Abstract
The aryl hydrocarbon receptor (AhR) repressor (AhRR) inhibits the AhR activity. AhRR acts by competing with AhR for heterodimer formation with the AhR nuclear translocator (Arnt) and preventing the AhR.Arnt complex from binding the xenobiotic-responsive elements. Here, we report that AhRR has three evolutionarily conserved SUMOylation consensus sequences within its C-terminal repression domain and that Lys-542, Lys-583, and Lys-660 at the SUMOylation sites are modified by SUMO-1 in vivo. Arginine mutation of the three lysines results in a significant reduction of transcriptional repression activity. SUMOylation of the three lysine residues is important for the interaction between AhRR and ANKRA2, HDAC4, and HDAC5, which are important corepressors for AhRR. Arnt, a heterodimer partner for AhRR, markedly enhanced the SUMOylation of AhRR. AhRR, but not AhR, also significantly enhanced the SUMOylation of Arnt. The SUMOylation of both AhRR and Arnt is important for the efficient transcriptional repression activity of the AhRR/Arnt heterodimer.
Highlights
Naling pathway [1, 3, 4]
Lysines at Amino Acids 542, 583, and 660 of aryl hydrocarbon receptor repressor (AhRR) Are Modified by SUMO-1—To examine whether these potential SUMO modification sites are SUMOylated in vivo, we replaced the three conserved lysines with arginines to create the AhRR 3KR mutant
When amino acid sequences of the C-terminal region of AhRR were compared among mammals and fish, three SUMOylation sites including minimal adjacent sequences were revealed to be distinctly conserved (Fig. 1B), suggesting that these sequences are involved in some fundamental biological processes
Summary
Plasmids—Constructions of pBOSGAL4DBD-AhRR, pBOSHA-AhRR, pBOSFLAG-ANKRA2, pG3TK-Luc, and plasmids encoding HDAC4-FLAG and HDAC5-FLAG was described previously [8]. pSG5His-SUMO-1 was generated by fusing full-length human SUMO-1 cDNA with the His tag sequence and cloning into the expression vector pSG5 (Stratagene). pBOSHA-SUMO-1 and pBOSHA-SUMO-2 were generated by ligating the full-length human SUMO-1 or SUMO-2 cDNA with the SmaI site of pBOST7-HA. pBOS-Ubc was generated by inserting the EcoRI/SalI fragment of pBSK-mUbc into the pEFBOS vector [11] cleaved with EcoRI/SalI. pCMV3xFLAG-AhRR was constructed by ligating the bluntended EcoRI/SalI fragment from pBSK-mAhRR with the bluntended p3xFLAG-CMV-10 (Sigma). PBOSHA-SUMO-1 and pBOSHA-SUMO-2 were generated by ligating the full-length human SUMO-1 or SUMO-2 cDNA with the SmaI site of pBOST7-HA. To construct pBOSHA-AhR and pBOSHA-Arnt, pBSK-mAhR and pBSKmArnt [1] were digested with HindIII/XbaI and NcoI/BamHI, respectively, and the blunt-ended fragments of mAhR and mArnt were ligated with the SmaI site of pBOST7-HA. Luciferase Assay—Hepa-1, COS-7, or HeLa cells (5.0 ϫ 104 cells/well) were grown in 24-well dishes for 24 h and transfected with the expression plasmids indicated in the figure legends, such as pG3TK-Luc or pXRE4TK-Luc, and the expression plasmids for sea pansy luciferase as an internal control. Immunoblot analysis was performed using anti-FLAG (Sigma), anti-HA (Sigma), anti-SUMO-1 (ALEXIS), anti-Arnt [13], and antiAhRR antibodies [8]. Fluorescence Analysis—Hepa-1 cells were cultured to subconfluency on coverslips and transfected with expression plasmids for EGFP-AhRR (wild type (WT) or 3KR), His-tagged SUMO-1, and Ubc, with or without Arnt. After 48 h of transfection, cells were incubated with Hoechst DNA stain, and fluorescent images were observed using fluorescence microscopy (magnification ϫ1,000)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
