phosphorylation in A431 cells was investigated. Kinase FA/GSK-3 alpha was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could be tyrosine-dephosphorylated and inactivated down to less than 15% of control values in a concentration-dependent manner by 50-400 nM okadaic acid (a specific inhibitor of protein phosphatase types 1 and 2A), as demonstrated by metabolic 32P labeling the cells, followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and by immunodetection in an anti-kinase FA/GSK-3 alpha immunoprecipitate kinase assay. Taken together, the results provide initial evidence that serine/threonine phosphatase(s) may play a role involved in the modulation of kinase FA/GSK-3 alpha activity in cells, suggesting an involvement of serine/threonine dephosphorylation in the modulation of tyrosine phosphorylation and activation of protein kinase FA/GSK-3 alpha, representing a new mode of signal transduction pathway for the regulation of this multisubstrate protein kinase in cells.
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PMID:Okadaic acid, a serine/threonine phosphatase inhibitor, induces tyrosine dephosphorylation/inactivation of protein kinase FA/GSK-3 alpha in A431 cells. 751 66

Glycogen Synthase Kinase-3 (GSK-3) was isolated from bovine heart tissue extracts by a procedure involving ammonium sulfate fractionation, followed by chromatography on phosphocellulose, Cibacron blue 3GA-agarose, DEAE-Sephacel, CM-Sepharose, heparin-agarose, myelin basic protein-Sepharose, and LiChrospher 1000 C00-. GSK-3 was identified by its activation of protein phosphatase-1i (PP-1i). The purified enzyme had a specific activity of 25,500 units of protein phosphatase-1i activated/mg protein. The enzyme is an asymmetric monomeric protein of 53 kDa. The molecular size and retention of activity after autophosphorylation indicated that the isolated enzyme was the GSK-3 alpha-isoform.
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PMID:Purification and characterization of bovine heart glycogen synthase kinase-3. 783 Dec 7

Although protein kinase FA/GSK-3 alpha (an activating factor of ATP.Mg-dependent protein phosphatase) has been established as a cytosolic enzyme in mammalian nonnervous tissues involved in the metabolic regulation, immunological and biochemical studies on tissue and subcellular distributions demonstrate that kinase FA/GSK-3 alpha is in fact a membrane-associated enzyme and most abundantly exists in brain particulate membrane fractions depending on the tissue homogenization conditions. For instance, when brain was homogenized in Polytron without 0.32 M sucrose, approximately 40% of the total FA/GSK-3 alpha was found in the cytosol. However, when brain was homogenized in buffer containing 0.32 M sucrose and in a glass homogenizer with Teflon pestle, more than 80% of the total FA/GSK-3 alpha was found associated with the particulate membrane fractions. By manipulating these findings, we have developed a simplified procedure for purification of homogeneous kinase FA/GSK-3 alpha in high recovery and in a substantial amount from brain tissue. The data explain why kinase FA/GSK-3 alpha cannot be isolated in a reasonable amount from most mammalian tissues for the past years. The specific pure antibody that can specifically recognize kinase FA/GSK-3 alpha from crude tissue extracts together with the high quantity purification of the enzyme as presented in this report provides an initial key step for studies on the role of kinase FA/GSK-3 alpha in the regulation of brain functions especially in the brain particulate membrane fractions.
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PMID:Immunological and biochemical study on tissue and subcellular distributions of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase): a simplified and efficient procedure for high quantity purification from brain. 813 17

Phosphorylation of inhibitor 2, the regulatory subunit of the ATP-Mg-dependent protein phosphatase, by glycogen synthase kinase 3 (GSK-3) causes activation of the phosphatase. Prior phosphorylation by casein kinase II has been shown to enhance both phosphorylation and activation of the phosphatase by GSK-3 (DePaoli-Roach, A. A. (1984) J. Biol. Chem. 259, 12144-12152). Reported here is a comparison of the phosphorylation of inhibitor 2 by two defined isoforms of GSK-3, GSK-3 alpha and GSK-3 beta. GSK-3 beta was a significantly better inhibitor 2 kinase than was GSK-3 alpha. The Vmax/Km value for GSK-3 beta was approximately 10-fold higher than that for GSK-3 alpha. GSK-3 beta phosphorylated inhibitor 2 to a stoichiometry of approximately 1.0 mol of phosphate/mol of inhibitor 2. The phosphorylation by GSK-3 beta was determined to be exclusively at Thr-72 on the basis of the inability of the enzyme to modify a mutant inhibitor 2 in which Thr-72 was changed to alanine. Prior phosphorylation by casein kinase II promoted the action of GSK-3 alpha in keeping with earlier reports using undefined GSK-3 preparations. Phosphorylation by GSK-3 beta, in contrast, was unaffected by the previous action of casein kinase II. These results suggest that there can be important differences in substrate recognition by different isoforms of the same protein kinase and may help explain why some reported GSK-3 substrates require prior phosphorylation whereas other do not.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isoform differences in substrate recognition by glycogen synthase kinases 3 alpha and 3 beta in the phosphorylation of phosphatase inhibitor 2. 828 31


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