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 trimeric form of protein phosphatase 2A (PP2A1 or polycation-stimulated protein phosphatase H1) was purified to homogeneity from rabbit skeletal muscle. Preparative SDS-polyacrylamide gel electrophoresis was used to purify the individual subunits with relative molecular masses of 36, 55, and 65 kDa. Sequence analysis of five peptides from the 65-kDa regulatory subunit (PR65) suggested that it was identical with the PR65 subunit derived from the dimeric protein phosphatase 2A2. Amino acid sequences derived from the 55-kDa regulatory subunit (PR55) were used to clone human and rabbit cDNAs encoding this protein. The PR55 subunit was found to be encoded by two genes, termed alpha and beta. The open reading frames of the PR55 alpha and beta cDNAs spanned 1341 and 1329 nucleotides, respectively, and predicted proteins with a molecular mass of about 52 kDa that are 86% identical. Comparison of the human PR55 amino acid sequences with the data obtained from the rabbit skeletal muscle protein and a partial rabbit PR55 beta cDNA clone indicated a high degree of conservation. Analysis of the mRNA expression in human cell lines revealed that the PR55 alpha isoform was encoded by two transcripts of about 2.3 and 2.5 kb and a less abundant 4.4-kb mRNA. Whereas a PR55 beta transcript of about 2.3 kb was detected at high levels in the neuroblastoma derived cell line LA-N-1, the level of the mRNA was very low in the other human cell lines analyzed. Interestingly, the PR55 sequence showed limited homology to the catalytic domain (domains VI-IX) of the c-abl protein tyrosine kinase.
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PMID:Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform. 184 34

Mouse epidermal cytosol contains a protein phosphatase with Mr 38,000, which dephosphorylates the elongation factor 2 (EF-2) of protein biosynthesis and is stimulated after topical application of TPA to mouse skin [(1988) Biochem. Biophys. Res. Commun. 153, 1129-1135]. Dephosphorylation of EF-2 by this phosphatase is inhibited by okadaic acid at concentrations as low as 10(-8) M, but not by heparin up to concentrations of 600.micrograms/ml. The catalytic subunit of protein phosphatase 2A (PP2Ac) with EF-2 as a substrate exhibits the same sensitivity towards okadaic acid and insensitivity towards heparin as the EF-2 phosphatase of epidermal cytosol. The catalytic subunit of protein phosphatase 1 (PP1c) is strongly suppressed by heparin and less sensitive towards okadaic acid than PP2Ac. PP2Ac is around 50 times more efficient in dephosphorylating EF-2 than PP1c. These data indicate that the TPA-stimulated EF-2 phosphatase in epidermal cytosol is a type 2A protein phosphatase.
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PMID:A type 2A protein phosphatase dephosphorylates the elongation factor 2 and is stimulated by the phorbol ester TPA in mouse epidermis in vivo. 255 20

We have shown previously (Nishimura, M., Fedorov, S., and Uyeda, K. (1994) (J. Biol. Chem. 269, 26100-26106) that the administration of high concentrations of glucose stimulates dephosphorylation of Fru-6-P,2-kinase: Fru-2,6-bisphosphatase in perfused liver, and xylulose (Xu) 5-P activates the dephosphorylation reaction. To characterize the protein phosphatase, we have purified the Xu 5-P-activated protein phosphatase to homogeneity from livers of rats injected with high glucose. Several protein phosphatases in the livers were separated by DEAE-cellulose chromatography, but only one peak of the enzyme was activated by Xu 5-P. The protein phosphatase was inhibited by okadaic acid (IC50 = 1-3 nM) and did not require Mg2+ or Ca2+, suggesting that the enzyme was type 2A. The enzyme was a heterotrimer (M(r) = 150,000) and consisted of structural (A, 65 kDa) catalytic (C, 36 kDa), and regulatory (B, 52 kDa) subunits. Amino acid sequences of five tryptic peptides derived from the B subunit showed similarity with those of the B alpha isoform of rat protein phosphatase 2A, but five out of 73 residues were different. The protein phosphatase catalyzed dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase, phosphorylase alpha, and pyruvate kinase, and the Km values were 0.8 microM, 3.7 microM, and 2.2 microM, respectively. Among these substrates dephosphorylation of only the bifunctional enzyme was activated by Xu 5-P, and the K alpha value for Xu 5-P was 20 microM. Xu 5-P was the only sugar phosphate which activated the PP2A among all the sugar phosphates examined. These results demonstrated the existence and isolation of a unique heterotrimeric protein phosphatase 2A in rat liver which catalyzed the dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase and was activated specifically by Xu 5-P. The Xu 5-P-activated protein phosphatase 2A explains the increased Fru 2,6-P2 level in liver after high glucose administration.
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PMID:Purification and characterization of a novel xylulose 5-phosphate-activated protein phosphatase catalyzing dephosphorylation of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. 759 45

Inhibitor-2 (I-2) is the regulatory subunit of the cytosolic ATP-Mg-dependent form of type 1 serine/threonine protein phosphatase and its phosphorylation at Thr-72 by glycogen synthase kinase-3 results in phosphatase activation. Activation of cytosolic type 1 phosphatase has been observed in cells treated with growth factors. Reported here is the phosphorylation and activation of the ATP-Mg-dependent phosphatase by mitogen-activated protein kinase (MAPK). Recombinant I-2 was phosphorylated by activated MAPK to an extent (approximately 0.3 mol of phosphate/mol of polypeptide) similar to that reported for phosphorylation by the alpha isoform of glycogen synthase kinase-3. The phosphorylation of I-2 by MAPK was exclusively at Thr-72, the site involved in the activation of phosphatase. Incubation of MAPK with purified ATP-Mg-dependent phosphatase resulted in phosphorylation of the I-2 component and activation of the phosphatase. Ribosomal S6 protein kinase II (p90rsk) was also able to phosphorylate the recombinant I-2; however, this phosphorylation occurred on serines and had no effect on phosphatase activation. Our data may explain growth factor-induced activation of the ATP-Mg-dependent phosphatase and suggest that MAPK may of cytosolic type 1 phosphatase in response to insulin and/or other growth factors.
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PMID:Phosphorylation and activation of the ATP-Mg-dependent protein phosphatase by the mitogen-activated protein kinase. 762 58

A colorimetric phosphatase-inhibition bioassay was developed for the quantitative measurement of okadaic acid (OA) the main diarrhetic toxin responsible for diarrhetic shellfish poisoning. The assay used an artificial substrate, paranitrophenylphosphate, and a semi-purified protein phosphatase PP2Ac containing extract prepared from rabbit muscle. Calibration dose-inhibition curves were constructed using standard OA and they permitted easy determination of the enzyme concentration Et in their linear portion. In the range of linearity, the slope increased when Et decreased, thus giving a detecting limit of 0.04 pmol in the reaction mixture (1 ml). The lowest assayable concentration of OA was 4 ng/ml in aqueous solutions and 40 ng/ml (i.e., 100 ng of OA per g of mussel tissue) in crude methanol mussels extracts. The intra and interassay coefficients of variation in the measurement of OA for the toxin spiked aqueous samples averaged, respectively, 7.7% and 3.7%, and interexperiments coefficients of variation for the toxin spiked mussel extracts averaged 4.6%. The presence of OA was ascertained by a method in which one assay was performed at two or three different levels of enzyme concentration. The rapidity, accuracy, reproducibility, specificity, and simplicity of the procedure provides a simple way to assay okadaic acid in buffered or complex solutions.
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PMID:Highly sensitive assay of okadaic acid using protein phosphatase and paranitrophenyl phosphate. 786 65

The gene expression for alpha and beta isoforms of type 2A protein phosphatase (PP2A-alpha and -beta) in the adult rat brain was examined by in situ hybridization analysis. No marked difference in the gene expression was discerned between the two isoforms in large portions of brain, except for the thalami in which the expression level for the alpha isoform was similar to that in the cerebral neocortex whereas that for the beta was lower than that in the neocortex. The gene expression was observed intensely in the piriform cortex, the cerebellar Purkinje and granule cell layers, and the hippocampal pyramidal and dentate granule cell layers, and the locus ceruleus, whereas the moderate levels of its expression were observed in the olfactory mitral cells and the pontine nuclei. The cerebral neocortex expressed the mRNA moderately to weakly without any laminar patterns, whereas the expression level in the caudate-putamen was very low. This expression pattern is basically similar to that of PP2C reported previously, except for the plexus choroideus and ependyma having no significant expression for PP2A.
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PMID:Localization of mRNA for protein phosphatase 2A in the brain of adult rats. 801 74

The catalytic subunit of the major protein phosphatase associated with bovine cardiac myofibrils was purified to homogeneity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme revealed only one band with an apparent molecular weight of 37,000. On gel filtration chromatography, the phosphatase activity and the protein co-eluted as a single peak with an apparent molecular weight of 37,000. The purified enzyme was identified as the catalytic subunit of protein phosphatase 1, as determined by sensitivity to inhibitor 1, inhibitor 2, okadaic acid and by specific immunostaining. Evidence obtained with specific antipeptide antibodies demonstrated that this myofibril protein phosphatase was predominantly the alpha isoform of protein phosphatase 1. The purified catalytic subunit was completely inactive. It was activated by pretreatment with Co2+/trypsin in the presence of high ionic strength. Treatment with trypsin alone did not activate the latent enzyme. The enzyme was also activated by Co2+ or Mn2+ alone but not by Ca2+, Mg2+, Ni2+, Cu2+ or Zn2+. Activation of the enzyme was not reversed by removal of Co2+, but Mn(2+)-activated phosphatase activity was partially reversed when Mn2+ was removed. The catalytic subunit could form a 1:1 complex with inhibitor 2 in vitro. The resulting holoenzyme was also activated by pretreatment with Co2+. Since phosphatase 1 alpha is the major phosphatase associated with cardiac myofibril, it is suggested that it is responsible for the dephosphorylation of myosin and other myofibril phosphoproteins.
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PMID:A latent form of protein phosphatase 1 alpha associated with bovine heart myofibrils. 808 38

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.
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PMID:Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases. 840 Apr 54

We examined the immunohistochemical distribution of the two mammalian isoforms of calcineurin catalyic subunits, A alpha and A beta, in central nervous system (CNS) tissues of cows, rats, and humans. Cryostat sections and paraffin sections of parformaldehyde-fixed tissues were stained with antipeptide antibodies for each isoform. The same localization pattern was observed in both cryostat and paraffin sections. In the latter, the intensity of the staining was dramatically enhanced by microwave irradiation. Calcineurin isoforms were localized in a variety of nerve cells but not in neuroglial cells. Their differential expression as the A alpha isoform in the nucleus and the A beta isoform in the cytoplasm was present in a variety of CNS nerve cells, most distinctively in Purkinje cells of the cerebellum and pyramidal cells of the cerebrum, irrespective of species. These results suggest that each isform has distinct intracellular sites of action in CNS neurons and that the phenomenon has been conserved during mammalian evolution.
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PMID:Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin) in central nervous system neurons: immunohistochemistry on formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation. 854 76

Phospholipase D (PLD) which was partially purified from membranes of porcine brain could be stimulated by multiple cytosolic components; these included ADP-ribosylation factor (Arf) and RhoA, which required guanine nucleotides for activity, and an unidentified factor which activated the enzyme in a nucleotide-independent manner (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Here, we report purification of the latter factor, its identification as the alpha isoform of protein kinase C (PKCalpha), and characterization of its regulation of PLD activity. Stimulation of PLD by purified PKCalpha or recombinant PKCalpha (rPKCalpha) occurred in the absence of any nucleotide and required activators such as Ca2+ or phorbol ester. This action was synergistic with stimulation of PLD evoked by either Arf or RhoA. Dephosphorylation of rPKC alpha with protein phosphatase 1 or 2A resulted in a loss of its kinase activity, but had little effect on its ability to stimulate PLD either alone or in conjunction with Arf. Staurosporine inhibited the kinase activity of PKCalpha without affecting activation of PLD. Finally, gel filtration of PKCalpha that had been cleaved with trypsin demonstrated that stimulatory activity for PLD coeluted with the regulatory domain of the enzyme. These data indicate that PKC may regulate signaling events through direct molecular interaction with downstream effectors as well as through its well characterized catalytic modification of proteins by phosphorylation.
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PMID:Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity. 862 5


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