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 present study demonstrated the presence within the myocardium of phosphoprotein phosphatase activity which can account for dephosphorylation of a 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum that has been associated with the stimulatory effects of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase on calcium transport (Tada, M., Kirchberger, M. A., and Katz, A. M. (1975) J. Biol. Chem. 250:2640-2647). Dog cardiac microsomes, consisting mainly of fragmented sarcomplasmic reticulum, were phosphorylated by incubation with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, and subsequently washed with trichloroacetic acid or buffered KCl. Phosphorylated microsomes contained approximately 1 nmole of 32P bound per mg of microsomal protein, 32P labeling occurring almost exclusively at the 22,000 dalton component. Soluble phosphoprotein phosphatases, isolated from the cytosol, catalyzed dephosphorylation of 32P-labeled microsomes. The existence of a phosphoprotein phosphatase that is associated with the microsomes was demonstrated by the ability of the microsomes to dephosphorylate 32P-histone. This membrane-associated phosphatase activity can also account for a rapid decrease in the amount of 32P-labeling of the 22,000 dalton protein. The dephosphorylation of the phosphorylated 22,000 dalton protein by phosphoprotein phosphatase satisfies an important requirement for the phosphorylation of the 22,000 dalton protein to serve a physiological role, namely, its reversibility.
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PMID:Phosphoprotein phosphatase-catalyzed dephosphorylation of the 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum. 17 94

The role of adenosine 3',5'-monophosphate (cyclic AMP)-dependent membrane phosphorylation in the regulation of microsomal calcium transport in rat aortic smooth muscle was studied. Cyclic AMP-dependent protein kinase augmented the phosphorylation of serine residues in a microsomal protein component with a molecular weight of about 44,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the majority of 32P incorporation was in serine residue(s). The phosphorylated protein had stability characteristics of a phosphoester. The phosphorylated substrate was not extracted from the trichloroacetic acid (TCA) precipitate with organic solvents or by suspension in hot TCA; and the demonstrated hydroxylamine insensitivity suggested that the substrate was not lipid or nucleic acid. Intrinsic phosphoprotein phosphatase cleaved the labeled phosphate from the cyclic AMP-stimulated microsomes in the first 5 min of incubation. Microsomes phosphorylated in the presence of 1 micron cyclic AMP or 1 micron cyclic AMP plus 0.1 mg/ml protein kinase exhibited enhanced calcium uptake. We suggest that reversible phosphorylation of microsomal membranes may play an important role in the regulation of aortic microsomal calcium transport by cyclic AMP.
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PMID:Role of cyclic AMP in rat aortic microsomal phosphorylation and calcium uptake. 20 57

A novel non-phorbol-ester-like tumor promoter, okadaic acid (OA) has been shown to be an inhibitor of protein phosphatase I and IIA and, thus, to cause an "apparent activation" of protein kinase C (PKC). We previously showed that cis-diamminedichloroplatinum(II) (CDDP)-resistant cells, PC-9/CDDP, were cross-resistant to OA and that the cross-resistance was not due to the increased efflux of OA. We hypothesized that the phosphorylation status of some cellular proteins might be important in CDDP-resistance. No significant difference in PKC activity or total protein phosphatase activity measured in vitro was seen between PC-9 and PC-9/CDDP cells, nor in their sensitivity to inhibition by OA, nor in the amount of phosphorylation of whole cells or TCA-insoluble material. By SDS-PAGE after incubation of intact cells with 32P, we detected a marked increase, compared to PC-9 cells, in phosphorylation of the nuclear proteins of MW 32 and 20 kDa in CDDP-resistant PC-9/CDDP cells with no apparent difference in protein content. When phosphorylation of nuclear proteins observed in PC-9/CDDP cells was analyzed by 2-dimensional SDS-PAGE, the 32-kDa protein had a PI of about 4.5. The 32-kDa and 20-kDa bands were increased in a dose-dependent manner by CDDP treatment. On the other hand, no increase in phosphorylation of these proteins was observed in parental PC-9 cells. These results demonstrate a marked difference in the phosphorylation status of specific nuclear proteins between parental and CDDP-resistant cell lines, which may be related to CDDP-resistance.
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PMID:Increased phosphorylation of nuclear phosphoproteins in human lung-cancer cells resistant to cis-diamminedichloroplatinum (II). 131 Apr 90

The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDA protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [35S]ATP-gamma-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-gamma-S (1 mM) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.
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PMID:Progesterone and cAMP-dependent protein kinase regulate in vivo the level of phosphorylation of two proteins (Mr 20,000 and Mr 32,000) in Xenopus oocytes. 300 91

In liver and muscle the major active phosphorylase and synthase phosphatase activity is associated with the glycogen particle. When we examined the effect of the inhibitor-1 and modulator protein on the enzyme present in crude glycogen fractions from dog liver, the phosphorylase phosphatase was not or only slightly affected. Since the enzyme isolated from the glycogen complex by DEAE-cellulose chromatography could be inhibited by inhibitor-1 as well as the modulator protein, it was assumed that an unknown mechanism or factor present in the glycogen fraction was responsible for this reduced sensitivity of the protein phosphatase. This led to the discovery (7) of the deinhibitor protein which has now been extensively purified from dog liver. The deinhibitor protein was shown to be thermostable, ethanol- and trichloroacetic acid-resistant, but non-dialyzable and it was destroyed by pronase or trypsin. The apparent molecular weight was estimated at about 17,500 in gel filtration, 8,300 in sodium dodecyl sulfate polyacrylamide gel electrophoresis and 5,500 in sucrose density gradient centrifugation, behavior which is consistent with the assumption that the deinhibitor protein may have little ordered structure. Glycogen synthesis requires both phosphorylase and glycogen synthase as dephosphorylated enzymes. The interaction of the deinhibitor protein with the protein phosphatase brings about several effects which, when considered together, could all facilitate the dephosphorylation of glycogen synthase and phosphorylase. The protein phosphatase present in a resuspended glycogen pellet dephosphorylates inhibitor-1 in the absence of Mn2+. This ability of the phosphatase, which is lost during purification of the enzyme, can be restored upon addition of the deinhibitor protein. Owing to the association of the deinhibitor protein with the active phosphatase the enzyme becomes insensitive to inhibition by inhibitor-1 and the modulator protein, and more resistant to the conversion into the FA-ATP,Mg-dependent form, brought about by the modulator protein. During the activation of the ATP,Mg-dependent phosphatase under conditions where kinase FA is rate limiting, the deinhibitor protein increases the level without affecting the rate of activation.
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PMID:Regulation of protein phosphatase activity by the deinhibitor protein. 608 15

A major impasse to understanding the physiologic role(s) of alkaline phosphatase (ALP) is uncertainty as to its natural substrates. Various in vitro studies have led other investigators to suggest that ALP functions as a plasma membrane phosphoprotein phosphatase, consistent with our demonstration of ecto-topography of ALP in a variety of cell types. Thus, we compared the phosphorylation of plasma membrane proteins from control fibroblasts to those from profoundly ALP-deficient fibroblasts of hypophosphatasia patients. Fibroblasts from 3 controls and 3 hypophosphatasia patients (ALP activity < 4% of control) were biosynthetically labeled with 32Pi for 2 h. 32P incorporation into total trichloroacetic acid (TCA)-precipitable material was not significantly different in control and patient cells. Plasma membranes were prepared from these cells by hypotonic shock, solubilized, and subjected to two-dimensional (2-D) gel electrophoretic separation. Video densitometric analysis of silver-stained 2-D gels failed to reveal any consistent difference in the protein profile between patient vs. control fibroblasts (i.e., unique species, altered pls, or increased abundance). Autoradiography of individual 2-D gels demonstrated 63 plasma membrane phosphoproteins with molecular weights ranging from 15 to 152 kDa and predominantly acidic pls. Although several of these phosphoproteins appeared to have had donor-specific labeling, none was unique or especially abundant in the hypophosphatasia group. Thus, in ALP-deficient fibroblasts, normal incorporation of 32P into total cellular protein and into all identifiable plasma membrane phosphoproteins indicates that ALP does not modulate the phosphorylation of plasma membrane proteins.
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PMID:Evidence against a role for alkaline phosphatase in the dephosphorylation of plasma membrane proteins: hypophosphatasia fibroblast study. 822 82

Protein phosphatase inhibitor-1 (PPI-1) has been shown to be present in heart tissue and smooth muscle. Whether PPI-1 is present in cardiomyocytes is not known. The purpose of this study was to determine whether PPI-1 is present and is hormonally regulated in cardiomyocytes. A trichloroacetic acid (TCA) extract enriched in PPI-1 was isolated from guinea pig ventricular cardiomyocytes. The TCA extract inhibited the activity of type 1 protein phosphatase by 20 +/- 4% (n = 3 expts). On phosphorylation by the catalytic subunit of adenosine 3', 5'-cyclic monophosphate-dependent protein kinase, the extent of this inhibition was augmented to 4.5-fold. Dephosphorylation of the phosphorylated TCA extract by type 2 protein phosphatase reduced inhibition to 2 +/- 0.2% (n = 3 expts). To determine whether isoproterenol increases phosphorylation of PPI-1 in cardiomyocytes, the TCA extracts were prepared from cardiomyocytes treated with 1 microM isoproterenol and from untreated cardiomyocytes. The inhibitory activity of the TCA extract in untreated cardiomyocytes was 25 +/- 3% (n = 3 expts) and increased to 75 +/- 2% (n = 3 expts) in isoproterenol-treated cardiomyocytes. With the use of a rabbit skeletal muscle PPI-1 antibody, immunoblots of the TCA extract of cardiomyocytes identified a 28-kDa protein. A 28-kDa protein was also immunoprecipitated from a TCA extract isolated from isoproterenol-treated 32P-labeled cardiomyocytes. The immunoprecipitation was blocked by the addition of excess amounts of purified rabbit skeletal muscle PPI-1. Isoproterenol-treated cardiomyocytes increased the phosphorylation of the 28-kDa protein by 232 +/- 20% (n = 3 expts) compared with untreated cardiomyocytes. We conclude that 1) the 28-kDa protein is PPI-1, 2) PPI-1 is present in ventricular cardiomyocytes, and 3) PPI-1 is hormonally regulated. A decrease in type 1 protein phosphatase activity through phosphorylation of PPI-1 may be an important pathway for augmenting cardiac contractility.
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PMID:Evidence for presence and hormonal regulation of protein phosphatase inhibitor-1 in ventricular cardiomyocyte. 896 52

Calcineurin is a major player in calcium-dependent signal transduction pathways of eukaryotes. Calcineurin acts on transcription factors (e.g. CRZ1 in Saccharomyces cerevisiae) and governs the expression of genes in a species-dependent fashion. In Candida albicans, the calcineurin pathway is involved in tolerance to antifungal agents, cation homeostasis and virulence. However, the components of the calcineurin pathway are still poorly investigated in this yeast species. Taking S. cerevisiae as a model to reconstitute this pathway, two CRZ1-like genes, CRZ1 and CRZ2 (for calcineurin-responsive zinc finger 1 and 2 genes), were found with C(2)H(2) zinc finger domains. Only CRZ1 was able to restore the calcium hypersusceptibility of a S. cerevisiae crz1Delta mutant and to mediate calcium-dependent gene expression in this yeast species. Several experiments showed that CRZ1 was dependent on calcineurin in C. albicans: (i) phenotypic analysis of a crz1Delta/Delta mutant showed impaired growth as compared with the wild type in the presence of cations (Ca(2+), Mn(2+)) as does a mutant lacking calcineurin subunit A (cnaDelta/Delta) and (ii) a green fluorescent protein (GFP)-Crz1p fusion protein showed a calcium- and calcineurin-dependent nuclear localization. To further analyse the relationship between calcineurin and CRZ1, a comprehensive analysis of calcineurin/Crz1p-dependent gene expression following addition of Ca(2+) (200 mM) was performed. Among the expression of 264 genes altered by at least twofold, the upregulation of 60 genes was dependent on both calcineurin and CRZ1. Interestingly, a motif [5'-G(C/T)GGT-3'] with similarity to the target sequence of Crz1p (GNGGCG/TCA) from S. cerevisiae was identified as a putative regulatory sequence in the upstream regions of these calcineurin/Crz1p-dependent genes. However, additional experiments showed that calcineurin may have other targets in addition to CRZ1. First, CRZ1 was not involved in tolerance to antifungal agents (fluconazole, terbinafine) on the opposite to calcineurin. Second, CRZ1 was only moderately influencing virulence in a mice model of infection which is in sharp contrast to the strong avirulence of cnaDelta/Delta mutant in the same animal model. Even though this work establishes CRZ1 as a calcineurin target, further studies are needed to identify other calcineurin-dependent elements in C. albicans.
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PMID:CRZ1, a target of the calcineurin pathway in Candida albicans. 1646 87

Significant changes usually take place in the internal metabolism of insects during metamorphosis. The glycolysis-tricarboxylic acid (glycolysis-TCA) pathway is important for energy metabolism. To elucidate its dynamics, the mRNA levels of genes involved in this pathway were examined in the midgut of Spodoptera litura during metamorphosis, and the pyruvate content was quantified. The expression patterns of these genes in response to starvation were examined, and the interaction between protein phosphatase 1 (PP1) and phosphofructokinase (PFK) was studied. The results revealed that the expression or activities of most glycolytic enzymes was down-regulated in prepupae and then recovered in some degree in pupae, and all TCA-related genes were remarkably suppressed in both the prepupae and pupae. Pyruvate was enriched in the pupal midgut. Taken together, these results suggest that insects decrease both glycolysis and TCA in prepupae to save energy and then up-regulate glycolysis but down-regulate TCA in pupae to increase the supply of intermediates for construction of new organs. The expression of all these genes were down-regulated by starvation, indicating that non-feeding during metamorphosis may be a regulator of glycolysis-TCA pathway in the midgut. Importantly, interaction between PP1 and PFK was identified and is suggested to be involved in the regulation of glycolysis.
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PMID:Dynamics and regulation of glycolysis-tricarboxylic acid metabolism in the midgut of Spodoptera litura during metamorphosis. 2668 13

Alternaria alternata (Fries) Keissler is a lethal pear pathogen that causes leaf black spot disease of pear in Southern China. Heat-stable activity factor (HSAF) is a polycyclic tetramate macrolactam (PTM) produced by Lysobacter enzymogenes and many other microbes with a broad-spectrum antifungal activity against many filamentous fungi. In this study, we evaluated the antifungal effect of HSAF against A. alternata and proposed its antifungal mechanism in A. alternata. We report that HSAF inhibited the mycelial growth of A. alternata in a dose-dependent manner. Transcriptomics analysis revealed that HSAF treatment resulted in an expression alteration of a wide range of genes, with 3729 genes being up-regulated, and 3640 genes being down-regulated. Furthermore, we observed that HSAF treatment disrupted multiple signaling networks and essential cellular metabolisms in A. alternata, including the AMPK signaling pathway, sphingolipid metabolism and signaling pathway, carbon metabolism and the TCA (tricarboxylic acid) cycle, cell cycle, nitrogen metabolism, cell wall synthesis and a key hub protein phosphatase 2A (PP2A). These observations suggest that HSAF breaches metabolism networks and ultimately induces increased thickness of the cell wall and apoptosis in A. alternata. The improved understanding of the antifungal mechanism of HSAF against filamentous fungi will aid in the future identification of the direct interaction target of HSAF and development of HSAF as a novel bio-fungicide.
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PMID:Transcriptomics Analysis of the Chinese Pear Pathotype of Alternaria alternata Gives Insights into Novel Mechanisms of HSAF Antifungal Activities. 2993 28


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