The immunosuppressive drugs cyclosporin A and FK506 interfere with the inducible transcription of cytokine genes in T cells and in other immune cells, in part by preventing the activation of NF-AT (nuclear factor of activated T cells). We show that transcription factor NFAT1 in T cells is rapidly dephosphorylated on stimulation, that dephosphorylation occurs before translocation of NFAT1 into the cell nucleus, and that dephosphorylation increases the affinity of NFAT1 for its specific sites in DNA. Cyclosporin A prevents the dephosphorylation and the nuclear translocation of NFAT1 in T cells, B cells, macrophages, and mast cells, delineating at least one mechanism that contributes to the profound immunosuppressive effects of this compound.The nuclear factor of activated T cells (NF-AT), a multisubunit protein, is thought to regulate transcriptional induction of the interleukin 2 (IL-2) gene and other cytokine genes in antigenstimulated T cells (1-3). An NF-AT binding activity is detected in cytosolic extracts of resting T cells and in nuclear extracts of cells that have been stimulated with T-cell-receptor ligands or with ionomycin (4-7). The immunosuppressive drugs cyclosporin A (CsA) and FK506 block the appearance of this DNA-binding activity in nuclear extracts (8, 9) and inhibit the transcription of several cytokine genes in activated T cells (10-16). The identification of the protein phosphatase calcineurin as the immediate target of the immunosuppressive drugs (17-21) led to the proposal that calcineurin is central in the signal transduction pathway leading to transcription of the IL-2 gene and other cytokine genes in T cells and that CsA and FK506 exert a major portion of their immunosuppressive effect by preventing the nuclear translocation of a cytosolic subunit of NF-AT (3,(22)(23)(24) (8,(29)(30)(31)(32)(33)(34).A protein (NFAT1/NFATp) that meets the defining criteria of the preexisting cyclosporin-sensitive subunit of NF-AT has been purified from cytosolic extracts of a murine T-cell clone (35), and cDNAs encoding three protein isoforms related by alternative splicing have been isolated (36). Additional family members (NFATc, NFATx/NFAT4, NFAT3) encoded by separate genes have been identified (37)(38)(39). The presence of the mRNAs for NFAT1, NFATc, and NFATx/NFAT4 in T cells or thymus (36)(37)(38)(39) indicates that these proteins could be involved in controlling the expression of the IL-2 gene and other cytokine genes in T cells. NFAT1 is expressed in certain cells of the immune system in addition to T cells (40) and could contribute to cytokine gene expression in these other cell types.Here we have used specific antisera against NFAT1 to examine the early steps in its activation, which include changes in its phosphorylation state, its subcellular localization, and its DNA-binding activity. MATERIALS AND METHODSCells. Splenic and peritoneal cells were collected from CB6F1 mice. T-cell blasts (>98% CD3+) were obtained as described (41). Purified splenic B cells (>96% B220+) were obtained by a...

We show here that NFAT1 is rapidly activated, then slowly deactivated, by stimulation of T cells through their antigen receptor. Within minutes of T-cell receptor stimulation, NFAT1 is dephosphorylated, translocates from the cytoplasm into the nucleus, and shows an increase in its ability to bind to DNA. These changes are dependent on calcium mobilization and calcineurin activation, since they are also elicited by ionomycin and are blocked by the immunosuppressive drug cyclosporin A. After several hours of T-cell receptor stimulation, the majority of the NFAT1 in the cell reverts to its original phosphorylated form, reappears in the cytoplasm, and again displays a low affinity for DNA. Deactivation of NFAT1 is facilitated by phorbol 12-myristate 13-acetate and inhibitors of capacitative calcium entry and most likely reflects the slow return of intracellular free calcium concentrations towards resting levels. Our results suggest that calcineurin-dependent signalling pathways mediate the early activation of NFAT1, while phorbol 12-myristate 13-acetate-dependent feedback pathways contribute to the late deactivation. Persistent NFAT-dependent cytokine gene transcription in activated T cells may be mediated by other NFAT family proteins in addition to NFAT1 during the immune response.