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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-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.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Tokumitsu H,Masuda ES,Tsuboi A,Arai K,Arai N

doi

10.1006/bbrc.1993.2311

subject

Has Abstract

pub_date

1993-10-29 00:00:00

pages

737-44

issue

2

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(83)72311-9

journal_volume

196

pub_type

杂志文章

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