Construction Of Deletion Mutants Of CUE Domain Containing 2(CUEDC2) And Preliminary Research Of Its Function

Until now, little is known about the function of CUEDC2 protein (GenBank: NM₀24040), and in NCBI, there is no research article about its function. The students in our lab has performed yeast two-hybrid screen using the PR as bait, identifing CUEDC2 as PRB-binding protein, and CUEDC2 can promotes PR degradation through ubiquitin-proteasome. This research shed light into the function of CUEDC2.The student of our lab have performed a yeast two-hybrid screen using CUEDC2 as bait, identifying multiple clones, two of which were found to encode IKKβ. To confirm the results of yeast two-hybrid screen, and also to identify the domain of CUEDC2 with which IKKβinteract, we first constructed many deletion mutant of CUEDC2. Since CUEDC2 only has the CUE domain(1-2), we first constructed C-terminal, N-terminal, containing-CUE domain and not-containing CUE domain deletion mutant of CUEDC2. To identify which domain of CUEDC2 can suppress the NF-κB signaling, we constructed many deletion mutant of CUEDC2 into pCDNA3.0-Flag plasmid. Also we constructed a series of deletion mutant of IKKβ. We confirmed the interaction between CUEDC2 and IKKβby using in vitro GST pull-down assays. Unexpectly, we also found the interaction between CUEDC2 and IKKα. Then we confirmed the C-terminal sequence of CUEDC2 was necessary for interaction with IKKα.Since IKK is very important protein in NF-κB signaling^3-13, we suspected that CUEDC2 may affect the activity of NF-κB. To determine the significance of the interation between CUEDC2 and IKKα/β, we established a luciferase reporter system in eukaryotic cells for analyzing transcriptional activity of NF-κB. NF-κB transcriptional activation was specifically inhibited by CUEDC2 in the presence of TNFαin 293T cells. Comparable results were obtained in HeLa cells. HEK293 cells were stimulated by IL-1β, and we found CUEDC2 also inhibited the transactivation of NF-κB by IL-1β. To determine in which level CUEDC2 suppress the NF-κB signaling, we co-transfected CUEDC2 and several members of the TNFR receptor families in 293T cells^14-16, and we confirmed CUEDC2 suppress NF-κB signaling at the level of IKK.