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)

FK506 inhibits the evolutionarily conserved, Ca(2+)-dependent phosphatase calcineurin, which in yeast is essential for growth during sodium stress. We undertook a chemical genetic modifier screen to identify small molecules that suppress the ability of FK506 to inhibit yeast growth in high NaCl. One of these small molecule suppressors, SFK1 (suppressor of FK506 1), causes a mitochondrially induced death in low salt, concomitant with the release of reactive oxygen species. Biochemically, SFK1 interacts with Por1p, a channel protein in the outer mitochondrial membrane, suggesting that SFK1 interacts with the mitochondria directly. A genome-wide screen of yeast deletion strains for hypersensitivity to SFK1 yielded several strains with impaired mitochondrial function, as well as several with reduced sodium tolerance. Our data link ionic balance to mitochondrial function and suggest a role for calcineurin in mediating this signaling network.
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PMID:A small molecule suppressor of FK506 that targets the mitochondria and modulates ionic balance in Saccharomyces cerevisiae. 1283 85

Regulating the intracellular Na+/K+ ratio is an essential process for salinity tolerance. The yeast mutant, can, which is deficient in calcineurin, can not grow on medium containing Na+ because it is unable to regulate the intracellular Na+/K+ ratio. Expression of the STO gene of Arabidopsis thaliana in the can mutant complements the salt-sensitive phenotype. A protein of Arabidopsis, an H-protein promoter binding factor (HPPBF-1), that binds to STO protein was isolated. HPPBF-1 cDNA has a sequence encoding a Myb DNA binding-motif and its gene expression is induced by salt stress. Furthermore, HPPBF-1 protein is localized in the nucleus. Although, the expression level of STO is not induced under salt-stress conditions, overexpression of STO in a transgenic Arabidopsis plant gave it a higher salt tolerance than was observed in the wild type. When STO transgenic plants and wild-type plants were subjected to salt stress, root growth was increased by 33-70% in the transgenic plants under salt stress. These results suggest that STO is involved in salt-stress responses in Arabidopsis.
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PMID:Salt tolerance-related protein STO binds to a Myb transcription factor homologue and confers salt tolerance in Arabidopsis. 1290 88

Calcineurin is a conserved Ca(2+)-calmodulin-activated, serine/threonine-specific protein phosphatase that regulates a variety of physiological processes, e.g., cell cycle progression, polarized growth, and adaptation to salt and alkaline pH stresses. In the pathogenic yeast Cryptococcus neoformans, calcineurin is also essential for growth at 37 degrees C and virulence. To investigate whether calcineurin plays a role in the virulence of Candida albicans, the major fungal pathogen of humans, we constructed C. albicans mutants in which both alleles of the CMP1 gene, encoding the calcineurin catalytic subunit, were deleted. The C. albicans Delta cmp1 mutants displayed hypersensitivity to elevated Na(+), Li(+), and Mn(2+) concentrations and to alkaline pH, phenotypes that have been described after calcineurin inactivation in the related yeast Saccharomyces cerevisiae. Unlike S. cerevisiae calcineurin mutants, which exhibit reduced susceptibility to high Ca(2+) concentrations, growth of C. albicans was inhibited in the presence of 300 mM CaCl(2) after the deletion of CMP1, demonstrating that there are also differences in calcineurin-mediated cellular responses between these two yeast species. In contrast to C. neoformans, inactivation of calcineurin did not cause temperature sensitivity in C. albicans. In addition, hyphal growth, an important virulence attribute of C. albicans, was not impaired in the Delta cmp1 mutants under a variety of inducing conditions. Nevertheless, the virulence of the mutants was strongly attenuated in a mouse model of systemic candidiasis, demonstrating that calcineurin signaling is essential for virulence in C. albicans.
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PMID:Calcineurin is essential for virulence in Candida albicans. 1293 82

SOS2 (salt overly sensitive 2) is a serine/threonine protein kinase required for salt tolerance in Arabidopsis thaliana. In this study, we identified the protein phosphatase 2C ABI2 (abscisic acid-insensitive 2) as a SOS2-interacting protein. Deletion analysis led to the discovery of a novel protein domain of 37 amino acid residues, designated as the protein phosphatase interaction (PPI) motif, of SOS2 that is necessary and sufficient for interaction with ABI2. The PPI motif is conserved in protein kinases of the SOS2 family (i.e., protein kinase S, PKS) and in the DNA damage repair and replication block checkpoint kinase, Chk1, from various organisms including humans. Mutations in the conserved amino acid residues in the PPI motif abolish the interaction of SOS2 with ABI2. We also identified a protein kinase interaction domain in ABI2 and examined the interaction specificity between PKS and the ABI phosphatases. We found that some PKSs interact strongly with ABI2 whereas others interact preferentially with ABI1. The interaction between SOS2 and ABI2 was disrupted by the abi2-1 mutation, which causes increased tolerance to salt shock and abscisic acid insensitivity in plants. Our results establish the PPI motif and the protein kinase interaction domain as novel protein interaction domains that mediate the binding between the SOS2 family of protein kinases and the ABI1/2 family of protein phosphatases.
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PMID:A novel domain in the protein kinase SOS2 mediates interaction with the protein phosphatase 2C ABI2. 1450 88

Saccharomyces cerevisiae strains lacking the Ppz1 protein phosphatase are salt tolerant and display increased expression of the ENA1 Na(+)-ATPase gene, a major determinant for sodium extrusion, while cells devoid of the similar Ppz2 protein do not show these phenotypes. However, a ppz1 ppz2 mutant displays higher levels of ENA1 expression than the ppz1 strain. We show here that the increased activity of the ENA1 promoter in a ppz1 ppz2 mutant maps to two regions: one region located at -751 to -667, containing a calcineurin-dependent response element (CDRE), and one downstream region (-573 to -490) whose activity responds to intracellular alkalinization. In contrast, the increased ENA1 expression in a ppz1 mutant is mediated solely by an intact calcineurin/Crz1 signaling pathway, on the basis that (i) this effect maps to a single region that contains the CDRE and (ii) it is blocked by the calcineurin inhibitor FK506, as well as by deletion of the CNB1 or CRZ1 gene. The calcineurin dependence of the increased ENA1 expression of a ppz1 mutant would suggest that Ppz1 could negatively regulate calcineurin activity. In agreement with this notion, a ppz1 strain is calcium sensitive, and this mutation does not result in a decrease in the calcium hypertolerance of a cnb1 mutant. It has been shown that ENA1 can be induced by alkalinization of the medium and that a ppz1 ppz2 strain has a higher intracellular pH. However, we present several lines of evidence that show that the gene expression profile of a ppz1 mutant does not involve an alkalinization effect. In conclusion, we have identified a novel role for calcineurin, but not alkalinization, in the control of ENA1 expression in ppz1 mutants.
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PMID:Regulation of ENA1 Na(+)-ATPase gene expression by the Ppz1 protein phosphatase is mediated by the calcineurin pathway. 1455 76

Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling. We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18. [Methylsarcosine(3)]CsA was shown to inhibit calcineurin, either in its free form with an IC(50) value of 10 microm, or in its complex form with cyclophilin 18 with an IC(50) of 500 nm. [Dimethylaminoethylthiosarcosine(3)]CsA ([Dat-Sar(3)]CsA) was found to inhibit calcineurin on its own, with an IC(50) value of 1.0 microm, but was not able to inhibit calcineurin after forming the [Dat-Sar(3)]CsA-cyclophilin 18 binary complex. Despite their different inhibitory properties, both CsA and [Dat-Sar(3)]CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression. Furthermore, to demonstrate that [Dat-Sar(3)]CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Delta12), in which all the 12 cyclophilins and FKBPs were deleted. [Dat-Sar(3)]CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Delta12 strain.
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PMID:Substitution in position 3 of cyclosporin A abolishes the cyclophilin-mediated gain-of-function mechanism but not immunosuppression. 1458 19

The effects of changing cytosolic [Mg(2+)] ([Mg(2+)](i)) on L-type Ca(2+) currents were investigated in rat cardiac ventricular myocytes voltage-clamped with patch pipettes containing salt solutions with defined [Mg(2+)] and [Ca(2+)]. To control [Mg(2+)](i) and cytosolic [Ca(2+)] ([Ca(2+)](i)), the pipette solution included 30 mM citrate and 10 mM ATP along with 5 mM EGTA (slow Ca(2+) buffer) or 15 mM EGTA plus 5 mM BAPTA (fast Ca(2+) buffer). With pipette [Ca(2+)] ([Ca(2+)](p)) set at 100 nM using a slow Ca(2+) buffer and pipette [Mg(2+)] ([Mg(2+)](p)) set at 0.2 mM, peak l-type Ca(2+) current density (I(Ca)) was 17.0 +/- 2.2 pA pF(-1). Under the same conditions, but with [Mg(2+)](p) set to 1.8 mM, I(Ca) was 5.6 +/- 1.0 pA pF(-1), a 64 +/- 2.8% decrease in amplitude. This decrease in I(Ca) was accompanied by an acceleration and a -8 mV shift in the voltage dependence of current inactivation. The [Mg(2+)](p)-dependent decrease in I(Ca) was not significantly different when myocytes were preincubated with 10 microM forskolin and 300 microM 3-isobutyl-L-methylxanthine and voltage-clamped with pipettes containing 50 microM okadaic acid, to maximize Ca(2+) channel phosphorylation. However, when myocytes were voltage-clamped with pipettes containing protein phosphatase 2A, to promote channel dephosphorylation, I(Ca) decreased only 25 +/- 3.4% on changing [Mg(2+)](p) from 0.2 to 1.8 mM. In the presence of 0.2 mM[Mg(2+)](p), changing channel phosphorylation conditions altered I(Ca) over a 4-fold range; however, with 1.8 mM[Mg(2+)](p), these same manoeuvres had a much smaller effect on I(Ca). These data suggest that [Mg(2+)](i) can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca(2+)](p) to 1, 100 or 300 nM also showed that the [Mg(2+)](p)-induced reduction of I(Ca) was smaller at the lowest [Ca(2+)](p), irrespective of channel phosphorylation conditions. This interaction between [Ca(2+)](i) and [Mg(2+)](i) to modulate I(Ca) was not significantly affected by ryanodine, fast Ca(2+) buffers or inhibitors of calmodulin, calmodulin-dependent kinase and calcineurin. Thus, physiologically relevant [Mg(2+)](i) modulates I(Ca) by counteracting the effects of Ca(2+) channel phosphorylation and by an unknown [Ca(2+)](i)-dependent mechanism. The magnitude of these effects suggests that changes in [Mg(2+)](i) could be critical in regulating L-type channel gating.
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PMID:Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes. 1461 71

FK506 inhibits the Ca2+/calmodulin-dependent protein phosphatase calcineurin, which plays a critical role in yeast subjected to salt stress. A chemical genetic screen for small molecules that suppress growth inhibition by high NaCl plus FK506 identified a structurally related class of suppressors of FK506 (SFKs) named SFKs 2-4. To identify possible protein targets for these small molecules, a genome-wide screen of approximately 4,700 haploid yeast deletion strains was undertaken for strains showing resistance to high NaCl plus FK506. This screen yielded a number of genes not previously implicated in salt stress, including ALD6, which encodes an NADP(+)-dependent aldehyde dehydrogenase, and UTR1, which encodes an NAD+ kinase. Transcriptional profiling of yeast treated with SFK2 indicated that the SFKs target the Ald6p pathway. In addition, screening of the deletion strains for hypersensitivity to SFK2 yielded ZWF1, encoding glucose-6-phosphate dehydrogenase, which has been shown to play an overlapping role with Ald6p in NADPH production. Furthermore, the SFKs inhibited the activity of Ald6p in vitro. Having established that the SFKs target Ald6p, they were used as tools to implicate systematically other gene products in the Ald6p pathway, including Utr1p, which may function by supplying Ald6p with its NADP+ cofactor. Furthermore, growth improvement by the SFKs on high NaCl plus FK506 was shown to require GPD1, which encodes an NADH-dependent glycerol-3-phosphate dehydrogenase that is important for the production of glycerol in response to osmotic stress.
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PMID:Identification of Ald6p as the target of a class of small-molecule suppressors of FK506 and their use in network dissection. 1514 68

The yeast gene VHS3 (YOR054c) has been recently identified as a multicopy suppressor of the G(1)/S cell cycle blockade of a conditional sit4 and hal3 mutant. Vhs3 is structurally related to Hal3, a negative regulatory subunit of the Ser/Thr protein phosphatase Ppz1 important for cell integrity, salt tolerance, and cell cycle control. Phenotypic analyses using vhs3 mutants and overexpressing strains clearly show that Vhs3 has functions reminiscent to those of Hal3 and contrary to those of Ppz1. Mutation of Vhs3 His(459), equivalent to the supposedly functionally relevant His(90) in the plant homolog AtHal3a, did not affect Vhs3 functions mentioned above. Similarly to Hal3, Vhs3 binds in vivo to the C-terminal catalytic moiety of Ppz1 and inhibits in vitro its phosphatase activity. Therefore, our results indicate that Vhs3 plays a role as an inhibitory subunit of Ppz1. We have found that the vhs3 and hal3 mutations are synthetically lethal. Remarkably, lethality is not suppressed by deletion of PPZ1, PPZ2, or both phosphatase genes, indicating that it is not because of an excess of Ppz phosphatase activity. Furthermore, a Vhs3 version carrying the H459A mutation did not rescue the synthetically lethal phenotype. A conditional vhs3 tetO:HAL3 double mutant displays, in the presence of doxycycline, a flocculation phenotype that is dependent on the presence of Flo8 and Flo11. These results indicate that, besides its role as Ppz1 inhibitory subunit, Vhs3 (and probably Hal3) might have important Ppz-independent functions.
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PMID:Functional characterization of the Saccharomyces cerevisiae VHS3 gene: a regulatory subunit of the Ppz1 protein phosphatase with novel, phosphatase-unrelated functions. 1519 4

PKR is a cellular protein kinase activated by double-stranded RNA (dsRNA) that phosphorylates eukaryotic initiation factor alpha (eIF2alpha) and inhibits protein translation. Activation of PKR is accompanied by Ser/Thr autophosphorylation on multiple sites. Because PKR negatively regulates cell growth, overexpression and purification of PKR are difficult to achieve. Here, we describe overexpression and purification of recombinant PKR protein from Escherichia coli under native conditions at the milligram level. Affinity, ion exchange, and gel filtration chromatographies revealed multiple fractions of PKR with distinctive biochemical characteristics. During gel filtration, a small amount of PKR was found in a high molecular weight (>300 kDa) fraction that also contained endogenous bacterial RNA. The PKR in this fraction has a constitutive substrate phosphorylation activity. The majority of PKR is found in fractions of lower molecular weight and is free of RNA but is differentially phosphorylated as examined by isoelectric focusing electrophoresis and can be further separated by gradient anion exchange chromatography. PKR eluted with low salt has a lower level of basal autophosphorylation, and its kinase activity can be induced by dsRNA. With an increasing NaCl gradient, the purified PKR exhibits an increased level of autophosphorylation and constitutive kinase activity but reduced dsRNA inducibility. The highest salt eluent of PKR exhibits little dsRNA-induced activation. The inducible activation of high salt eluent PKR by dsRNA can be partially restored by treatment with protein phosphatase 1. The production of multiple fractions of PKR with different biochemical properties in E. coli suggests that the spectrum of PKR activity and regulation in mammalian cells is likely to be similarly complex.
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PMID:Biochemical analyses of multiple fractions of PKR purified from Escherichia coli. 1545 Jan 28


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