The association of the NF-B p65/p50 dimer with IB␣ plays a pivotal role in regulating its nuclear translocation and gene transcription. In addition, serine phosphorylation at various sites of the p65 subunit has been shown to be important in initiating transcription. Here we demonstrate that the regulation of nuclear translocation of p65 phosphorylated at serine 536 is not dependent on IB␣. Stimulation of either Jurkat or normal human T cells resulted in the nuclear translocation of phospho-p65 (Ser 536 ). In addition, the phospho-p65 (Ser 536 ) was not associated with either IB␣ or p50, and the nuclear translocation of phospho-p65 (Ser 536 ), but not total p65, was unaffected by the proteosome inhibitor MG-132, which blocks IB protein degradation and prevents p65/p50 dimer nuclear translocation. Accordingly, the co-expression of a dominant negative mutant of IB␣ blocked the transcriptional activity mediated by wild type but not the dominant positive p65 mutant (S536D). Furthermore, the transfection of the S536D form of p65 led to the induction of interleukin-8 transcription following stimulation, whereas the S536A form, which cannot be phosphorylated at this site, did not. Together, the findings suggest that p65 phosphorylated on serine 536 is not associated with or regulated by IB␣, that it has a distinct set of target genes, and that it may represent a noncanonical NF-B pathway that is independent of IB␣ regulation.The NF-B signaling pathway responds rapidly to a wide range of stimuli (1). Activation leads to the translocation of the transcription factors from the cytoplasm to the nucleus. The NF-B transcription factor consists of two subunits of either homo-or heterodimers of RelA/ p65, c-Rel, and p50. The complexes are held in the cytoplasm and prevented from activating transcription by a class of proteins referred to as inhibitors of NF-B or IB proteins. Upon stimulation, the IB proteins are phosphorylated by one of a number of IB kinases (IKK-␣, -, and -␥), ubiquitinylated, and degraded, which thereby allows the NF-B complex to translocate into the nucleus (2). However, recent findings have demonstrated the shuttling of the NF-B complex in and out of the nucleus in the absence of stimulation (3, 4). In addition to nuclear translocation of the NF-B complex, several studies have shown that the NF-B proteins are modified post-translationally, and those changes influence transcriptional activity. Examples of activation-induced posttranslational modifications include the acetylation of p65 to facilitate the retention of the NF-B complex in the nucleus (5, 6). In addition, the S-nitrosylation of cysteine 62 of p50 has been shown to affect the NF-B binding to DNA (7, 8).We have previously described the phosphorylation of various Rel proteins following the stimulation of T cells, and the phosphorylation of p50 increased the DNA binding capacity (9). Several studies have demonstrated the phosphorylation of p65 in response to various stimuli (10). Serine 276 of p65 is phosphorylated by protein kinase A during IB d...
NFAT proteins constitute a family of transcription factors involved in mediating signal transduction. Using a panel of specific antisera in immunoprecipitation assays, we found that NFATp (135 kDa) is constitutively expressed in normal human T cells, while synthesis of NFATc (predominant form of 86 kDa) is induced by ionomycin treatment. NFAT4/x was very weakly expressed in unstimulated cells, and its level did not increase upon treatment with activating agents. NFAT3 protein was not observed under any conditions. Highermolecular-weight species of NFATc (of 110 and 140 kDa) were also detected. In addition, translation of NFATc mRNA apparently initiates at two different AUG codons, giving rise to proteins that differ in size by 36 amino acids. Additional size heterogeneity of both NFATc and NFATp results from phosphorylation. In contrast to ionomycin treatment, exposure of cells to phorbol myristate acetate (PMA) plus anti-CD28 did not induce NFATc, indicating that under these conditions, interleukin-2 synthesis by these cells is apparently independent of NFATc. In DNA binding assays, both PMA plus anti-CD28 and PMA plus ionomycin resulted in nuclear NFAT. Surprisingly, the PMA-ionomycin-induced synthesis of NFATc that was detected by immunoprecipitation was not mirrored in the DNA binding assays: nearly all of the activity was due to NFATp. This is the first study of expression of all family members at the protein level in normal human T cells.NFAT (nuclear factor of activated T cells) is implicated in regulation of interleukin-2 (IL-2) gene transcription (for reviews, see references 13 and 30). In addition, NFAT-binding sites have been identified in the regulatory regions of various other cytokine genes, including the IL-4 (3, 33, 38), tumor necrosis factor alpha (7, 23), and IL-3/granulocyte-macrophage colony-stimulating factor (4, 22) genes. Though originally found in T cells, NFAT DNA-binding activity and/or protein has now been found in other cell types, including B cells (2,40,41,45), mast cells (29), natural killer (NK) cells (1), and a neuronal cell line and certain regions of the brain (8). Thus, NFAT is likely to play an important role in the regulation of a variety of genes in a number of different cell types.To date, cDNAs from four different NFAT genes (NFATp, NFATc, NFAT3, and NFAT4/NFATx) have been cloned, and they constitute a related but quite divergent family (9,11,18,21,24,26,27). The family in turn is weakly related to the Rel/NF-B family of transcription factors over a 300-aminoacid region called the Rel homology domain (RHD). NFAT sequences in this region govern DNA binding and association with the AP-1 transcription factor (12), and within the RHD, sequence conservation among the NFAT proteins is very high. Upstream of the RHD, NFAT proteins are less closely related, but they do share several serine-and proline-rich segments. Downstream of the RHD, NFAT proteins are variable in length and in sequence.The hallmark of NFAT activity is its inducibility by agents that increase intracellular Ca...
Optimal activation of T celis requires at least two signals. One signal can be delivered by the antigen-specific T-cell receptor, and the second signal is provided by the costimulatory molecule(s) delivered by the antigen-presenting cell. CD28 is a T-cell surface molecule and stimulation through this protein plays an important role in delivering the second activation signal. In this report, we show that in human peripheral blood T cells, CD28-mediated signal transduction involves the rel family proteins-c-Rel, p50, and p65. Treatment of peripheral blood T cells with phorbol 12-myristate 13-acetate (PMA) and anti-CD28 monoclonal antibody (mAb) results in augmentation of nuclear c-Rel, p50, and p65, and this augmentation can occur in the presence of the immunosupressant cyclosporin A. It is also shown in this report that, in response to PMA/anti-CD28 mAb or anti-CD3/anti-CD28 mAb, c-Rel, p50, and p65 are associated with CD28-responsive element present in the promoter of the human interleukin 2 gene. The functional significance of c-Rel involvement in the CD28-responsive complex is demonstrated by transient transfection analysis, where cotransfection of c-Rel augments the level of expression of a chloramphenicol acetyltransferase reporter gene linked to the CD28-responsive element.
Interferon-gamma (IFN-gamma) is an immunoregulatory cytokine expressed in large granular lymphocytes and T cells. However, the molecular mechanisms underlying IFN-gamma gene transcription have not been fully defined. Here, we analyze the mechanisms responsible for the inhibition of IFN-gamma promoter activity by the glucocorticoid hormone dexamethasone. Cotransfection assays performed in Jurkat T cells demonstrated that the activity of the initial 108 base pairs of the IFN-gamma promoter was down-regulated in the presence of dexamethasone. Furthermore, utilizing electrophoretic mobility shift analysis, we identified activator protein 1 AP-1-cAMP response element binding protein-activating transcription factor (CREB-ATF) binding elements situated in positions of the IFN-gamma promoter previously identified as essential for promoter activity. Moreover, dominant negative mutants of the c-Jun proto-oncogene were able to mimic the same down-regulatory effect exerted by dexamethasone, and mutations that abolished the binding of the AP-1 CREB-ATF factors were able to block the glucocorticoid effect. These results suggest a model involving the inhibition of IFN-gamma AP-1 CREB-ATF DNA binding complexes as one of the mechanisms involved in the negative regulatory action of glucocorticoids on IFN-gamma gene expression and support the relevance of AP-1 CREB-ATF binding factors during the transcriptional activation of the IFN-gamma promoter in T cells.
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