RING Finger Protein AO7 Supports NF-κB-mediated Transcription by Interacting with the Transactivation Domain of the p65 Subunit

Kaori Asamitsu, Toshifumi Tetsuka, Satoshi Kanazawa, Takashi Okamoto
2003 Journal of Biological Chemistry  
In this study, a novel interactor of the p65 subunit (RelA) of NF-B 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
more » ... ble for the interaction with p65 TAD. AO7 was predominantly localized in the nucleus and activated the NF-B-dependent gene expression upon stimulation with IL-1␤ or TNF or overexpression of NF-B-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. NF-B plays a crucial role in many cellular events such as inflammation, host defense, cell survival, and proliferation (1-4). The members of the NF-B family in mammalian cells include the proto-oncogene c-Rel, RelA (p65), RelB, NFkB1 (p50/105), and NFkB2 (p52/p100). Rel family members form hetero-and homodimers with distinct specificities in various combinations and the combination of p65 and p50 represents a major form of NF-B (1-4). NF-B exists in the cytoplasm in an inactive form associated with IB inhibitor proteins (1, 2) and stimulation of cells with inducers such as phorbol esters, interleukin-1␤ (IL-1␤), 1 and tumor necrosis factor (TNF) leads to the activation of extracellular signal-regulated kinase kinase kinase 1/3, NF-B-inducing kinase (NIK), and IB kinase (IKK) complex containing IKK␣ and IKK␤. Once phosphorylated by IKK complex, IBs are ubiquitinated and degraded by 26 S proteasome, resulting in nuclear translocation of NF-B (1-4) . The protein region responsible for the transcriptional activation (called the "transactivation domain" (TAD)) of p65 has been mapped in their unique C-terminal region containing at least two TADs within its C-terminal 120 amino acids, termed TA1 and TA2 (5-7). It has been revealed that the p65 TAD interacts with transcriptional coactivators, such as p300/ CREB-binding protein (8, 9) and FUS/TLS (10), and general transcription factors, including TBP (11) and TFIIB (7, 12). Interaction of p65 with these factors stimulates transcription by initiating chromatin remodeling or by recruiting RNA polymerase II. In an unexpected scenario, the involvement of ubiquitination has recently been implicated in the regulation of TADs of some transcriptional activators such as VP16 (13, 14), Myc (13), and nuclear receptors (15-17), either directly or indirectly (for an excellent review, see Conaway et al. (18)). In this context, we became interested in AO7, which we have identified as one of the interacting proteins with the p65 TAD in the CytoTrap TM yeast two-hybrid screen. We have adopted this alternative screening method instead of the commonly used method utilizing the Gal4 transcription system, because the p65 TAD is functional in the yeast (10, 19). AO7 encodes a protein containing a RING finger domain and is ubiquitously expressed in various tissues (20). AO7 was initially identified in the yeast two-hybrid screen of a murine T cell library by using UbcH5b, an E2 enzyme, as bait (20) . Although the target protein for the uniquitination complex involving AO7 and UbcH5b still remains to be determined, AO7 has been shown to act as a putative E3 ligase at least in vitro (20). We found that AO7 acts as a mediator of p65 transactivation. A possible mechanism of its action is discussed. , containing the C-terminal TAD of p65, a bait plasmid for the Cytotrap yeast two-hybrid screen, was generated by inserting the p65 C-terminal region corresponding to amino acids 262-551 into pSos (Stratagene). pCMV-p65, pGal4-p65, and its mutants, pGal4-p65-(1-551), pGal4-p65N (containing amino acid positions 1-286 of p65), pGal4-p65C (containing amino acid positions 286 -551 of p65), pGal4-p65C1 (containing amino acid positions 286 -520 of p65), and pGal4-p65C3 (containing amino acid positions 521-551 of p65) were described previously (10, 19). pGal4-p65C2, containing amino acid positions 428 -551 of p65, was constructed by PCR using 5Ј and 3Ј oligonucleotide primers containing an EcoRI site and HindIII site, respectively. pCMV-NIK (21) is a generous gift from D. Wallach (Weitzmann Institute of Science, Rehovot, Israel). To generate pcDNA-AO7-(1-459), pcDNA-AO7-(1-219), and pcDNA-AO7-(212-459), each containing a FLAG epitope tag in the N terminus, the various portions of AO7 cDNA were amplified by PCR using pGEMAO7, containing the full-length AO7 cDNA, as a template with 5Ј and 3Ј oligonucleotide primers containing an EcoRI site and HindIII site, respectively. These EXPERIMENTAL PROCEDURES Plasmids
doi:10.1074/jbc.m211831200 pmid:12748188 fatcat:oz6t6w7qmrfy7aezr4xkyem354