Mapping of the immunophilin-immunosuppressant site of interaction on calcineurin.
The interaction of the immunosuppressive complexes cyclosporin A-cyclophilin A and FK506 binding protein-FK506 with the Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin has been investigated by means of photoaffinity labeling and chemical cross-linking. Photolabeling of purified bovine brain calcineurin with the affinity label [O-[4-[4-(1-diazo-2,2,2-trifluoroethyl)benzoyl]aminobutanoyl]-D- serine8]cyclosporin in the presence of cyclophilin A results, in addition to the labeling of cyclophilin itself, in the transfer of some of the chemical probe to both the catalytic subunit A and the regulatory subunit B of calcineurin. Chemical cross-linking studies with disuccinimidyl suberate in the presence of either cyclophilin A, B, or C in complex with cyclosporin A or FK506 binding protein-FK506 result on the other hand in the apparently exclusive and strictly immunosuppressant-dependent formation of covalent immunophilin-calcineurin B subunit products. Cross-linking of immunophilins to calcineurin B subunit requires the presence of subunit A. In the present study, using a set of recombinant maltose-binding protein fusion products representing different stretches of the catalytic subunit A, we were able to map the minimal calcineurin A sequence necessary for immunophilin-ligand-calcineurin B interaction to occur.
Husi
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Laboratory of Biochemistry, Sandoz Pharma Ltd, Basel, Switzerland.
Luyten
M A
MA
Zurini
M G
MG
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents; D016572:Cyclosporine / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007166:Immunosuppressive Agents / Q000378:metabolism*; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010777:Photochemistry; D011994:Recombinant Proteins / Q000378:metabolism; D016559:Tacrolimus / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins
D000345:Affinity Labels; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D006360:Heat-Shock Proteins; D007166:Immunosuppressive Agents; D011994:Recombinant Proteins; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus