The subcellular localization of the transcription factor NFATc is tightly regulated by the calcium-regulated phosphatase calcineurin, which acts to directly dephosphorylate NFATc, causing its rapid translocation from the cytoplasm to the nucleus. The calcineurin-mediated nuclear localization of NFATc is opposed by poorly defined protein kinases that act either to directly antagonize nuclear import or, alternatively, to promote nuclear export. Here, we provide evidence that the cellular protein kinases JNK, ERK, p38, and CK2 (formerly casein kinase II) are involved in the regulation of NFATc subcellular localization. We show that JNK, ERK, and p38 physically associate with the NFATc N-terminal regulatory domain and can directly phosphorylate functionally important residues involved in regulating NFATc subcellular localization, namely Ser 172 and the conserved NFATc Ser-Pro repeats. Moreover, we found that overexpression of JNK, ERK, or p38 is able to block ionomycin-induced NFATc nuclear translocation, whereas treatment of cells with both PD98059 and SB202190, which inhibit MAPK/SAPK signaling pathways, is sufficient to trigger NFATc nuclear localization. Finally, we show that CK2 also binds the N terminus of NFATc and phosphorylates functionally important amino acid residues, including a conserved amino acid motif located downstream of each of the NFATc Ser-Pro repeats that appears to be important for regulating NFATc nuclear export. Collectively, these studies identify functionally important amino acid residues and protein kinases involved in the regulation of NFATc subcellular localization.Antigenic stimulation of T lymphocytes initiates a complex series of intracellular signal transduction pathways that lead to the expression of a panel of immunoregulatory genes, whose function is critical to the initiation and coordination of the immune response (1-3). The NFAT (nuclear factor of activated T cells) transcription factor family plays a pivotal role in this process and is involved in the expression of a number of immunologically important genes, including the cytokines IL-2, 1 IL-3, IL-4, IL-5, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-␣, as well as several cellsurface molecules such as CD40L and FasL (2, 4). Although originally described in T cells, it is now apparent that NFAT proteins are also expressed in other immune system cells, including B cells, mast cells, basophils, and natural killer cells, as well as a variety of nonimmune cell types and tissues such as skeletal muscle, neurons, heart, and adipocytes (4 -14). In most cases, NFAT target genes have not been identified in these latter cell types, although it is apparent that NFAT acts as a calcium-dependent transcription factor and serves to couple gene expression to changes in intracellular calcium levels.NFAT family members appear to be regulated primarily at the level of their subcellular localization and are located exclusively in the cytoplasm of resting T cells (2,4,15). In response to antigen receptor signaling...

T cell activation is known to be critically regulated by the extent and duration of TCR-induced signaling pathways. The NFAT family of transcription factors is believed to play an important role in coupling these quantitative differences in TCR-induced signaling events into changes in gene expression. In this study we have specifically investigated the effects of sustained NFAT signaling on T cell activation by introducing a constitutively active mutant version of NFATc1 (caNFATc1) into primary murine CD4(+) T cells and examining its effects on gene expression. We now report that ectopic expression of caNFATc1 partially mimics TCR signaling, resulting in enhanced expression of CD25 and CD40 ligand and down-regulation of CD62L. More importantly, we find that expression of caNFATc1 in T cells maintained under either nonpolarizing or Th1-skewing conditions leads to a marked selective increase in the number of cells expressing the prototypical Th1 cytokine, IFN-gamma. Furthermore, when expressed in Th2-skewed cells, caNFATc1 appears to attenuate Th2 differentiation by decreasing production of IL-4 and promoting the expression of IFN-gamma. Finally, we find that caNFATc1 enhances expression of functional P-selectin glycoprotein ligand-1, up-regulates Fas ligand expression, and increases susceptibility to activation-induced cell death, cellular traits that are preferentially associated with Th1 effector cells. Taken together, these results suggest that sustained NFAT signaling, mediated by ectopic expression of caNFATc1, acts to promote a Th1-like pattern of gene expression and thereby serves to highlight the important relationship between the degree of NFAT signaling and the qualitative pattern of gene expression induced during T cell activation.

We demonstrate that baboon Treg cells suppress immune responses to xenogeneic stimulation. These studies suggest that adoptive transfer of expanded Treg cells into transplant recipients may provide an approach to prevent cell-mediated rejection of grafts and potentially induce tolerance in the pig to baboon xenotransplantation preclinical model.

Due to the shortage of human organs, xenotransplantation is being explored as an alternative to allotransplantation, but immune rejection remains a major hurdle to its implementation. We tested the ability of human CD4