RING finger protein AO7 supports NF-kappaB-mediated transcription by interacting with the transactivation domain of the p65 subunit.
In this study, a novel interactor of the p65 subunit (RelA) of NF-kappaB has been explored by performing yeast two-hybrid screen using the transactivation domain (TAD) of p65 located in the C terminus as bait. We have isolated a RING finger motif-containing protein, AO7, previously identified as an interacting protein with a ubiquitin-conjugating enzyme, Ubc5B. We confirmed the protein-protein interaction between p65 and AO7 in vitro and in vivo and found that the C-terminal region of AO7 is responsible for the interaction with p65 TAD. AO7 was predominantly localized in the nucleus and activated the NF-kappaB-dependent gene expression upon stimulation with IL-1beta or TNF or overexpression of NF-kappaB-inducing kinase. We found that both the RING finger and the C-terminal regions of AO7 were necessary for the transcriptional activation. When cotransfected with plasmids expressing Gal4-p65 fusion proteins containing various functional domains of p65, we found that p65 TAD was essential for the transcriptional activation mediated by AO7. Furthermore, the p65-mediated transactivation was suppressed by a ubiquitination-defective AO7 mutant in which the essential Cys residue within the RING finger motif was substituted by Ser. These data suggest that AO7 interacts with the p65 TAD and modulates its transcriptional activity.
Asamitsu
Kaori
K
Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.
Tetsuka
Toshifumi
T
Kanazawa
Satoshi
S
Okamoto
Takashi
T
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051996:Transcription Factor RelA; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases; D016335:Zinc Fingers / Q000235:genetics
D016328:NF-kappa B; D011994:Recombinant Proteins; D015534:Trans-Activators; D051996:Transcription Factor RelA; C476616:RNF25 protein, human; D044767:Ubiquitin-Protein Ligases