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 Ca2+-calmodulin-depe ndent protein phosphatase that regulates signal transduction, We have characterized CsA- or FK506-resistant mutants isolated from a CsA-FK50 6-sensitive Saccharomyces cerevisiae strain, Three mutations that conf er dominant CsA resistance are single amino acid substitutions (T350K, T350R, Y377F) in the calcineurin A catalytic subunit CMP1. One mutati on that confers dominant FK506 resistance alters a single residue (W43 0C) in the calcineurin A catalytic subunit CMP2. In vitro and in vivo, the CsA-resistant calcineurin mutants bind FKBP12-FK506 but have redu ced affinity for cyclophilin A-CsA. When introduced into the CMP1 subu nit, the FK506 resistance mutation (W388G) blocks binding by FKBP12-FK 506, but not by cyclophilin A-CsA. Go-expression of CsA-resistant and FK506-resistant calcineurin A subunits confers resistance to CsA and t o FK506 but not to CsA plus FK506, Double mutant calcineurin A subunit s (Y377F, W388C CMP1 and Y419F, W430C CMP2) confer resistance to CsA, to FK506 and to CsA plus FK506, These studies identify cyclophilin A-C sA and FKBP12-FK506 binding targets as distinct, highly conserved regi ons of calcineurin A that overlap the binding domain for the calcineur in B regulatory subunit.