Abstract
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.
Highlights
The finding that calcineurin is a common target for both the very dissimilar complexes CsA-CypA and FK506-FKBP12 [13] has provided a quantum leapin our understanding of the mechanism of action
Main intracellular recep- ing site [25],an autoinhibitory domain (261, which is a typical tors to which these drugsbind with high affinity are a growing feature of calmodulin-dependent enzymes, and a putativesubfamily of proteins collectively named immunophilins: cyclophi- unit B binding site [27] have been identified within the large lins for cyclosporin and FK506 binding proteins (FKBPs)’ for subunit A
The eluted material was concentrated hy ultrafiltration ence of cyclophilinbecause no signalscanhedetectedafter photolysis in the absence of the latter at equimolar concrntration. These results suggest that the interaction sitefor the solvent-c.xposcd domain of thc. immunophilin-bound cyclosporin lies a t o r very near the interface between the two calcineurin subunits
Summary
Cinmrrin-The nature of the calcineurin-immunophilin-ligand interactionwasfurtherinvestigated by means of chemical. In thc for thecharacterization of cyclosporin-bindingproteinshas experiment shown, 50 pmol of purifid bovine hrain been described [36].The recent structural studies on the cy- calcineurin were incuhated with equimolar concentrations of closporin A-cyclophilin A complex [18] showedposition 8 of either CypA, CypR, CypC, or FKRP in the presence of their cyclosporin not to be buried within the cyclophilin molecule, cognate ligands CsAor FK506 and treated with0.1 mv DSS as and provided a longflexiblesidechain is introduced at this described under “Experimental Procedures.”. C specific immunophilin ligands, as is best exemplified in lone I , where FKRP and calcineurin were cross-linked in the absence of FK506. Cross-linking nf Co/rinc~rrrinR t o ~ r l ~ r l ~ r r r ~ o p h iI /ni tt~h .os/'rvsk n a visible in Fig. 3 ( C and D, l o w 1 ) correspondstothe cnce of Short Rccomhinnnt C'olcinvurIn ASlrc ~ l ~ ~ i ~ c ~ . ~ - - l ~ ~ ~ c ; l u . s c
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