ABSTRACT
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.
Subject(s)
Calmodulin-Binding Proteins/metabolism , Conserved Sequence/genetics , Cyclosporine/metabolism , Phosphoprotein Phosphatases/metabolism , Tacrolimus/metabolism , Amino Acid Isomerases/genetics , Amino Acid Isomerases/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Calcineurin , Calmodulin-Binding Proteins/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA Mutational Analysis , DNA-Binding Proteins/metabolism , Drug Resistance, Microbial/genetics , Genes, Fungal/genetics , Genetic Complementation Test , Heat-Shock Proteins/metabolism , Molecular Sequence Data , Peptidylprolyl Isomerase , Phosphoprotein Phosphatases/genetics , Point Mutation/physiology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Tacrolimus Binding ProteinsSubject(s)
Antifungal Agents/pharmacology , Calmodulin-Binding Proteins/antagonists & inhibitors , Carrier Proteins/metabolism , Cyclosporine/pharmacology , Heat-Shock Proteins/metabolism , Phosphoprotein Phosphatases/antagonists & inhibitors , Saccharomyces cerevisiae/drug effects , Tacrolimus/pharmacology , Amino Acid Isomerases/biosynthesis , Amino Acid Isomerases/genetics , Animals , Calcineurin , Calmodulin-Binding Proteins/biosynthesis , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Drug Resistance, Microbial , Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/genetics , Mammals , Models, Biological , Mutagenesis , Peptidylprolyl Isomerase , Phosphoprotein Phosphatases/biosynthesis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Tacrolimus Binding ProteinsABSTRACT
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.
