RESUMEN
Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. We have identified two new isoforms of the transcription factor NFAT1 (previously termed NFATp) that are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive agent cyclosporin A (CsA). Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol 12-myristate 13-acetate; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol 12-myristate 13-acetate. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response.
Asunto(s)
Citocinas/biosíntesis , Proteínas de Unión al ADN/metabolismo , Linfocitos T/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Chlorocebus aethiops , Clonación Molecular , Cartilla de ADN , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The cyclosporin-sensitive factor NFATp cooperates with Fos and Jun family proteins to regulate transcription of the interleukin 2 gene in activated T cells. We have defined a 187-amino-acid fragment of NFATp, located centrally within the protein sequence, as the minimal region required for DNA binding and for complex formation with Fos and Jun. The sequence of this region of NFATp shows a low degree of similarity to the Rel homology region. One specific short sequence in NFATp (RAHYETEG), located near the NH2 terminus of the DNA-binding domain, resembles a highly conserved sequence (RFRYxCEG) that is located near the NH2 terminus of the Rel homology region and that has been implicated in DNA binding by Rel family proteins. Mutational analysis demonstrates that the residues in this sequence that are identical in NFATp and Rel family proteins contribute to DNA binding by NFATp. Further, mutation of the threonine residue in this sequence to cysteine, as in Rel proteins, confers on NFATp a sensitivity to sulfhydryl modification similar to that of Rel family proteins. The results suggest that NFATp and Rel family proteins bind to DNA using similar structural motifs.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Nucleares , Proteínas Oncogénicas de Retroviridae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión al ADN/genética , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Proteínas Oncogénicas v-rel , Unión Proteica , Proteínas Oncogénicas de Retroviridae/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
Nuclear factor of activated T cells (NFAT) is a transcription factor that regulates expression of the cytokine interleukin-2 (IL-2) in activated T cells. The DNA-binding specificity of NFAT is conferred by NFATp, a phosphoprotein that is a target for the immunosuppressive compounds cyclosporin A and FK506. Here, the purification of NFATp from murine T cells and the isolation of a complementary DNA clone encoding NFATp are reported. A truncated form of NFATp, expressed as a recombinant protein in bacteria, binds specifically to the NFAT site of the murine IL-2 promoter and forms a transcriptionally active complex with recombinant protein fragment react with T cell NFATp. The molecular cloning of NFATp should allow detailed analysis of a T cell transcription factor that is central to initiation of the immune response.