Purification of the 120 kDa component of the human nuclear factor of activated T cells (NF-AT): reconstitution of binding activity to the cis-acting element of the GM-CSF and IL-2 promoter with AP-1.
The cis-acting element of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter, CLE0, is required for stimulation dependent expression of the GM-CSF gene by phorbol ester (PMA) and calcium ionophore (A23187) in T cells. We recently obtained evidence that NF-CLE0 gamma, one of the CLE0-binding factors, is similar to the nuclear factor of activated T cells, NF-AT. In the present study, we show that the affinity-purified NF-AT from nuclear extracts of human Jurkat T cells stimulated with both PMA and A23187 bound strongly to the CLE0 element and formed a NF-CLE0 gamma complex. This DNA-protein complex was competitively inhibited by oligonucleotides containing NF-AT and AP-1 binding sites, suggesting that the CLE0 gamma complex is identical to NF-AT and contains AP-1 proteins. Here, one component of NF-AT with an apparent molecular mass of 120 kDa on SDS-polyacrylamide gel electrophoresis was purified to near homogeneity by Mono Q chromatography. The purified 120 kDa protein reconstitutes NF-AT binding in combination with recombinant cJun/cFos heterodimer. Furthermore, we demonstrate that binding of this 120 kDa protein to both the NF-AT and the CLE0 sequences can be reconstituted with the addition of affinity-purified Jurkat AP-1 proteins. These results indicate that NF-AT (NF-CLE0 gamma), which is composed of the 120 kDa nuclear protein and AP-1 proteins, regulates the stimulation-dependent expression of the GM-CSF gene as it does the IL-2 gene.
Tokumitsu
H
H
Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304.
Masuda
E S
ES
Tsuboi
A
A
Arai
K
K
Arai
N
N
D001483:Base Sequence; D001665:Binding Sites; D000001:Calcimycin / Q000494:pharmacology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D016207:Cytokines / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D016207:Cytokines; D004268:DNA-Binding Proteins; D007376:Interleukin-2; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D014157:Transcription Factors; D000001:Calcimycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013755:Tetradecanoylphorbol Acetate