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

Hiroshi Tokumitsu, E. S. Masuda, A. Tsuboi, K. Arai, N. Arai

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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-CLE0γ, 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 CLEO element and formed a NF-CLE0γ complex. This DNA-protein complex was competitively inhibited by oligonucleotides containing NF-AT and AP-1 binding sites, suggesting that the CLE0γ 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-CLE0γ), 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.

Original languageEnglish
Pages (from-to)737-744
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume196
Issue number2
DOIs
Publication statusPublished - Oct 29 1993
Externally publishedYes

Fingerprint

NFATC Transcription Factors
Transcription Factor AP-1
Granulocyte-Macrophage Colony-Stimulating Factor
Interleukin-2
Purification
Proteins
T-cells
Genes
Calcimycin
TCF Transcription Factors
T-Lymphocytes
Jurkat Cells
Calcium Ionophores
Molecular mass
Phorbol Esters
Nuclear Proteins
Chromatography
Cell Extracts
Electrophoresis
Oligonucleotides

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

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title = "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",
abstract = "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-CLE0γ, 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 CLEO element and formed a NF-CLE0γ complex. This DNA-protein complex was competitively inhibited by oligonucleotides containing NF-AT and AP-1 binding sites, suggesting that the CLE0γ 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-CLE0γ), 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.",
author = "Hiroshi Tokumitsu and Masuda, {E. S.} and A. Tsuboi and K. Arai and N. Arai",
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T1 - Purification of the 120-kDa Component of the Human Nuclear Factor of Activated T Cells (NF-AT)

T2 - Reconstitution of Binding Activity to the cis-Acting Element of the GM-CSF and IL-2 Promoter with AP-1

AU - Tokumitsu, Hiroshi

AU - Masuda, E. S.

AU - Tsuboi, A.

AU - Arai, K.

AU - Arai, N.

PY - 1993/10/29

Y1 - 1993/10/29

N2 - 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-CLE0γ, 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 CLEO element and formed a NF-CLE0γ complex. This DNA-protein complex was competitively inhibited by oligonucleotides containing NF-AT and AP-1 binding sites, suggesting that the CLE0γ 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-CLE0γ), 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.

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