Screening Of CUEDC2-interacting Proteins By Yeast Two-hybrid Assasy And Functional Analysis Of The Protein Complex

CUEDC2 is a CUE domain-contained protein. Until now, little is known about the function of CUEDC2 protein (GeneBank: NM₀24040). Identified as ubiquitin binding motifs, the CUE domains are small, moderately-conserved domains of about 40 amino acid residues that are found in a variety of eukaryotic proteins. CUE domains interact with both mono- and poly-ubiquitin, and have a dual role in mono-and poly-ubiquitin recognition as well as in facilitating intramolecular monoubiquitination. Many important cellular processes are regulated through posttranslational modification by ubiquitin. In this study, we performed yeast two-hybrid screen to identify CUEDC2-interacting proteins and preliminary analysis on the biological functions of the interaction, for investigate the function of CUEDC2.We used full length of CUEDC2 as baits respectively, to screen human mammary cDNA library. From 185 candidate clones, we finally identified 7 clones that interacted with CUEDC2. Sequencing analysis showed that the seven clones were four different proteins, Homo sapiens THAP domain containing, apoptosis associated protein 3 (THAP3 ); Homo sapiens suppressor of cytokine signaling 3 (SOCS3); Homo sapiens apoptosis associated protein, growth arrest and DNA damage-inducible gene 34 (GADD34) ; Homo sapiens IkB kinase-beta (IKKβ). Full-length cDNA for these proteins was then cloned from a mammary library. The interaction between CUEDC2 and these proteins were confirmed by in vitro GST pull-down assays and in vivo co-immunoprecitipation analysis.To further research the function of CUEDC2, we determine the significance of the interation between CUEDC2 and GADD34, basing on the result of screens. The GADD34 protein was reported to up-regulate the activity of p53. We used luciferase reportor system in eukaryotic cells for analyzing transcriptional activity of p53. Luciferase assays showed that GADD34 specifically up-regulates p53 transcriptional activity in, but CUEDC2 falls to affect the p53 activity. After co-transfection with CUEDC2, GADD34 was more up-regulate the transcriptional activity of p53 in MCF-7 cells. We further demonstrated that CUEDC2 could induce the GADD34 expression specifically in 293T cells. These data suggest that CUEDC2 could up-regulate p53 activity indirectly by induce the GADD34 expression through the interaction with it.In conclusion, our data demonstrated that CUEDC2 interacts with GADD34 and induced the expression of GADD34, then regulate p53 activity indirectly. Therefore, we found the CUEDC2-GADD34 complex providing important insight into the function of CUEDC2 in cell signaling.