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)

Plasma membranes have been prepared from porcine thyroid glands using sucrose gradients. The fractions having a density in sucrose of 1.18 g/ml mainly contained plasma membranes and were moderately contaminated with other subcellular components as shown by marker enzyme data. Purified plasma membranes incubated in the presence of [32-P]gamma ATP incorporated 32-P. Kinetics of incorporation of 32-P into endogenous substrates studied in various buffers and with increasing ATP concentration suggest a phosphodephosphorylating system related to cAMP-dependent protein kinase and phosphoprotein phosphatase activities. The two enzymatic activities associated with plasma membranes have been demonstrated using exogenous substrates. cAMP increases and fluoride ions decrease the extent of membrane phosphorylation. The specific activity of protein kinase was 10-12 times higher than in the initial homogenate and was only slightly enhanced in the presence of 0.5% Nonidet as compared to microsomal fraction. cAMP binding to membrane proteins was 3 times higher than to the other particulate fractions. TSH present in the incubating medium or added after 5 min of 32-P labelling induced a rapid stimulation of endogenous phosphorylation followed by a rapid decrease. Phosphorylated membrane substrates were analyzed: high voltage paper electrophoresis after partial hydrolysis indicated that [32-P]phosphate is incorporated into serine and threonine residues as o-phosphate derivatives. SDS-polyacrylamide gel electrophoresis showed several 32--labelled fractions. When enhanced by cAMP, no specific phosphorylation of protein components was observed.
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PMID:Phosphorylation of purified thyroid plasma membranes incubated with [32-P]ATP. 16 13

1. Various proteins isolated from bovine tracheal smooth muscle were examined as phosphate acceptor substrates for a cyclic AMP-dependent protein kinase isolated from the same tissue. A fraction prepared in a manner similar to that of skeletal muscle troponin was the best substrate of the presumptive contractile proteins isolate. Actomyosin and tropomyosin were relatively poor substrates. 2. An assay was developed for the rapid detection in a large number of samples of the muscle specific substrate for the protein kinase on which we reported previously. 3. Using this assay, the muscle specific substrate found in bovine tracheal smooth muscle was partially purified resulting in a preparation which when resolved by polyacrylamide gel electrophoresis showed a single peak of 32P incorporated, and which could be further characterized. 4. Our findings suggest that the substrate contains a protein subunit of molecular weight 19 000, which can be phosphorylated at serine and threonine residues, in the presence of cyclic AMP and protein kinase. The phosphate is in a covalent ester linkage with these residues. 5. A phosphoprotein phosphatase was isolated from the bovine tracheal smooth muscle. 6. Bovine tracheal smooth muscle contains cyclic AMP dependent protein kinase and phosphoprotein phospahatase activity as well as the muscle specific substrate, suggesting that these elements may be part of a mechanism which regulates smooth muscle tone.
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PMID:Cyclic AMP-stimulated phosphorylation of bovine tracheal smooth muscle contractile and non-contractile proteins. 18 31

Incubation of Swiss 3T3 or L929 cells with tumor necrosis factor (TNF) leads to the rapid stimulation of several cytosolic Ser/Thr kinases active toward myelin basic protein, the S6 peptide (RRLSSLR), the G peptide (SPQPSRRGSESSEE), and Kemptide (LRRASLG). This confirms the hypothesis that kinases other than protein kinases A and C may be involved in the TNF signal transduction. Chromatography on Mono Q resolved multiple kinase peaks with each substrate tested and moreover revealed a TNF-mediated casein kinase-2 activation in both cell lines, measurable with the specific RRREEESEEE peptide or with the G peptide. The TNF-stimulated myelin basic protein kinases-1 and -2 were identified as extracellular signal-regulated kinases-2 and -1, respectively, based on their elution pattern on Mono Q chromatography, their inactivation by protein phosphatase action, their reaction with phosphothreonine and phosphotyrosine antibodies, and by their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 42- and 44-kDa proteins recognized by anti-extracellular signal-regulated kinase antibodies.
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PMID:Tumor necrosis factor stimulates multiple serine/threonine protein kinases in Swiss 3T3 and L929 cells. Implication of casein kinase-2 and extracellular signal-regulated kinases in the tumor necrosis factor signal transduction pathway. 128 78

Cell surface tyrosine kinase receptors are subject to a rapid activation by their ligand, which is followed by secondary regulatory processes. The IHE2 cell line is a unique model system to study the regulation of EGF binding to EGF receptors after activation of the EGF receptor kinase. IHE2 cells express both a chimeric insulin-EGF receptor kinase (IER) and a kinase-deficient EGF receptor (HER K721A). We have previously reported that IER is an insulin-responsive EGF receptor tyrosine kinase that activates one or several serine/threonine kinases, which in turn phosphorylate(s) the unoccupied HER K721A. In this article we show that insulin through IER activation induces a decrease in 125I-EGF binding to IHE2 cells. Scatchard analysis indicates that, as for TPA, the effect of insulin can be accounted for by a loss of the high affinity binding of EGF to HER K721A. Since this receptor transmodulation persists in protein kinase C downregulated IHE2 cells, it is likely to be due to a mechanism independent of protein kinase C activation. Using an in vitro system of 125I-EGF binding to transmodulated IHE2 membranes, we illustrate that the inhibition of EGF binding induced by IER activation is related to the phosphorylation state of HER K721A. Further, studies with phosphatase 2A, or at a temperature (4 degrees C) where only IER is functional, strongly suggest that the loss of high affinity EGF binding is related to the serine/threonine phosphorylation of HER K721A after IER activation. Our results provide evidence for a "homologous desensitization" of EGF receptor binding after activation of the EGF receptor kinase of the IER receptor.
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PMID:Activation of insulin-epidermal growth factor (EGF) receptor chimerae regulates EGF receptor binding affinity. 130 16

TSH regulation of insulin and insulin-like growth factor-I (IGF-I) receptor kinases has been studied in FRTL5 cultured thyroid cells. Preincubation of intact cells with TSH increased by 2-fold insulin and IGF-I receptor autophosphorylation and phosphorylation of the p175 endogenous substrate for the receptors. Enhanced phosphorylations reached a maximum within 30 min, were maintained for 30 min more, and vanished after 120 min of TSH incubation. TSH dose-responses exhibited half-maximal and maximal effects at 1 and 10 pM, respectively. In vitro, insulin as well as IGF-I receptors purified from cells treated with 10 pM TSH also exhibited 2-fold enhanced receptor autophosphorylation and kinase activity toward the exogenous substrate poly(Glu,Tyr) (4:1). At variance with TSH, cell incubation with either 8-bromo-cAMP or the protein kinase-C activator 12-O-tetradecanoylphorbol-13-acetate inhibited insulin and IGF-I receptor kinases. In intact cells, TSH stimulation of insulin and IGF-I receptor kinases was accompanied by enhanced turnover of phosphate on autophosphorylated receptors, increased receptor tyrosine phosphorylation, and decreased receptor serine/threonine phosphorylation in response to insulin. Incubation of in vivo labeled insulin and IGF-I receptors with extracts from TSH-treated cells also decreased receptor phosphoserine and phosphothreonine content. Furthermore, preincubation of insulin and IGF-I receptors with extracts from TSH-treated cells enhanced in vitro autophosphorylation. The latter effect was inhibited by the serine/threonine phosphatase inhibitors fluoride and okadaic acid, but not by the tyrosine phosphatase inhibitor vanadate. The data suggest that in FRTL5 cells, TSH induces the activity of a Ser/Thr protein phosphatase, which dephosphorylates insulin and IGF-I receptors and enhances their endogenous kinases.
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PMID:Thyrotropin regulates autophosphorylation and kinase activity in both the insulin and the insulin-like growth factor-I receptors in FRTL5 cells. 131 Dec 44

A sphingomyelin cycle has been identified whereby the action of certain extracellular agents results in reversible sphingomyelin hydrolysis and the concomitant generation of ceramide. Moreover, a cell-permeable ceramide, C2-ceramide (N-acetylsphingosine), is a potent modulator of cell proliferation and differentiation. We report herein that C2-ceramide, C6-ceramide, and natural ceramides activate a cytosolic serine/threonine protein phosphatase in a dose-dependent manner. Initial activation is observed at concentrations of ceramide as low as 0.1 microM with peak response occurring at 5-10 microM. However, other closely related sphingolipids, sphingosine and sphingomyelin, were largely inactive. Ceramide-stimulated phosphatase was inhibited by okadaic acid, an inhibitor of protein phosphatases, with an IC50 of 0.1-1 nM, depending on the concentration of ceramide. Ceramide-stimulated phosphatase was insensitive to Mg2+ and Mn2+ cations. Using sequential anion exchange chromatography, ceramide-stimulated phosphatase activity could be resolved from ceramide-nonresponsive phosphatases. The activity of partially purified enzyme was stimulated 3.5-fold by ceramide. The identification of a phosphatase as a molecular target for the action of ceramide defines a novel intracellular signaling pathway with potential roles in the regulation of cell proliferation and differentiation.
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PMID:Ceramide stimulates a cytosolic protein phosphatase. 131 82

Okadaic acid and calyculin A, specific and cell permeable inhibitors of protein phosphatase 1 and 2A, inhibited aggregation, secretion and delta [Ca++]i in thrombin stimulated platelets. The inhibitory effect of calyculin A (IC50: 3.6-4.8nM) was about two hundred times more potent than that of okadaic acid (IC50: 0.8-1.3 microM), which is consistent with the difference of the reported Ki values for protein phosphatase 1. These phosphatase inhibitors and PGI2 synergistically enhanced the phosphorylation of 50kDa protein (P50), which is solely related to the inhibition of platelet reaction. These results indicate that serine/threonine protein phosphatase 1 might play a role in platelet activation.
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PMID:The effects of okadaic acid and calyculin A on thrombin induced platelet reaction. 131 73

Simian virus 40 (SV40) large tumor antigen (T) is an oncoprotein whose biological and biochemical functions appear to be modulated by phosphorylation. Recently, SV40 DNA replication in vitro has been shown to be activated by dephosphorylation involving the activity of a serine/threonine phosphoprotein phosphatase belonging to the type 2A class (PP2A) [Virshup, D.M., Kauffman, M.G. & Kelly, T.J. (1989) EMBO J., 8, 3891-3898]. To address the question of how specificity of PP2A activity towards T is regulated, an in vitro assay to study the process of T dephosphorylation was developed. Unlabeled extracts from cells enriched for various stages of the cell cycle were incubated with 32P-labeled, immunocomplexed T. Extracts from a population of cells enriched for S phase demonstrated a selective ability to dephosphorylate this labeled protein when compared with extracts prepared from G1- and M-phase cells. The time course of release from growth arrest demonstrated that this T-specific phosphatase activity occurred at the onset of host-cell DNA synthesis. In contrast, when using 32P-labeled phosphorylase a as the substrate, phosphatase activity appeared to be present throughout the cell cycle. The data presented here are consistent with the notion that PP2A activity towards T is regulated in a cell cycle-dependent manner.
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PMID:Selective ability of S-phase cell extracts to dephosphorylate SV40 large T antigen in vitro. 131 15

Two antipeptide antibodies, one against the peptide corresponding to residues 307-327 (alpha Y91) and one against the peptide corresponding to the C-terminal portion (alpha C92) of the deduced amino acid sequence of the extracellular signal-regulated kinase 1 (ERK1), precipitated two 41-kDa and/or two 43-kDa phospho-proteins from mitogen-stimulated Swiss 3T3 cells. Electrophoretic mobilities on two-dimensional gels of the immunoprecipitated 41- and 43-kDa phosphoproteins were similar to those of the 41- and 43-kDa cytosol proteins, whose increased tyrosine phosphorylation we and others had originally identified in various mitogen-stimulated cells (Cooper, J. A., Sefton, B. M., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37; Kohno, M. (1985) J. Biol. Chem. 260, 1771-1779); phosphopeptide map analysis revealed that they were respectively identical molecules. All those phosphoproteins contained phosphotyrosine, and the more acidic forms contained additional phosphothreonine. Immunoprecipitated 41- and 43-kDa phosphoproteins had serine/threonine kinase activity toward myelin basic protein (MBP) and microtuble-associated protein 2 (MAP2). With the combination of two-dimensional gel electrophoresis and the kinase assay in MBP-containing polyacrylamide gels of the alpha Y91 immunoprecipitates, with or without phosphatase 2A treatment, we showed that only their acidic forms were active. These results clearly indicate that 41- and 43-kDa proteins, the increased tyrosine phosphorylation of which is rapidly and commonly induced by mitogen stimulation of fibroblasts, are family members of ERKs/MAP2 kinases and that phosphorylation both on tyrosine and threonine residues is necessary for their activation.
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PMID:Mitogen-induced tyrosine-phosphorylated 41- and 43-kDa proteins are family members of extracellular signal-regulated kinases/microtubule-associated protein 2 kinases. 131 74

The Drosophila retinal degeneration C (rdgC) gene is required to prevent light-induced retinal degeneration. Molecular analysis shows that the rdgC transcription unit encodes a novel serine/threonine protein phosphatase. Amino acids 153-393 define a domain that has 30% identity with the catalytic domains of types 1, 2A, and 2B serine/threonine protein phosphatases. A putative regulatory domain is appended that contains multiple potential Ca(2+)-binding sites or "EF hand motifs." Thus, the analysis suggests that the rdgC protein is a novel type of serine/threonine protein phosphatase that is directly regulated by Ca2+. rdgC is expressed in the visual systems of the fly, as well as in the mushroom bodies of the central brain.
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PMID:Drosophila retinal degeneration C (rdgC) encodes a novel serine/threonine protein phosphatase. 131 7


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