Early growth response proteins EGR-4 and EGR-3 interact with immune inflammatory mediators NF-kappaB p50 and p65.
Here, we characterize the basis for the T-cell-specific activity of the human zinc-finger protein early growth response factor 4 (EGR-4). A yeast two-hybrid screen showed interaction of EGR-4 with NF-kappaB p50. Using recombinant proteins, stable physical complex formation was confirmed for EGR-4 and EGR-3 with p50 and with p65 using glutathione-S-transferase pull-down assays and surface-plasmon-resonance and peptide-spot analyses. In vivo interaction of EGR-4 and EGR-3 with NF-kappaB p65 was demonstrated by immunoprecipitation experiments and fluorescence-resonance-energy transfer (FRET) analysis showing interaction in the nucleus of transfected Jurkat T cells. In transfection assays, EGR-p50 complexes were transcriptionally inactive and EGR-p65 complexes strongly activated transcription of the promoters of the human genes encoding the cytokines interleukin 2, tissue necrosis factor alpha and ICAM-1. The EGR-p65 complexes increased reporter-gene activity about 100-fold and thus exceeded the transcriptional activities of the p65 homodimer and the p65/p50 heterodimers. The major interaction domain for p65 was localized within the third zinc finger of EGR-4 using deletion mutants for pull-down assays and peptide-spot assays. By computer modeling, this interaction domain was localized to an alpha-helical region and shown to have the central amino acids surface exposed and thus accessible for interaction. In summary, in T cells, the two zinc-finger proteins EGR-4 and EGR-3 interact with the specific nuclear mediator NF-kappaB and control transcription of genes encoding inflammatory cytokines.
Wieland
Gerhard D
GD
Department of Infection Biology, Leibniz-Institute for Natural Products, Research and Infection Biology, Hans-Knoell-Institute, Butenbergstrasse 11a, 07745 Jena, Germany.
Nehmann
Nina
N
Müller
Doreen
D
Eibel
Hermann
H
Siebenlist
Ulrich
U
Sühnel
Jürgen
J
Zipfel
Peter F
PF
Skerka
Christine
C
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D051776:Early Growth Response Protein 3 / Q000096:biosynthesis / Q000378:metabolism*; D051765:Early Growth Response Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D007668:Kidney / Q000166:cytology; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D052002:NF-kappa B p50 Subunit / Q000276:immunology / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D051996:Transcription Factor RelA / Q000276:immunology / Q000378:metabolism*; D014162:Transfection; D016335:Zinc Fingers / Q000502:physiology
C111028:EGR4 protein, human; D051765:Early Growth Response Transcription Factors; D052002:NF-kappa B p50 Subunit; D011994:Recombinant Proteins; D051996:Transcription Factor RelA; D051776:Early Growth Response Protein 3