Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A

EMBO J. 1995 Jun 15;14(12):2772-83.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Isomerases / genetics
  • Amino Acid Isomerases / metabolism
  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Calcineurin
  • Calmodulin-Binding Proteins / genetics
  • Calmodulin-Binding Proteins / metabolism*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Conserved Sequence / genetics*
  • Cyclosporine / 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
  • Phosphoprotein Phosphatases / metabolism*
  • Point Mutation / physiology
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / genetics
  • Tacrolimus / metabolism*
  • Tacrolimus Binding Proteins

Substances

  • Calmodulin-Binding Proteins
  • Carrier Proteins
  • DNA-Binding Proteins
  • Heat-Shock Proteins
  • Recombinant Fusion Proteins
  • Cyclosporine
  • Calcineurin
  • Phosphoprotein Phosphatases
  • Amino Acid Isomerases
  • Tacrolimus Binding Proteins
  • Peptidylprolyl Isomerase
  • Tacrolimus