Expression of NFAT-Family Proteins in Normal Human T Cells

Abstract

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...

The Duration of Nuclear Residence of NFAT Determines the Pattern of Cytokine Expression in Human SCID T Cells

“…Polyclonal antiserum anti-67.1 against an N-terminal peptide common to all NFAT1 isoforms or anti-T2B1 against a C-terminal peptide of NFAT1c (Y. Wang et al, NYAS), mAb 7A6 against NFAT2 (Alexis Biochemicals, San Diego, CA), and polyclonal antisera against NFAT3 and -4 (gift from T. Hoey, Tularik) were used to detect NFAT family members. Consistent with previous results showing that PBL do not contain NFAT3 (28,29), we were unable to detect NFAT3 by immunoblotting of either control or patient T cells. Therefore, the experiments shown here report only on the activation status of NFAT1, -2, and -4.…”

“…No attempt was made to sequence NFAT3, because we could not detect NFAT3 proteins by immunoblotting, and mRNA expression was absent in immune cells as shown by others (28,29). The DBD and N-terminus of NFAT1, -2, and -4 of our two SCID patients did not reveal any mutation that would result in an amino acid exchange, frame shift, insertion, deletion, or premature stop codon.…”

Cooperation between SMAD and NF-κB in growth factor regulated type VII collagen gene expression

“…The protein concentration in the extracts was determined using a commercial assay kit (Bio-Rad). For supershift experiments, the following antibodies were used: rabbit polyclonal antibodies against NF-kB1 and RelA , NF-kB2 (p52) (Lanoix et al, 1994), Rel (p75) (Tan et al, 1992), RelB (p68) (Nancy Rice, unpublished; raised against a synthetic 17-amino acid peptide covering the C-terminus of human RelB: RE-AAFGGGLLPGPEAT), a pan-NF-AT antibody, raised against an internal peptide of human NF-AT common to all members of the NF-AT family (Lyakh et al, 1997). Nuclear extracts were incubated overnight with 1 ml of either antisera prior to the binding reaction.…”

A Role for the p38 MAP Kinase Pathway in the Nuclear Shuttling of NFATp

“…In T cells, NFATp and NFATc appear to be the major NFAT proteins expressed and regulated during the T cell activation process. NFAT3, on the other hand, has not been detected in the immune system, and NFAT4, whose mRNA levels are elevated in thymus and in nonlymphoid tissues (1), is found at low levels in resting or activated peripheral blood T cells (23). Whereas the putative kinase that could regulate the nuclear exit of NFATp has not yet been identified, GSK3 has been reported to enhance the nuclear export of NFATc (24).…”