CYCLOSPORINE-MEDIATED INHIBITION OF BOVINE CALCINEURIN BY CYCLOPHILIN-A AND CYCLOPHILIN-B

The Ca2+- and calmodulin-dependent protein phosphatase calcineurin is inhibited by the immunosuppressant drug cyclosporin A in the presence of cyclophilin A or B. Of the two isoforms, cyclophilin B is more potent by a factor of 2-5 when either the phosphoprotein [P-32]casein or the [P-32]phosphoserine [Ser(P-32)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide R(II), [Ser(P-32)15]R(II), is used as substrate. With [Ser(P-32(15)]R(II) as substrate, the concentrations of the cyclosporin A.cyclophilin A and cyclosporin A.cyclophilin B complexes, which cause 50 % inhibition of calcineurin activity, are 120 and 50 nM, respectively. Lowering the concentration of calcineurin 80% with [P-32]Casein as substrate lowered the apparent inhibition constant for each complex even further; 50% inhibition of calcineurin was observed at 40 nM for cyclosporin A.cyclophilin A, whereas it was < 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [P-32]casein or [Ser(P-32)15]R(II), the concentration of calcineurin required for measurable phosphatase activity is such that these complexes behave as tight-binding inhibitors of calcineurin, and steady-state kinetics cannot be used to assess inhibition patterns or K(i) values. Limited trypsinization of calcineurin produces a fragment that is still inhibited, indicating that the interaction of cyclosporin.cyclophilin with calcineurin does not require either calmodulin or Ca2+.