FIG. 3.
Enhancement of transactivation capability of p45/NF-E2 by sumoylation. (A) Reporter transactivation by myc-p45 (black bars) and myc-p45 (K368R) (white bars), respectively. 293 cells were transfected with 2 μg of pRBGP2-Luc (map shown on top) and different amounts of the expression plasmid pEF-myc-p45 or pEF-myc-p45 (K368R). The luciferase activities were measured and calculated. Their relative values are shown in the bar histogram. Data shown are expressed as means ± standard errors of the means from at least three independent assays, each carried out in duplicate. The amounts of myc-p45 and myc-p45 (K368R) in different transfectants were compared by immunoblotting. (B) Enhancement of p45-mediated transactivation by coexpressed UBC9. 293 cells were transfected with 2 μg of pRBGP2-Luc, 2 μg of pEF-myc-p45, or 2 μg of pEF-myc-p45 (K368R) and different amounts of the expression plasmid pcDNA-UBC9. Note that the transactivation mediated by myc-p45 (black bars), but not myc-p45 (K368R) (white bars), was enhanced with coexpression of UBC9. The amounts of myc-p45 and myc-p45 (K368R) in different transfectants were compared by immunoblotting. (C) Enhancement of p45-mediated transactivation by coexpressed SUMO-1. 293 cells were transfected with 2 μg of pRBGP2-Luc, 2 μg of pEF-myc-p45, and different amounts of the expression plasmid pCMV-SUMO-1 GG or pCMV-SUMO-1 AA. Note that the transactivation mediated by myc-p45 was enhanced by coexpression of SUMO-1 GG (black bars) but not by that of SUMO-1-AA (white bars). (D) Requirement of K368 for enhancement of p45-mediated transactivation by SUMO. Parallel transfections were done as described above for panel C except that pEF-myc-p45 was replaced with pEF-myc-p45 (K368R). The amounts of myc-p45 (K368R) in different transfectants were estimated by immunoblotting. We have also analyzed the transactivation capability of p45/NF-E2 in K562 cells as affected by coexpression of UBC9 or SUMO-1. Results similar to those seen with 293 cells were obtained, but the effects were smaller due to the low activity of the cytomegalovirus promoter in K562 cells (data not shown).