Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B.
The Ca(2+)- 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 [32P]casein or the [32P]phosphoserine [Ser(32P)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide RII, [Ser(32P)15]RII, is used as substrate. With [Ser(32P15]RII 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 [32P]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 less than 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [32P]casein or [Ser(32P)15]RII, 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 Ki 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+.
Swanson
S K
SK
Section of Hematology Research, Mayo Clinic and Foundation, Rochester, MN 55905.
Born
T
T
Zydowsky
L D
LD
Cho
H
H
Chang
H Y
HY
Walsh
C T
CT
Rusnak
F
F
D000591:Amino Acid Isomerases / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D016572:Cyclosporine / Q000378:metabolism / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D019696:Peptidylprolyl Isomerase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D014357:Trypsin; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase