2-SITE INTERACTION OF NUCLEAR FACTOR OF ACTIVATED T-CELLS WITH ACTIVATED CALCINEURIN

Transcription factors belonging to the nuclear factor of activated T c ells (NFAT) family regulate the expression of cytokine genes and other inducible genes during the immune response. The functions of NFAT pro teins are directly controlled by the calcium- and calmodulin-dependent phosphatase calcineurin, Here we show that the binding of calcineurin to NFAT is substantially increased when calcineurin is activated with calmodulin and calcium. FK506-FKBP12 drug-immunophilin complexes inhi bited the interaction of NFAT with activated calcineurin much more eff ectively than they inhibited the interaction with inactive calcineurin , suggesting that part of the interaction with activated calcineurin i nvolved the enzyme active site. We have previously shown that NFAT is targeted to inactive calcineurin at a region distinct from the calcine urin active site (Aramburu, J,, Garcia-Cozar, F, J,, Raghavan, A, Okam ura, H,, Rao, A., and Hogan, P, G, (1998) Mel. Cell 1, 627-637); this region is also involved in NFAT binding to activated calcineurin, sinc e binding is inhibited by an NFAT peptide spanning the calcineurin doc king site on NFAT, The interacting surfaces are located on the catalyt ic domain of the calcineurin A chain and on an 86-amino acid fragment of the NFAT regulatory domain. NFAT binding to the calcineurin catalyt ic domain was inhibited by the calcineurin autoinhibitory domain and t he RII substrate peptide, which bind in the calcineurin active site, a s well as by the NFAT docking site peptide, which binds to a region of calcineurin distinct from the active site.; We propose that, in resti ng cells, NFAT is targeted to a region of the calcineurin catalytic do main that does not overlap the calcineurin active site, Upon cell acti vation, displacement of the autoinhibitory domain by calmodulin bindin g allows NFAT to bind additionally to the calcineurin active site, thu s positioning NFAT for immediate dephosphorylation at functional phosp hoserine residues.