COMPETITIVE-INHIBITION OF CALCINEURIN PHOSPHATASE-ACTIVITY BY ITS AUTOINHIBITORY DOMAIN

Carcineurin (protein phosphatase 2B), a calmodulin and calcium-depende nt serine/threonine phosphatase, appears to be regulated by a C-termin al autoinhibitory domain. A 25 amino acid peptide derived from this do main inhibits calcineurin phosphatase activity in vitro. Here we show that a 97 amino acid fragment of the calcineurin A alpha C-terminus is approx. 8-fold more potent than the shorter peptide in calcineurin in hibition experiments. Mutation of an evolutionarily conserved Asp to A sn, previously shown to disrupt calcium-dependent signalling and calci neurin regulation in T-lymphocytes, greatly reduced inhibition by the autoinhibitory domain in vitro. Kinetic analysis of wild-type and muta ted autoinhibitory domains show that both are competitive inhibitors o f calcineurin phosphatase activity with K-i values of 5.0+/-0.2 mu M a nd 36.0+/-3.7 mu M respectively. This suggests intrasteric regulation of calcineurin, with the autoinhibitory domains interacting at the act ive site of the enzyme. The competitive behaviour of the autoinhibitor y domains contrasts with the mechanism of calcineurin inhibition by im munosuppressant-immunophilin complexes, which have been shown to bind to calcineurin at a region removed from the active site.