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 peptidyl-prolyl isomerases FKBP12 and cyclophilin A (immunophilins) form complexes with the immunosuppressants FK506 and cyclosporin A that inhibit the phosphatase calcineurin. With the yeast two hybrid system, we detect complexes between FKBP12 and the calcineurin A catalytic subunit in both the presence and absence of FK506. Mutations in FKBP12 surface residues or the absence of the calcineurin B regulatory subunit perturb the FK506-dependent, but not the ligand-independent, FKBP12-calcineurin complex. By affinity chromatography, both FKBP12 and cyclophilin A bind calcineurin A in the absence of ligand, and FK506 and cyclosporin A respectively potentiate these interactions. Both in vivo and in vitro, the peptidyl-prolyl isomerase active sites are dispensable for ligand-independent immunophilin-calcineurin complexes. Lastly, by genetic analyses we demonstrate that FKBP12 modulates calcineurin functions in vivo. These findings reveal that immunophilins interact with calcineurin in the absence of exogenous ligands and suggest that immunosuppressants may take advantage of the inherent ability of immunophilins to interact with calcineurin.
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PMID:Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands. 752 75

Several disciplines, including chemical ecology, seek to understand the molecular basis of information transfer in biological systems, and general molecular strategies are beginning to emerge. Often these strategies are discovered by a careful analysis of natural products and their biological effects. Cyclosporin A, FK506, and rapamycin are produced by soil microorganisms and are being used or considered as clinical immunosuppressive agents. They interrupt the cytoplasmic portion of T-cell signaling by forming a complex with a binding protein--FKBP12 in the case of FK506 and rapamycin and cyclophilin A (CyPA) in the case of cyclosporin A (CsA). This complex in turn inhibits a protein target, and the best understood target is calcineurin, which is inhibited by FK506-FKBP12 and CyPA-CsA. Mutational and structural studies help define how FK506-FKBP12 interacts with calcineurin, and the results of these studies are summarized. The existence of strong FK506-FKBP12 binding suggests that FK506 is mimicking some natural ligand for FKBP12. Synthetic and structural studies to probe this mimicry are also described.
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PMID:The chemistry of signal transduction. 752 14

FKBP12 is an 11.8-kDa protein that binds the potent immunosuppressants FK506 and rapamycin. When bound to FK506, FKBP12 forms an inhibitory complex with calcineurin and interferes with signal transduction in activated T lymphocytes. In studying human FKBP12 cDNAs and the human FKBP12 gene, we found that three distinct transcripts can encode human FKBP12. The transcripts, which we designate FKBP 12A, 12B and 12C, contain identical open reading frames, but vary in abundance and are distinguished by unique 3' untranslated regions. The mature transcripts derive from either four or five exons and are generated by the differential use of one splice junction and three cleavage-polyadenylation sites within FKBP12. FKBP12A and 12B populations increase in abundance and/or stability when T-cell populations are mitogenically activated in vitro, implying that one result of T-cell stimulation is increased demand for the FKBP12 message. These transcripts are also present in a variety of human tissues, suggesting that FKBP12 and/or the mRNAs encoding it might affect physiological function(s) in a diverse array of cells.
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PMID:Three distinct messenger RNAs can encode the human immunosuppressant-binding protein FKBP12. 752 39

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

FK506 and cyclosporin A (CsA) are immunosuppressive agents that inhibit IL-2 production by activated T cells, but only CsA inhibits IgE activation-induced cytokine transcripts in mouse IL-3-dependent, bone marrow-derived mast cells (BMMC). We previously associated the resistance of BMMC to FK506 with a deficiency in the expression of FK506 binding protein (FKBP) 12, a molecule that forms a complex with FK506 capable of inhibiting calcineurin phosphatase activity in vitro. In this report, we establish that FKBP12 mediates FK506 inhibition of both calcineurin phosphatase activity and IgE activation-induced cytokine transcripts in a Kirsten murine sarcoma virus-immortalized mast cell line that is FKBP12 deficient. Overexpression of FKBP12 by transfection enhanced the ability of FK506 to inhibit calcineurin phosphatase activity (IC50 = 2 nM), compared with cells transfected with the expression vector alone (IC50 > 30 nM). The IC50 value for FK506 inhibition of IgE activation-induced transcripts for TNF-alpha decreased from 40 nM in vector control cells to 10 nM in FKBP12 transfectants. Similarly, the IC50 value for inhibition of IL-6 transcripts decreased from > 1000 nM in vector control cells to 35 nM in FKBP12 transfectants. In contrast, activation-elicited release of the secretory granule mediator beta-hexosaminidase was only partially inhibited by FK506 at 1000 nM, regardless of the levels of FKBP12 expressed by the cells. Thus, FKBP12 is the dominant cytosolic protein that mediates FK506 inhibition of TNF-alpha and IL-6 transcripts.
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PMID:The complex of FK506-binding protein 12 and FK506 inhibits calcineurin phosphatase activity and IgE activation-induced cytokine transcripts, but not exocytosis, in mouse mast cells. 753 Jul 43

The immunosuppressive drugs FK506 and rapamycin bind to a family of intracellular proteins termed FK506-binding proteins (FKBP). FK506 and rapamycin inhibit lymphocyte-activation pathways by forming complexes with an FKBP; subsequently, the drug/FKBP complexes interact with target molecules involved in signal transduction. A key target of FK506/FKBP12 complexes is calcineurin, a calcium- and calmodulin-dependent serine/threonine phosphatase. In mammalian cells, rapamycin treatment is associated with inhibition of the activity of several cellular serine/threonine kinases, including p70 S6 kinase. These kinases may function in signaling pathways involving TOR gene producs, which have been shown to interact with rapamycin/FKBP12 complexes in vitro. To determine if FKBP12 mediates the effects of both FK506 and rapamycin in mammalian cells, we overexpressed FKBP12 in a murine mast cell line. Increased expression of FKBP12 resulted in increased sensitivity to FK506 and rapamycin, as measured by inhibition of calcineurin activity and p70 S6 kinase activity, respectively. In contrast, overexpression of FKBP25 had no effect on sensitivity to either drug. Two distinct point mutations in FKBP12, one altering a hydrophobic residue within the drug-binding pocket and the other changing a charged surface residue of FKBP12, abrogated its ability to mediate sensitivity to FK506 and rapamycin. These results establish that FKBP12 can mediate sensitivity to both FK506 and rapamycin in mammalian cells.
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PMID:FK506 binding protein 12 mediates sensitivity to both FK506 and rapamycin in murine mast cells. 753 90

The immunosuppressive complexes cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 inhibit calcineurin, a heterodimeric Ca(2+)-calmodulin-dependent protein phosphatase that regulates signal transduction. We have characterized CsA- or FK506-resistant mutants isolated from a CsA-FK506-sensitive Saccharomyces cerevisiae strain. Three mutations that confer dominant CsA resistance are single amino acid substitutions (T350K, T350R, Y377F) in the calcineurin A catalytic subunit CMP1. One mutation that confers dominant FK506 resistance alters a single residue (W430C) in the calcineurin A catalytic subunit CMP2. In vitro and in vivo, the CsA-resistant calcineurin mutants bind FKBP12-FK506 but have reduced affinity for cyclophilin A-CsA. When introduced into the CMP1 subunit, the FK506 resistance mutation (W388C) blocks binding by FKBP12-FK506, but not by cyclophilin A-CsA. Co-expression of CsA-resistant and FK506-resistant calcineurin A subunits confers resistance to CsA and to FK506 but not to CsA plus FK506. Double mutant calcineurin A subunits (Y377F, W388C CMP1 and Y419F, W430C CMP2) confer resistance to CsA, to FK506 and to CsA plus FK506. These studies identify cyclophilin A-CsA and FKBP12-FK506 binding targets as distinct, highly conserved regions of calcineurin A that overlap the binding domain for the calcineurin B regulatory subunit.
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PMID:Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A. 754 Sep 76

Calcineurin is a calcium-dependent protein phosphatase that plays a pivotal role in antigen-stimulated T cell activation. The complexes formed between the immunosuppressants cyclosporin A and FK506 and their respective intracellular binding proteins (immunophilins) block T cell activation by binding to calcineurin. Recent studies have shown that the immunophilin-immunosuppressant complexes interact with the latch region of the calcineurin B subunit (Milan, D., Griffith, J., Su, M., Price, E. R., and McKeon, F. (1994) Cell 79, 437-447). Mutations in the B subunit-binding domain of the calcineurin A subunit result in a reduction of calcineurin activity that correlates with B binding affinity. Calcineurin A subunit mutants D348A, F350A, W352A, S353A, and E359A lost greater than 90% of their activity to activate the transcription factor NF kappa B in Jurkat T cells. Furthermore, calcineurin A subunit mutants of residues Thr351, Leu354, and Lys360 showed NF kappa B transactivation activity and phosphatase activity with increased resistance to FKBP12-FK506 but displayed no or minimal increase in resistance for cyclosporin A inhibition. Together, these results strongly suggest that the B subunit-binding domain is required for calcineurin activity intracellulary and interacts with the FKBP12-FK506 complex.
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PMID:Interaction of FKBP12-FK506 with calcineurin A at the B subunit-binding domain. 754 Oct 44

The X-ray structure of the ternary complex of a calcineurin A fragment, calcineurin B, FKBP12, and the immunosuppressant drug FK506 (also known as tacrolimus) has been determined at 2.5 A resolution, providing a description of how FK506 functions at the atomic level. In the structure, the FKBP12-FK506 binary complex does not contact the phosphatase active site on calcineurin A that is more than 10 A removed. Instead, FKBP12-FK506 is so positioned that it can inhibit the dephosphorylation of its macromolecular substrates by physically hindering their approach to the active site. The ternary complex described here represents the three-dimensional structure of a Ser/Thr protein phosphatase and provides a structural basis for understanding calcineurin inhibition by FKBP12-FK506.
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PMID:X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. 754 69

Calcineurin is a heterodimeric Ca2+/calmodulin-dependent protein phosphatase that regulates signal transduction and is the target of immunophilin-immunosuppressive drug complexes in T-lymphocytes and in yeast. Calcineurin is composed of a catalytic A subunit and a regulatory B subunit that is myristoylated at its amino terminus. We employed genetic and biochemical approaches to investigate the functional roles of myristoylation of calcineurin B (CNB1) in Saccharomyces cerevisiae. A calcineurin B mutant in which glycine 2 was substituted by alanine (CNB1-G2A) did not incorporate [3H]myristate when expressed in yeast. Both wild-type calcineurin B and the CNB1-G2A mutant protein are partially associated with membranes and cytoskeletal structures; hence, myristoylation is not required for these associations. In several independent genetic assays of calcineurin functions (recovery from alpha-factor arrest, survival during cation stress, and viability of a calcineurin-dependent strain), the nonmyristoylated CNB1-G2A mutant protein exhibited full biological activity. In vitro, both wild-type and CNB1-G2A mutant proteins formed complexes with both cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 that contained calcineurin A. Interestingly, expression of the nonmyristoylated CNB1-G2A mutant protein rendered yeast cells partially resistant to the immunosuppressant CsA, but not to FK506. This study demonstrates that calcineurin B myristoylation is not required for function, but may participate in inhibition by the cyclophilin A-CsA complex.
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PMID:Myristoylation of calcineurin B is not required for function or interaction with immunophilin-immunosuppressant complexes in the yeast Saccharomyces cerevisiae. 755 4


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