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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three forms of protein phosphatase-1 were isolated from rabbit skeletal muscle that had Mr values of 37 000, 34 000 and 33 000 determined by sodium dodecyl sulphate (SDS) gel electrophoresis. Each species dephosphorylated the beta-subunit of phosphorylase kinase very much faster than the alpha-subunit, was inhibited by inhibitors 1 and 2 with equal potency, and was converted to a form dependent on glycogen synthase kinase-3 and Mg-ATP for activity by incubation with inhibitor-2. Digestion with cyanogen bromide or Staphylococcus aureus proteinase followed by SDS gel electrophoresis showed a very similar pattern of cleavage products for all three forms. The Mr-37 000 and Mr-34 000 species were converted to the Mr-33 000 form by incubation with chymotrypsin. It is concluded that the Mr-33 000 and Mr-34 000 forms are derived from the Mr-37 000 component by limited proteolysis. Conversion of the Mr-37 000 to the Mr-33 000 form was accompanied by a two-fold increase in activity, indicating that an Mr-4000 fragment at one end of the polypeptide is an inhibitory domain that decreases enzyme activity. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle had an Mr of 36 000 determined by SDS gel electrophoresis and its specific activity (3 kU/mg) was much lower than that of the Mr-37 000 (15-20 kU/mg) or Mr-33/34 000 (40-50 kU/mg) forms of protein phosphatase-1. It dephosphorylated the alpha-subunit of phosphorylase kinase 4-5-fold faster than the beta-subunit, was unaffected by inhibitor-1 or inhibitor-2, and preincubation with the latter protein did not result in the production of a glycogen synthase kinase-3 and Mg-ATP-dependent form of the enzyme. Digestion with chymotrypsin did not alter the electrophoretic mobility of protein phosphatase 2A under conditions that caused quantitative conversion of the Mr-37 000 form of protein phosphatase-1 to the Mr-33 000 species. Digestion with cyanogen bromide or S. aureus proteinase, followed by SDS gel electrophoresis, showed a quite different pattern of cleavage products to those observed with protein phosphatase 1. Antibody to protein phosphatase-2A raised in sheep did not cross-react with any of the forms of protein phosphatase-1, as judged by immunoelectrophoretic and immunotitration experiments. It is concluded that protein phosphatase-1 and protein phosphatase-2A are distinct gene products.
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PMID:The catalytic subunits of protein phosphatase-1 and protein phosphatase 2A are distinct gene products. 631 40

NIPP-1 was originally isolated as a potent and specific nuclear inhibitory polypeptide (16-18 kDa) of protein phosphatase-1. We report here the cDNA cloning of NIPP-1 from bovine thymus and show that the native polypeptide consists of 351 residues and has a calculated mass of 38.5 kDa. The bacterially expressed central third of NIPP-1 completely inhibited the type-1 catalytic subunit, but displayed a reduced inhibitory potency after phosphorylation by protein kinase A and casein kinase 2. Translation of NIPP-1 mRNA in reticulocyte lysates resulted in the accumulation of both intact NIPP-1 and a smaller polypeptide generated by alternative initiation at the codon corresponding to Met143. A data base search showed that the COOH terminus of NIPP-1 is nearly identical to the human ard-1 protein (13 kDa), which has been implicated in RNA processing (Wang, M., and Cohen, S. N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10591-10595). Comparison of the cDNAs encoding ard-1 and NIPP-1 suggests that their mRNAs are generated by alternative splicing of the same pre-mRNA. Western blotting with antibodies against the COOH terminus of NIPP-1, however, showed a single polypeptide of 47 kDa, which was enriched in the nucleus. Northern analysis revealed a single transcript of 2.2 kilobases in bovine thymus and of 2.4 kilobases in various human tissues.
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PMID:Molecular cloning of NIPP-1, a nuclear inhibitor of protein phosphatase-1, reveals homology with polypeptides involved in RNA processing. 749 93

The Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis. The complete PPZ1 protein has been successfully expressed as a soluble glutathione-S-transferase fusion protein. The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase. It was also active towards p-nitrophenylphosphate. The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis. The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2. The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2. Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase.
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PMID:Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance. 761 85

The activity of protein phosphatase-1 in rat liver nuclei (PP-1N) was decreased by up to 97% by associated inhibitory polypeptides, depending on the assay and extraction conditions. These inhibitors were rapidly degraded by endogenous proteases, resulting in the accumulation of active heat-stable intermediates. Two major species of PP-1N could be differentiated by fractionation of a nuclear extract. PP-1NR111 contained, besides the delta-isoform of the catalytic subunit, an inhibitory polypeptide of 111 kDa. PP-1NR41 was found to be an inactive heterodimer between the delta-isoform of the catalytic subunit and NIPP-1, a nuclear inhibitor of PP-1, which in its undegraded form is heat labile and migrates during SDS-polyacrylamide gel electrophoresis as a polypeptide of 41 kDa. Native hepatic NIPP-1 displayed a reduced affinity for the catalytic subunit after phosphorylation by protein kinase A in vitro and after glucagon-induced phosphorylation in vivo.
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PMID:Subunit structure and regulation of protein phosphatase-1 in rat liver nuclei. 761 25

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) is a potent inhibitor of protein phosphatase-1 when it is phosphorylated on Thr-34 by cAMP-dependent protein kinase. DARPP-32 is highly enriched in some specific cell populations such as striatonigral neurons and choroid plexus epithelial cells. Here we show that recombinant rat DARPP-32 is phosphorylated by casein kinase I on seryl residues to a stoichiometry of approximately 2 mol of phosphate/mol of protein. DARPP-32 is one of the best known substrates for casein kinase I (Km = 3.4 +/- 0.3 microM), whereas the homologous phosphatase-1 inhibitor, inhibitor-1, is not. Phosphorylation of DARPP-32 by casein kinase I does not alter its ability to inhibit protein phosphatase-1. Residues phosphorylated by casein kinase I were identified as Ser-137 and Ser-189 by site-directed mutagenesis and by protein sequencing. Ser-137 and the preceding stretch of 16-18 acidic residues are conserved in DARPP-32 among all species examined, whereas Ser-189 is not. Phosphorylation of Ser-137 induces an unusual increase in DARPP-32 electrophoretic mobility in polyacrylamide gels in the presence of SDS. In striatonigral neurons, DARPP-32 is phosphorylated on Ser-137 and the stoichiometry of phosphorylation on this residue in vivo appears to be higher in the substantia nigra (axon terminals) than in the striatum (soma and dendrites). These results indicate that casein kinase I is highly active in striatonigral neurons in which it may play important roles, including in protein phosphatase-1 modulation via phosphorylation of DARPP-32.
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PMID:Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase I in vitro and in vivo. 772 83

The mechanism of inhibition of protein phosphatase-1 catalytic subunit (PP-1c) by recombinant DARPP-32 and synthetic peptides was studied. DARPP-32 was expressed in Escherichia coli as a non-fusion protein using a pEt-3a plasmid, purified to homogeneity and shown to have physicochemical properties similar to those of the protein purified from bovine brain. Recombinant DARPP-32 phosphorylated on threonine-34 by cAMP-dependent protein kinase inhibited PP-1c with an IC50 approximately 0.5 nM, comparable to that obtained with bovine DARPP-32. Non-phosphorylated DARPP-32, and mutated forms in which threonine-34 was replaced by an alanine or a glutamic acid, inhibited PP-1c with an IC50 approximately 1 microM. Surface plasmon resonance analysis showed binding of PP-1c to nonphospho- and phospho-DARPP-32-(8-38) synthetic peptides with apparent Kd values of 1.2 and 0.3 microM, respectively, supporting the existence of an interaction between non-phosphorylated DARPP-32 and PP-1c that is increased by phosphorylation of DARPP-32 at threonine-34. These results suggest a model in which DARPP-32 interacts with PP-1c by at least two low affinity sites, the combination of which is responsible for the high affinity (nM) inhibition.
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PMID:Mechanism of inhibition of protein phosphatase 1 by DARPP-32: studies with recombinant DARPP-32 and synthetic peptides. 782 84

The effect of modulators of protein phosphorylation on the transcriptional activity of the androgen receptor (AR) was studied under transient expression conditions. Activators of protein kinase-A [8-bromo-cAMP (8-Br-cAMP)] and protein kinase-C (phorbol 12-myristate 13-acetate) or an inhibitor of protein phosphatase-1 and -2A (okadaic acid) influenced minimally pMMTV-chloramphenicol acetyl-transferase (CAT) activity in CV-1 cells cotransfected with an AR expression plasmid in the absence of androgen. In the presence of testosterone, however, all compounds enhanced AR-mediated transactivation by 2- to 4-fold. A nonsteroidal antiandrogen, Casodex, behaved as a pure antagonist; it blunted the action of testosterone and was not rendered agonistic by activators of protein kinase-A. A reporter plasmid containing two androgen response elements (AREs) in front of the thymidine kinase promoter (pARE2tk-CAT) was also used to examine promoter specificity. It was activated by 8-Br-cAMP, forskolin, or okadaic acid even without AR or androgen. However, when forskolin or okadaic acid was used together with androgen and AR, the resulting AR-dependent transactivation of pARE2tk-CAT was more than additive. Intact DNA- and ligand-binding domains, but not the N-terminal amino acid residues 40-147, of the receptor were mandatory for the synergism between protein kinase-A activators and androgen. Immunoreactive AR content in transfected COS-1 cells was not influenced by exposure to 8-Br-cAMP. Similar results were obtained by ligand binding assays. Quantitative or qualitative differences were not observed in DNA-binding characteristics between receptors extracted from cells treated with testosterone with or without protein kinase-A activator. Collectively, the synergistic stimulation of AR-dependent transactivation by androgen and protein kinase activators is not due to changes in cellular AR content or affinity of the receptor for the cognate DNA element; rather, this phenomenon seems to result from altered interaction of ligand-activated AR with other proteins in the transcription machinery.
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PMID:Stimulation of androgen-regulated transactivation by modulators of protein phosphorylation. 792 97

In the medium-sized spiny neurons of the striatonigral pathway, a cascade of events involving the activation of dopamine D1 receptors, an increase in cyclic AMP, and activation of cyclic AMP-dependent protein kinase causes the phosphorylation of DARPP-32 on Thr34, converting DARPP-32 into a powerful inhibitor of protein phosphatase-1. In the present study, the incubation of striatal or substantia nigra slices with GABA also increased the phosphorylation of DARPP-32 on Thr34. GABA did not significantly increase cyclic AMP levels in slices. The phosphorylation of DARPP-32 by GABA was blocked in both brain regions by pretreatment of slices with the GABAA receptor antagonist, bicuculline, but not with the GABAB receptor antagonist, phaclofen. Moreover, the threonine phosphorylation of DARPP-32 produced by maximally effective doses of either forskolin (in striatum) or L-3,4-dihydroxyphenylalanine (in substantia nigra) was increased further by GABA. The data are consistent with a model in which GABA increases the phosphorylation state of DARPP-32 by inhibiting dephosphorylation of the protein by the calcium/calmodulin-dependent protein phosphatase, calcineurin.
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PMID:Phosphorylation of DARPP-32 is regulated by GABA in rat striatum and substantia nigra. 793 32

Glycogen synthase kinase-3 (GSK3) is inactivated in vitro by p70 S6 kinase or MAP kinase-activated protein kinase-1 beta (MAPKAP kinase-1 beta; also known as Rsk-2). Here we show that GSK3 isoforms are inhibited by 40% within minutes after stimulation of the rat skeletal-muscle cell line L6 with insulin-like growth factor-1 (IGF-1) or insulin. GSK3 was similarly inhibited in rabbit skeletal muscle after an intravenous injection of insulin. Inhibition resulted from increased phosphorylation of GSK3, probably at a serine/threonine residue(s), because it was reversed by incubation with protein phosphatase-2A. Rapamycin blocked the activation of p70 S6 kinase by IGF-1 in L6 cells, but had no effect on the inhibition of GSK3 or the activation of MAPKAP kinase-1 beta. In contrast, wortmannin, a potent inhibitor of PtdIns 3-kinase, prevented the inactivation of GSK3 and the activation of MAPKAP kinase-1 beta and p70 S6 kinase by IGF-1 or insulin. Wortmannin also blocked the activation of p74raf-1. MAP kinase kinase and p42 MAP kinase, but not the formation of GTP-Ras by IGF-1. The results suggest that the stimulation of glycogen synthase by insulin/IGF-1 in skeletal muscle involves the MAP-KAP kinase-1-catalysed inhibition of GSK3, as well as the previously described activation of the glycogen-associated form of protein phosphatase-1.
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PMID:The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. 794 42

A novel procedure for detection and assay of protein kinase and phosphatase activities in complex biological mixtures was developed. By means of capillary zone electrophoresis (CZE) methodology, the phosphorylated and dephosphorylated forms of the peptide Kemptide, a 46-amino-acid fragment from protein phosphatase inhibitor-1 and a peptide fragment corresponding to the RII subunit of cAMP-dependent protein kinase (PKA), were rapidly resolved. This facilitated nonradioactive detection of PKA and protein phosphatase-2B (calcineurin) in rabbit skeletal muscle extracts. In addition, the CZE procedure enabled a site-specific assay of a 14-amino-acid peptide from the glycogen-binding subunit of protein phosphatase-1 monophosphorylated on distinct sites by PKA and casein kinase-II. These results suggest that CZE may prove to be extremely useful for the analysis of peptides that are phosphorylated at multiple sites in vivo.
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PMID:A capillary electrophoresis-based assay for protein kinases and protein phosphatases using peptide substrates. 797 76


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