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)

Calcineurin, or phosphoprotein phosphatase type 2B (PP2B), is a calmodulin-regulated phosphoprotein phosphatase. We isolated a gene encoding a yeast PP2B homolog (CNA1) by screening a yeast genomic DNA library in the expression vector lambda gt11, first with 125I-labeled yeast calmodulin and then with a human cDNA encoding the catalytic (or A) subunit of calcineurin. The predicted CNA1 gene product is 54% identical to its mammalian counterpart. Using the polymerase chain reaction (PCR) with oligonucleotide primers based on sequences conserved between CNA1 and mammalian PP2B genes, we isolated a second gene, CNA2. CNA2 is identical to PP2Bw, a partial cDNA clone previously described by others as originating from rabbit brain tissue. Our findings demonstrate that a unicellular eukaryote contains phosphoprotein phosphatases of the 2B class. Haploid cells containing a single cna1 or cna2 null mutation, or both mutations, were viable. MATa cna1 cna2 double mutants were more sensitive than wild-type cells or either single mutant to growth arrest induced by the mating pheromone alpha factor and failed to resume growth during continuous exposure to alpha factor. Thus, calcineurin action antagonizes the mating-pheromone response pathway.
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PMID:Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase. 165 3

The immunosuppressants FK506 and cyclosporin A (CsA) bound to their receptors, FKBP12 or cyclophilin, inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, preventing T cell activation or, in yeast, recovery from alpha-mating factor arrest. Vegetative growth of yeast does not require calcineurin, and in strains sensitive to FK506 or CsA, growth is inhibited by concentrations of drug much higher than those required to inhibit T cell activation or recovery from mating factor arrest. We now describe the isolation of a mutant of Saccharomyces cerevisiae which is 100-1000-fold more sensitive to the growth inhibitory properties of these drugs. The mutation (fks1) also confers a slow growth phenotype which is partially suppressed by exogenously added Ca2+ and exacerbated by EGTA. Simultaneous disruption of the two genes (CNA1 and CNA2) encoding the alternative forms of the catalytic A subunit of calcineurin, or of the gene (CNB1) encoding the regulatory B subunit, is lethal in an fks1 mutant. Disruption of the gene encoding FKBP12 (FKB1) or the major, cytosolic cyclophilin (CPH1) in fks1 cells results in the loss of hypersensitivity to the relevant drug. Overexpression of CNA1 or CNA2, in conjunction with CNB1, results in a significant decrease in hypersensitivity to FK506 and CsA. The results show that the hypersensitivity of the fks1 mutant is due to the inhibition of calcineurin phosphatase activity by the receptor-drug complexes. The growth dependence of the mutant on the Ca2+/calcineurin signal pathway provides an important tool for studying in yeast certain aspects of immune suppression by these drugs.
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PMID:Calcineurin-dependent growth of an FK506- and CsA-hypersensitive mutant of Saccharomyces cerevisiae. 751 Mar 23

The Ca(2+)-calmodulin dependent protein phosphatase, calcineurin, is thought to mediate the action of the two immunosuppressants, cyclosporin A (CsA) and FK506. Calcineurin from all species consists of a catalytic A subunit and a regulatory peptide B, which plays an essential role in catalysis. The enzymatic function is probably also regulated by an autoinhibitory domain (AID) present in the catalytic subunit. We have used the yeast two-hybrid system to show that the putative AID of the yeast catalytic subunit Cna1 binds only to truncated Cna1, devoid of AID. Although deletion of the genes encoding the yeast catalytic subunits of calcineurin (CNA1 and CNA2) maintain the interaction, absence of the regulatory subunit Cnb1 prevents binding. Interestingly, both CsA and FK506 disrupt this interaction, whereas binding of Cna1 to calmodulin remains unaffected. This indicates that a simple cellular system, developed in yeast, could provide further insight into an understanding of calcineurin inhibition.
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PMID:The interaction between the catalytic A subunit of calcineurin and its autoinhibitory domain, in the yeast two-hybrid system, is disrupted by cyclosporin A and FK506. 752 90

FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca(2+)-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100-1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973-2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combination with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.
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PMID:The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth. 753 Feb 27

Calcineurin (also called protein phosphatase-2B) is a calmodulin-regulated protein phosphatase which plays an important role in signal transduction. The enzyme is a heterodimer of a 58-59 kDa calmodulin-binding catalytic subunit (calcineurin A) and a small (i.e. 19 kDa) Ca(2+)-binding regulatory subunit (calcineurin B). The highly conserved calcineurin B is encoded by a single gene in all tissues except testes, whereas there are three isoforms of calcineurin A (alpha, beta and gamma) encoded by genes on three different chromosomes. This enzyme can play a critical role in transcriptional regulation and growth control in T lymphocytes by a mechanism believed to involve dephosphorylation of the nuclear factor NF-AT which is essential for transcription of the interleukin-2 gene. To better evaluate the potential role of the calcineurin genes in human genetic disorders, we have studied their chromosome locations. Calcineurin B (PPP3R1) is located on human chromosome 2p16-->p15 and calcineurin A beta (PPP3CB, previous gene symbol CALNB) is present on 10q21-->q22. We confirm the localization of calcineurin A alpha (PPP3CA, previous gene symbol CALNA) to chromosome 4 without regional localization.
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PMID:Calcineurin A alpha (PPP3CA), calcineurin A beta (PPP3CB) and calcineurin B (PPP3R1) are located on human chromosomes 4, 10q21-->q22 and 2p16-->p15 respectively. 897 85

The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit of calcineurin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction of the target function had been achieved in antisense transformants with multiple construct integrations. Induction conditions caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the distinctive tip-high Ca2+ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for calcineurin in the precise regulation of apical growth, a common form of cellular proliferation. In vitro inhibition of N. crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants to the calcineurin-specific drugs CsA and FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype very similar to that of the cna-1 antisense mutants is consistent with this idea.
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PMID:Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient. 934 1

Calcineurin a calmodulin-dependent phosphatase plays a critical role in calcium-dependent activation of T-lymphocytes and is the major target for the inhibitory actions of the immunosuppressive drugs Tacrolimus (FK506) and Cyclosporin A (CsA). Calcineurin is a dimeric protein consisting of distinct A (catalytic) and B (regulatory) subunits. In humans two separate genes, CNA1 and CNA2, encode the calcineurin A (CNA) subunit. The region of CNA that interacts with Calcineurin B, calmodulin, and immunosuppressive drugs bound to their receptors--the immunophilins--has been identified to amino acids 281-414 (Greengard P, Allen PB, Nairin AC. Beyond the dopamine receptor: the DARPP-32/protein phosphatase-1 cascade. Neuron 1999;23:435). Our working hypothesis was that the differences in patient response to calcineurin inhibitors could be a consequence of inherited variations within their CNA genes. Single-strand conformational polymorphism (SSCP) analysis of cDNAs derived from the coding region for amino acids 281-414 of CNA1 and CNA2 in 32 healthy Caucasians did not detect polymorphic variations within these genes. These results suggest that this region is highly conserved and cannot account for individual variation in response of patients to FK506 and CsA treatment.
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PMID:Genetic conservation of the immunophilin-binding domains of human calcineurin A1 and A2. 1100 20

Antisense repression was used as a method to alter gene function in the human-pathogenic fungus Cryptococcus neoformans. The calcineurin A gene (CNA1) and the laccase gene (LAC1) were targeted since disruption of these loci results in phenotypes that are easy to screen (temperature sensitivity and lack of melanin, respectively). Serotype D yeasts were transformed with a plasmid containing the CNA1 cDNA in an antisense orientation under the control of the inducible GAL7 promoter, and serotype A yeasts were transformed with a plasmid containing the LAC1 cDNA in an antisense orientation under the control of the constitutive actin promoter. The calcineurin transformants demonstrated a temperature-sensitive phenotype only when grown on galactose, and the laccase transformants had decreased melanin production. Northern blot analysis of the calcineurin antisense transformants confirmed that the inducible phenotype was associated with a decrease in the native CNA1 transcript levels. Furthermore, it was possible to modestly impair growth of C. neoformans at 37 degrees C by using a 30 bp antisense oligonucleotide targeting CNA1. Antisense repression is now available as a tool for molecular studies in this organism, and may be applicable to other human-pathogenic fungi that have less amenable genetic systems.
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PMID:Antisense repression in Cryptococcus neoformans as a laboratory tool and potential antifungal strategy. 1178 13

CNA1 and CNA2 encode isoforms of the catalytic subunit of calcineurin, a Ser/Thr-specific phosphoprotein phosphatase regulated by Ca(2+)/calmodulin. The relative abundance of both transcripts was evaluated during growth of Saccharomyces cerevisiae in glucose by reverse-transcription polymerase chain reaction using PDA1 mRNA as a novel internal standard. CNA1 and CNA2 were concomitantly transcribed with different average expression ratios at the exponential and stationary growth phases and both showed a remarkable drop in the expression at diauxie. Prolonged hyper-osmotic shock resulted in a moderate induction of CNA1, whereas CNA2 expression was not affected.
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PMID:Expression of the yeast calcineurin subunits CNA1 and CNA2 during growth and hyper-osmotic stress. 1272 27

A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals.
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PMID:Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis. 1915 25


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