A SUMOylation/acetylation switch at K13 controls ΔLf transcriptional activity.
(A) K13 is the main acetylation site. Cells were co-transfected by WT, the mutant constructs or the null vector and then lysed 24 h later. Lysates were immunoprecipitated with anti-acetyllysine antibodies and immunoblotted with M2. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control (n = 3). B) Relative transcriptional activity of K13 and K379 mutants compared to WT. Cells were co-transfected with pGL3-S1Skp1-Luc reporter vector and WT, K13 or K379. His-SUMO-1 expression vector and/or the deacetylase inhibitor Trichostatin A (TSA, 15 ng/mL) were used to modulate the acetylation/SUMOylation ratio. Relative luciferase activities are expressed as described in Materials and Methods (n≥3; p < 0.05 (*)). C-D) Modulation of the SUMOylation level was performed either by knocking down Ubc9 using siUbc9 or by overexpressing His-SUMO-1 peptides (SUMO-1). Cells were reverse transfected or not with RNAiMax using 5 nM of siUbc9/siCtrl for 24 h before being transfected for 24 h with WT or K13 plasmid with or without His-SUMO-1 plasmid. Before cell lysis, the acetylation level was altered or not by an overnight treatment with TSA (15 ng/mL). Cells were then incubated with 10 μM of the proteasomal inhibitor MG132 for 2 h prior to lysis. NEM was added to lysis, IP and WB buffers. (C) Input was immunoblotted with anti-Ubc9 (upper panel) or anti-GAPDH (lower panel) antibodies. (D) Samples were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 (upper panel), then with anti-acetyllysine (middle panel) or finally with M2 (lower panel) antibodies. The acetylation/SUMOylation ratio (RAc/SUMO) was assayed as described in Material and Methods. The data presented correspond to one representative experiment of two conducted.