Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A.
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.
Cardenas
M E
ME
Department of Genetics, Duke University Medical Center, Durham, NC 27710, USA.
Muir
R S
RS
Breuder
T
T
Heitman
J
J
D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics*; D016572:Cyclosporine / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D005816:Genetic Complementation Test; D006360:Heat-Shock Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D016559:Tacrolimus / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000591:Amino Acid Isomerases; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus