INHIBITION OF NF-AT SIGNAL-TRANSDUCTION EVENTS BY A DOMINANT-NEGATIVEFORM OF CALCINEURIN

An inhibitory, ''dominant-negative,'' form of the calcineurin catalyti c (A) subunit was prepared, which lacks the calmodulin-binding domain. autoinhibitory domain and most of its catalytic core but possesses th e regulatory (B) subunit binding domain. When tested for its ability t o block calcineurin-dependent signaling in Jurkat cells, expression of this ''B-subunit knock-out'' (BKO) construct suppressed reporter gene activity driven by NF-AT, the pivotal promoter element for interleuki n (IL)-2 gene induction. Immunoprecipitation of epitope-labeled BKO de monstrated for the formation of a tight complex with endogenous B subu nit in Jurkat cells, consistent with an inhibitory mechanism that invo lves the sequestration of the B subunit. Furthermore, the sharply redu ced NF-AT activity produced by co-transfecting BKO could be ''rescued' ' by overexpression of transfected B subunit, suggesting that depletio n of this subunit was responsible for the inhibition. These data sugge st the potential utility of agents that disrupt calcineurin-mediated s ignal transduction pathways by blocking formation of the catalytically active dimer of calcineurin A and B subunits. (C) 1996 Academic Press , Inc.