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First published online July 12, 2005
doi: 10.1242/10.1242/jcs.02445

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Early growth response proteins EGR-4 and EGR-3 interact with immune inflammatory mediators NF-B p50 and p65

Gerhard D. Wieland^1,*, Nina Nehmann^1,*, Doreen Müller¹, Hermann Eibel², Ulrich Siebenlist³, Jürgen Sühnel⁴, Peter F. Zipfel¹ and Christine Skerka^1,

¹ Department of Infection Biology, Leibniz-Institute for Natural Products, Research and Infection Biology, Hans-Knoell-Institute, Butenbergstrasse 11a, 07745 Jena, Germany
² Research Group for Rheumatology, Albert-Ludwig-University, Hugstetter Strasse 55, 79106 Freiburg, Germany
³ Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD 20892, USA
⁴ Biocomputing Group, Institute of Molecular Biotechnology, Beutenbergstrasse 11, 07745 Jena, Germany

View larger version (38K): [in a new window]   Fig. 1. EGR-4 and EGR-3 bind NF-B p50 and p65 proteins. (A) Recombinant p50 and p65 expressed in E. coli as fusion proteins were coupled to GST matrix, and cell extract containing recombinant or Jurkat-cell-derived native EGR-4 or EGR-3 was applied to this matrix. After loading, the columns were extensively washed and bound proteins were eluted with glutathione. Cell lysates (Ly) and eluates (el) were separated by SDS-PAGE and analysed by western blotting. Use of surface-plasmon-resonance assays to identify binding of NF-B p50 (B) and p65 (C) to immobilized EGR-4. NF-B p50 and p65 were injected into a flow cell precoupled with recombinant EGR-4. The control values obtained by measuring absorbance of NF-B proteins to an uncoupled chip were subtracted from the profile with immobilized proteins. Representative experiments are shown. (D) Jurkat-cell extract was used to immunoprecipitate EGR-4 using p65 antiserum. The fractions (flow through, wash and eluate) were separated by SDS-PAGE and used for western blotting. p65 and EGR-4 were detected in the fraction obtained after loading the column (lane 2), the first wash fraction (lane 3) and in the eluate (lane 5). Cell extract with recombinant EGR-4 or p65 proteins are shown in lane 1.

View larger version (59K): [in a new window]   Fig. 2. In vivo localization and interaction of EGR-3 or EGR-4 and NF-B p65 proteins. Jurkat T cells were transiently transfected with EGR-3-ECFP and p65-EYFP or EGR-4-ECFP and p65-EYFP expression vectors, and fluorescence images were recorded 24 hours after transfection. Images were acquired under CFP, YFP and FRET filter settings (top) or upon photobleaching of the acceptor protein (Bleach, bottom). EGR-3-CFP and EGR-4-CFP are localized predominantly to the nucleus and p65-YFP is present in the cytoplasm. Upon photobleaching of the acceptor, the fluorescence signal of the donor increases because cross-talk between the proteins is eliminated. The fluorescence signal of the acceptor and the FRET signals are completely abrogated. Staining of DNA with DAPI indicates the position of the nuclei and the Jurkat T cells are shown with digital interference contrast (DIC) microscopy. Bar, 10 µm.

View larger version (10K): [in a new window]   Fig. 3. Transcriptional activity of EGR and NF-B complexes for inflammatory gene promoters. Human 293 kidney cells were transfected with reporter constructs containing the human promoters for the genes encoding (A) IL-2,(B) TNF- and (C) ICAM-1. The reporter constructs were transfected together with the indicated expression vectors. The transcriptional activity is shown as fold induction of the activity of the reporter construct alone, which was set to 1. Mean values and standard deviations are shown from at least three independent experiments performed on different days and measured in triplicate.

View larger version (11K): [in a new window]   Fig. 4. Localization of functional domains in the EGR-4 protein. Human 293 kidney cells were co-transfected with a human TNF--encoding reporter plasmid and the indicated expression vectors coding for p65 and truncated EGR-4 proteins. The transcriptional activity is shown as fold induction of the activity of the reporter construct alone, which was set to 1. Each column represents the mean value of four independent experiments performed at different days and measured in triplicate. Mean values and standard deviations are indicated.

View larger version (30K): [in a new window]   Fig. 5. Mapping of the NF-B p65 interaction regions within the zinc-finger domains of EGR-4 and EGR-3. 27 immobilized peptides with a length of 13 amino acids and an overlap of ten amino acids spanning the zinc-finger domain of EGR-3 (amino acids 272-361) (A), EGR-4 (amino acids 377-468) (B) and mutated EGR-4 (amino acids 377-468 with amino acid changes K455G, K456G, K457 G, K460 G, K464 G, K466 G and R468 G) (C) were incubated with purified p65. Binding was detected with specific antiserum. The linear amino acid stretches that bind p65 are shown.

View larger version (23K): [in a new window]   Fig. 6. Inhibitory effect of synthetic peptides. Synthetic peptides representing either the major interaction domain of EGR-4 (peptide 1, amino acids 455-468) or an unspecific scrambled sequence (peptide 2) were used as inhibitors for EGR-4/p65 complex formation. NF-B p65 was preincubated with peptide 1 (A) or peptide 2 (B) and bound to an EGR-4 matrix. (C) The specific inhibitory effect of peptide 1 was confirmed by surface plasmon resonance. p65 preincubated with peptide 1 or 2 was applied to the fluid phase and binding to immobilized EGR-4 was analysed. One representative experiment out of five is shown.

View larger version (43K): [in a new window]   Fig. 7. Localization of the p65 interaction domain in the crystal structure of the EGR zinc-finger domain bound to DNA. The structure of the zinc-finger domain of Zif268 was used as a template to localize the p65 interaction domain of EGR-4. (A) Conservation of amino acids in zinc finger III between EGR-1 and EGR-4. Positively charged amino acids involved in interaction are shown in blue. The conserved amino acids that complex the zinc ion and form the zinc-finger structure are highlighted in yellow. (B) Zinc finger I is shown in light gray, zinc finger II in dark gray and zinc finger III in black. The positions of the amino acids of EGR-4 that bind p65 are shown in red.

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