The flow of information from calcium-mobilizing receptors to nuclear factor of activated T cells (NFAT)-dependent genes is critically dependent on interaction between the phosphatase calcineurin and the transcription factor NFAT. A high-affinity calcineurin-binding peptide was selected from combinatorial peptide libraries based on the calcineurin docking motif of NFAT. This peptide potently inhibited NFAT activation and NFAT-dependent expression of endogenous cytokine genes in T cells, without affecting the expression of other cytokines that require calcineurin but not NFAT. Substitution of the optimized peptide sequence into the natural calcineurin docking site increased the calcineurin responsiveness of NFAT. Compounds that interfere selectively with the calcineurin-NFAT interaction without affecting calcineurin phosphatase activity may be useful as therapeutic agents that are less toxic than current drugs.

NFAT transcription factors are highly phosphorylated proteins that are regulated by the calcium-dependent phosphatase calcineurin. We show by mass spectrometry that NFAT1 is phosphorylated on fourteen conserved phosphoserine residues in its regulatory domain, thirteen of which are dephosphorylated upon stimulation. Dephosphorylation of all thirteen residues is required to mask a nuclear export signal (NES), cause full exposure of a nuclear localization signal (NLS), and promote transcriptional activity. An inducible phosphorylation site in the transactivation domain contributes to transcriptional activity. Our data suggest that dephosphorylation promotes NFAT1 activation by increasing the probability of an active conformation, in a manner analogous to that by which depolarization increases the open probability of voltage-gated ion channels. This conformational switch paradigm may explain modification-induced functional changes in other heavily phosphorylated proteins.

NFAT transcription factors are related to NF-B͞Rel proteins and form cooperative complexes with Fos and Jun on DNA. We have identified an NFAT-related protein, NFAT5, which differs from the conventional NFAT proteins NFAT1-4 in its structure, DNA binding, and regulation. NFAT5 contains a NFAT-like Rel homology domain, conserves the DNA contact residues of NFAT1-4, and binds DNA sequences similar to those found in the regulatory regions of well-characterized NFAT-dependent genes. However, it lacks the majority of Fos͞Jun contact residues and does not bind cooperatively with Fos and Jun to DNA. Unlike NFAT1-4, whose nuclear import is tightly regulated by calcineurinmediated dephosphorylation, NFAT5 is a constitutively nuclear phosphoprotein regardless of calcineurin activation. These features suggest that unlike the conventional NFAT proteins, NFAT1-4, which activate gene transcription by integrating inputs from calcium͞calcineurin and protein kinase C͞mitogen-activated protein kinase signaling pathways, NFAT5 participates in as-yet-unidentified signaling pathways in diverse immune and nonimmune cells.The NFAT family of transcription factors contains four members, NFAT1 (NFATp), NFAT2 (NFATc), NFAT3, and NFAT4 (NFATx), each expressed as several isoforms related by alternative splicing (1-6) (reviewed in ref. 7). Genes encoding NFAT proteins are transcribed in almost every tissue tested, although the expression of individual proteins is fairly tissue restricted (5, 7). NFAT1, NFAT2, and NFAT4 are expressed in immune cells, and their roles in the immune response have been well characterized (8-15). Additionally, NFAT2 has been implicated in cardiac valve development (16,17) and NFAT3 in cardiac hypertrophy (18).The transcriptional function of NFAT proteins is regulated at several levels. When activated by a rise in intracellular calcium, the calmodulin-dependent phosphatase calcineurin dephosphorylates NFAT proteins in the cytoplasm, exposing their nuclear localization sequences and inducing their translocation into the nucleus (19)(20)(21)(22). Calcineurin binds to NFAT proteins by recognizing a conserved targeting motif (PxIxIT) present near the N terminus of the NFAT regulatory domain (19). The regulatory domain also contains a nuclear localization sequence (NLS), a serine-rich region ([SP]SPxSS[S]xSSxSxxS[D͞E]), and three ''SP motifs'' (SPxxSPxxSPxx- [SPxx]xxx [D͞E][D͞E]) (reviewed in refs. 7 and 23). Once NFAT proteins are in the nucleus, their central Rel-like domain dictates their binding specificity for DNA (7). A major level of regulation is conferred by the ability of NFAT proteins to form stable, cooperative DNA-binding complexes with dimers of the AP-1 (Fos͞Jun) family at composite NFAT:AP-1 DNA elements that have been identified in multiple NFATregulated genes (7,24,25). This combinatorial mechanism results in integration of the calcium͞calcineurin and protein kinase C͞mitogen-activated protein kinase signaling pathways that are activated by stimulation of the T cell receptor as well as other im...