Only in the Presence of Immunophilins Can Cyclosporine and FK506 Disrupt in Vivo Binding of Calcineurin A to Its Autoinhibitory Domain yet Strengthen Interaction Between Calcineurin A and B Subunits

Abstract

The two immunosuppressants cyclosporin A (CsA) and FK506 exert their major therapeutic effect by inhibiting T-cell activation. It is believed that the drugs first bind to their cellular receptors, known as immunophilins, and then target the protein phosphatase calcineurin for inhibition. The catalytic activity of calcineurin is regulated by its autoinhibitory domain (AID) and by the calcium-binding proteins calcineurin B (CnB) and calmodulin. We have used the yeast two-hybrid system to show that AID, CnB and calmodulin can only bind to a truncated catalytic subunit of yeast calcineurin (i.e., CnA1Δ), devoid of AID, but not to full-length CnA1. Both CsA and FK506 cause disruption of the CnAlΔ-AID interaction,whereas their presence permits CnAΔ1to bind more strongly to CnB. In contrast, the binding of CnA1Δ to calmodulin is unaffected by the immunosuppressants. Significantly, in the absence of its cognate cytosolic receptor, neither CsA nor FK506 inhibits or stimulates the CnA1Δ-AID, CnA1Δ-CnB interactions. These in vivo observations not only provide supportive evidence for the mechanism by which drug-receptor complexes could modulate calcineurin activity but also unveil the possibility of identifying novel immunophilin-independent calcineurin inhibitors which may disturb the association of CnA1Δ to AID.