Moreover, ERK2 is not involved in the S118 phosphorylation that takes place following the hER N-terminal region ligand-dependent association with the C-terminal part of the protein (Mtivier phosphorylation assays (Figures?4 and ?and5)

Moreover, ERK2 is not involved in the S118 phosphorylation that takes place following the hER N-terminal region ligand-dependent association with the C-terminal part of the protein (Mtivier phosphorylation assays (Figures?4 and ?and5).5). the activation domain (AD) or DBD of the yeast transcriptional factor Gal4. Subsequent assays shown in Figure?2A confirm the ligand independence of the interaction between the two NRs. A dominant-negative form of COUP-TFI (Adam labeled hER and COUP-TFI using the GSTCCOUP-TFI 57C423 or GSTChER DF proteins. During the incubation, increasing amounts of DR-1, ERE (25C200?ng) and AP-1 as non-specific control (50C200?ng) Benzo[a]pyrene oligonucleotides were added. To obtain further information on the specific mechanisms of interaction between the two NRs, we identified their contacting surfaces. The different domains of COUP-TFI were fused to the Gal4AD, while the CCF (CF) or DCF (DF) domains of hER fused to the Gal4DBD were used as bait proteins (Figure?2B, left). These hER proteins exhibit no intrinsic transcriptional activity that might have interfered Benzo[a]pyrene in these assays. This property allowed us to show the ligand independence of all detected interactions (data not shown). The hER DBD?+?LBD bait (hER CF/Gal4DBD) associates with COUP-TFI N-terminal (amino acids 1C57), C-terminal (amino acids 153C423) and DBD (amino acids 57C153) regions. Performing the two-hybrid assays with the hER LBD alone (hER DF/Gal4DBD) showed that it associates with COUP-TFI N- and C-terminal regions (Figure?2B, right). The absence of the DBD in this hER DF/Gal4DBD bait and its failure to bind the COUP-TFI DBD demonstrate that the DBDs of the two NRs form direct contacts (Figure?2B, right). We next wanted to see if the hER N-terminal domain was able to contact COUP-TFI. When fused to the Gal4DBD, the hER N-terminal domain activates transcription in yeast through its AF-1 (Mtivier et al., 2001). Since such PTGS2 a protein is not informative in two-hybrid assays, we therefore conducted inverse assays. hER AB domains were fused to the Gal4AD, and COUP-TFI domains to the Gal4DBD. These assays demonstrate that the hER N-terminal region associates with the C-terminal part of COUP-TFI (Figure?2C). In conclusion, three surfaces of interaction are identified within the two NRs: one mediated by their DBD, one by their C-terminal region and one involving their N- and C-terminal regions. pull-down assays confirmed the yeast two-hybrid data (see below), indicating that the interaction surfaces used are specific. Correlation between physical interaction and transcriptional cooperation between hER and COUP-TFI We next identified the domains of COUP-TFI involved in the enhancement of hER transcriptional activity to look for a relationship between physical interaction and Benzo[a]pyrene transcriptional cooperation. This direct relationship is suggested first by the fact that the COUP-TFI C141S mutant is not able to enhance hER activity (Figure?2D). The partial or total deletion of the COUP-TFI N-terminal region (57C423 and 87C423 constructs) does not affect the increase in hER activity (Figure?2D). In contrast, deleting the COUP-TFI C-terminal region (57C153 protein) reduces the transcriptional cooperation by 60%. This points to the involvement of the COUP-TFI DBD and LBD in the transcriptional cooperation with hER. This is demonstrated further by the hER activity enhancement by the COUP-TFI DBD alone (residues 57C153) with hER. In the case of the COUP-TFI LBD, we had to fuse the COUP-TFI 153C423 region to the SV40 nuclear localization signal (NLS; Figure?2D) to obtain a correct localization of the protein, as revealed by green fluorescent protein (GFP; not shown). Expression of this GFPCNLS/COUP-TFI 153C423 protein increases hER-mediated transcription on the ERE-TK-Luc reporter by 2-fold. In conclusion, both an intact DBD.