Mechanisms of transactivation by nuclear factor of activated T cells-1.

Abstract

SummaryNuclear factor of activated T cells-family proteins (NFAT1/NFATp, NFATc, NFAT3, and NFAT4/NFATx/NFATc3) play a key role in the transcription of cytokine genes and other genes during the immune response. We have defined the mechanisms of transactivation by NFAT1. NFAT1 possesses two transactivation domains whose sequences are not conserved in the other NFAT-family proteins, and a conserved DNA-binding domain that mediates the recruitment of cooperating nuclear transcription factors even when it is expressed in the absence of other regions of the protein. The activity of the NH2-terminal transactivation domain is modulated by an adjacent regulatory region that contains several conserved sequence motifs represented only in the NFAT family. Our results emphasize the multiple levels at which NFAT-dependent transactivation is regulated, and predict significant differences in the architecture of cooperative transcription complexes containing different NFAT-family proteins.T he nuclear factor of activated T cells (NFAT) 1 is a calcium-regulated transcription factor that is a major target for the immunosuppressive drugs cyclosporin A (CsA) and FK506 (1-3). The DNA-binding specificity of NFAT is conferred by a novel family of transcription factors, of which four members (NFAT1/NFATp, NFATc, NFAT3, and NFATx/NFAT4/NFATc3, each containing several isoforms) have so far been described (4-9). NFAT1 protein is expressed in several classes of immune system cells (10-12), consistent with the detection of nuclear NFAT DNA-binding activity in activated T, B, mast, and NK cells (13)(14)(15)(16)(17)(18)(19). The distribution of the other NFAT-family proteins has not yet been assessed at the protein level; however the mR.NAs encoding these proteins differ in their cell and tissue distributions and their representation in resting versus activated cells (5-7), suggesting that each protein may subserve a different function. NFATc mlLNA is expressed at high levels in skeletal muscle and in activated lymphoid cells, NFATx/ NFAT4 mP,.NA is predominantly expressed in the thymus, and NFAT3 mR.NA is most strongly expressed outside the immune system (5-7).An important component of transactivation by NFATfamily proteins is their cooperation in the nucleus with Fos and Jun (20,21). This cooperation is mediated by interac1Abbreviations used in this paper: CAT, chloramphenicol acetyltransferase; CsA, cyclosporin A; DBD, DNA-binding domain; hGH, human growth hormone; NFAT, nuclear factor of activated T cells; NHP,., NFAT homology region; TAD, transactivation domain. tion of the highly conserved DNA-binding domains (DBDs) of NFAT-family proteins with Fos-Jun and Jun-Jun dimers (6,7,22); the bZIP (basic region-leucine zipper) regions of Fos and Jun suffice for this interaction (21), although an acidic region of Fos has also been implicated (23). Overexpression of Fos-and Jun-family proteins in T cells greatly augments transcription driven by tandem copies of the IL-2 and IL-4 promoter NFAT sites in activated T cells (20,(24)(25)(26). It has al...

NFAT2 Isoforms Differentially Regulate Gene Expression, Cell Death, and Transformation through Alternative N-Terminal Domains

“…One-hybrid assays have shown that the region spanning aa 1 to 30 of NFAT2␤ containing the acidic domain is necessary and sufficient for eliciting a high activation signal (28). Similarly, the regions spanning aa 1 to 144 and aa 1 to 217 of NFAT1 and NFAT3, respectively, showed high transactivation activity (40,41). Accordingly, the NFAT2␣ fragments spanning aa 1 to 44 and aa 1 to 106 that do not contain the N-terminal acidic domain were unable to induce transcription (28,37).…”

Expression of NFAT-Family Proteins in Normal Human T Cells

“…To date, there is very little functional information about the region downstream of the RHD in the NFAT proteins. A transactivation domain has been found in this region in NFATp (19), but the 86-kDa form of NFATc, which terminates at essentially the C terminus of the RHD, can activate transcription in reporter assays (6,11,26). Interaction of NFATp with AP-1 appears to be governed by sequences within the RHD (12,25), and interaction with calcineurin apparently occurs in the region upstream of the RHD (17,20,37,43).…”

Blockade of T-Cell Activation by Dithiocarbamates Involves Novel Mechanisms of Inhibition of Nuclear Factor of Activated T Cells

“…The GAL4 plasmids RSV-GAL4-DBD (RSV is Rous sarcoma virus) and GAL4-hNFAT1(1-415) have been described previously (32). The GAL4-hNFAT1(1-415) plasmid encodes the transactivation domain of NFAT1/p (amino acids 1 to 415) in frame with the GAL4 DNA binding domain (DBD).…”

“…Gel retardation assays were performed as previously described (18) cpm/g) of 32 P-labeled double-stranded oligonucleotides (2 l) was added, and this mixture was incubated at room temperature for 40 min. In competition experiments, 30-fold molar excess of unlabeled homologous oligonucleotides was added to the binding reaction mixture prior to the addition of the probe.…”