RESUMEN
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.
Asunto(s)
Proteínas de Unión a Calmodulina/metabolismo , Secuencia Conservada/genética , Ciclosporina/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Tacrolimus/metabolismo , Isomerasas de Aminoácido/genética , Isomerasas de Aminoácido/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Calcineurina , Proteínas de Unión a Calmodulina/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Farmacorresistencia Microbiana/genética , Genes Fúngicos/genética , Prueba de Complementación Genética , Proteínas de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil , Fosfoproteínas Fosfatasas/genética , Mutación Puntual/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Unión a TacrolimusAsunto(s)
Antifúngicos/farmacología , Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Ciclosporina/farmacología , Proteínas de Choque Térmico/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Tacrolimus/farmacología , Isomerasas de Aminoácido/biosíntesis , Isomerasas de Aminoácido/genética , Animales , Calcineurina , Proteínas de Unión a Calmodulina/biosíntesis , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Farmacorresistencia Microbiana , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Mamíferos , Modelos Biológicos , Mutagénesis , Isomerasa de Peptidilprolil , Fosfoproteínas Fosfatasas/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a TacrolimusRESUMEN
The immunophilin-immunosuppressant complexes cyclophilin-cyclosporin A (CsA) and FKBP12-FK506 inhibit the phosphatase calcineurin to block T-cell activation. Although cyclophilin A, FKBP12, and calcineurin are highly conserved from yeast to man, none had previously been shown to be essential for viability. We find that CsA-sensitive yeast strains are FK506 hypersensitive and demonstrate that calcineurin is required for viability in these strains. Mutants lacking cyclophilin A or FKBP12 are resistant to CsA or FK506, respectively. Thus, both the immunosuppressive and the antifungal actions of CsA and FK506 result from calcineurin inhibition by immunophilin-drug complexes. In yeast strains in which calcineurin is not essential, calcineurin inhibition or mutation of calcineurin confers hypersensitivity to LiCl or NaCl, suggesting that calcineurin regulates cation transport.