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...