A second calcineurin binding site on the NFAT regulatory domain

NFATc (a member of the family of nuclear factors of activated T cells) is a transcriptional factor responsible for the Ca2+-inducible activation of cy tokine genes during the immune response. in resting T cells, NFATc is retai ned in the cytoplasm by a mechanism that depends on multiple phosphorylatio ns in an N-terminal regulatory domain. Physical interaction with and dephos phorylation by Ca2+-activated calcineurin (Cn) allows the protein to enter the nucleus, where it binds to specific sites in cytokine gene promoters. P revious studies had identified a peptide segment in NFATc that binds Cn sta bly. Here we report the identification of a second Cn-binding element in NF ATc, which synergizes with the previously identified element. Although thes e sequences are conserved in all isoforms of NFAT. we find that the two sit es contribute differentially to the overall affinity for Cn in an isoform-d ependent manner. The regulatory domain of NFAT also was found to be entirel y devoid of structure, both in the phosphorylated and unphosphorylated stat e. This finding suggests that the NFAT regulatory domain does not undergo p hosphorylation-induced conformational switching, but instead requires partn er proteins to control accessibility of the NFAT nuclear localization and n uclear export signals.