Abstract
TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation
Highlights
22924 JOURNAL OF BIOLOGICAL CHEMISTRY have been shown to promote ubiquitination events, which are required for activation of their downstream pathways [3,4,5,6]
One of them encoded a portion of TRAF7 spanning amino acidic residues 266 – 670 (TRAF(266 – 670)), consisting of a polypeptide containing a portion of the zinc finger domain, the coiled-coil domain, and the seven WD40 repeats
We have identified TRAF7 as a protein that physically interacts with NF-B essential modulator (NEMO) and with the p65 member of the NF-B transcription factor family
Summary
Two-hybrid Screening—The two-hybrid screening was performed using the Matchmaker system (Clontech) as described previously [12]. Yeast AH109 expressing GAL4DBD-NEMO[1–339] was transformed with a human cDNA library cloned in pACT2 vector (Clontech) in fusion with GAL4TAD. Cells were lysed in lysis buffer, and immunocomplexes were bound to protein A/G (Roche Applied Science), resolved by SDS-PAGE, and analyzed by immunoblot assay. GST Pulldown Assay—To analyze NEMO interaction with TRAF7, NEMO-glutathione S-transferase (GST) fusion protein was generated in Escherichia coli DH5␣ cells using pGEX-5x-1 as plasmid (GE Healthcare). NEMO-GST fusion protein expression was assessed by SDS-PAGE. Samples were boiled for 5 min, and supernatants were separated by SDS-PAGE followed by blotting and incubation with TRAF7 antibody. Luciferase Assay—To assess NF-B and AP-1 activation, HEK293 cells were transfected with the indicated plasmidic DNAs together with pNF-B-luc or pAP-1-luc (Clontech) in 6-well plates. Yeast colonies were scored as positive when a growth developed within 24 –36 h; a negative was scored when growth failed to develop within 1 week
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