Ligand binding reduces SUMOylation of the peroxisome proliferator-activated receptor γ (PPARγ) activation function 1 (AF1) domain

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor regulating adipogenesis, glucose homeostasis and inflammatory responses. The activity of PPARγ is controlled by post-translational modifications including SUMOylation and phosphorylation that affects its biological and molecular functions. Several important aspects of PPARγ SUMOylation including SUMO isoform-specificity and the impact of ligand binding on SUMOylation remain unresolved or contradictory. Here, we present a comprehensive study of PPARγ1 SUMOylation. We show that PPARγ1 can be modified by SUMO1 and SUMO2. Mutational analyses revealed that SUMOylation occurs exclusively within the N-terminal activation function 1 (AF1) domain predominantly at lysines 33 and 77. Ligand binding to the C-terminal ligand-binding domain (LBD) of PPARγ1 reduces SUMOylation of lysine 33 but not of lysine 77. SUMOylation of lysine 33 and lysine 77 represses basal and ligand-induced activation by PPARγ1. We further show that lysine 365 within the LBD is not a target for SUMOylation as suggested in a previous report, but it is essential for full LBD activity. Our results suggest that PPARγ ligands negatively affect SUMOylation by interdomain communication between the C-terminal LBD and the N-terminal AF1 domain. The ability of the LBD to regulate the AF1 domain may have important implications for the evaluation and mechanism of action of therapeutic ligands that bind PPARγ.

Figure 1. Analyzing SUMOylation of PPARγ.

(A) PPARγ domain structure. PPARγ2 differs from PPARγ1 by a 30 amino acid extension at the N-terminus. The activation function 1 and 2 domains (AF1 and AF2), the DNA-binding domain (DBD) and the ligand-binding domain (LBD) are indicated. Positions of lysines (K) and serines (S) refer to PPARγ2 and PPARγ1, respectively. (B) Schematic outline of the experimental procedure for detecting SUMOylated PPARγ1. HA-PPARγ1 was transfected along with untagged SUMO1, His-SUMO1 or His-SUMO2 in HEK293 or HeLa cells. His-SUMO-conjugated proteins were subsequently purified from cell lysates by Ni-NTA affinity chromatography. SUMOylated HA-PPARγ1 was detected by immunoblotting for the HA-tag. (C) SUMOylation of PPARγ was analyzed as outlined in Figure 1B. PPARγ is SUMOylated by His-SUMO1 and His-SUMO2. (D) SUMOylation of PPARγ by His-SUMO1 in HEK293 or HeLa cells was analyzed as outlined in Figure 1B in the absence and presence of 1 µM rosiglitazone. (E) Upper panel: SUMOylation of PPARγ by His-SUMO2 in HEK293 cells was analyzed as outlined in Figure 1B in the absence and presence of 1 µM GW1929 or 1 µM rosiglitazone. The asterisk indicates a cross-reacting protein. Lower panel: To control for loading, the blot was re-probed with an anti His antibody. S1, untagged SUMO1, His-S1 and His-S2, His-tagged SUMO1 and His-tagged SUMO2; I, Input: 1% of cell lysate; P, Ni-pulldown: 90% of cell lysate.

Figure 2. Ligand binding to the C-terminal LBD reduces SUMOylation of lysine 33 within the N-terminal AF1 domain.

PPARγ mutants were transfected in HEK293 cells and analyzed for His-SUMO2 or His-SUMO1 modification in the absence and presence of ligands as outlined in the legend to Figure 1. (A) SUMOylation of wild type PPARγ and of the PPARγ K77R mutant in the absence and presence of 1 µM rosiglitazone (Rosi). (B) Summary of quantitative Western blot analyses. SUMOylation of wild type PPARγ by His-SUMO2 and of the PPARγ K77R mutant by His-SUMO2 or His-SUMO1 in the absence and presence of rosiglitazone (Rosi) or GW1929 (GW) was analyzed by imager quantification using fluorescence-labeled secondary antibodies. Wild type PPARγ and the PPARγ K77R mutant were analyzed separately. The values obtained for SUMOylated wild type PPARγ or for the PPARγ K77R mutant relative to the input signal in the absence of ligands were arbitrarily set to 1. (C) Analysis of the N-terminal (amino acid 1-256) and the C-terminal domain (amino acid 247-475) of PPARγ for modification by His-SUMO1 or His-SUMO2. (D) Analysis of the PPARγ Δ1-68 K77R mutant for SUMOylation by His-SUMO1 or His-SUMO2 in the absence or presence of rosiglitazone. (E) Analysis of the PPARγ mutants PPARγ K33R, PPARγ K77R, PPARγ K33/77R and PPARγ K33/64/68/77R for modification by His-SUMO2. (F) Analysis of the PPARγ K33/64/68/77R mutant for modification by His-SUMO2 in the absence or presence of rosiglitazone. (G) Analysis of the PPARγ K77R and PPARγ K33/64/68R mutants for modification by His-SUMO2 in the absence or presence of rosiglitazone. (H) Model depicting interdomain communication regulating SUMOylation of PPARγ. Ligands reduce SUMOylation of K33 and potentially of K64 and K68 but not of K77.

Figure 3. PPARγ S82A and S82D mutations do not affect SUMOylation.

The indicated PPARγ K33R, K77R, S82A, S82D, K33R/S82A, K33R/S82D, K77R/S82A and K77R/S82D mutants were transfected in HEK293 cells and analyzed for SUMO modification in the absence and presence of ligands as outlined in the legend to Figure 1. (A) The phosphorylation blocking S82A and the phosphorylation mimicking S82D mutations did not significantly affect SUMOylation of PPARγ at K33 and K77. (B) PPARγ S82A and S82D mutations did not affect rosiglitazone-induced reduction of PPARγ SUMOylation at K33.

Figure 4. Lysine 365 within the LBD is essential for ligand-induced reduction of PPARγ SUMOylation.

(A) (B) and (C) The PPARγ K365R (A) and PPARγ K77/365R (B) mutants were transfected in HEK293 cells and analyzed for His-SUMO2 and His-SUMO1 modification in the absence and presence of ligands as outlined in the legend to Figure 1. The blot shown in the upper left panel of figure 4B was re-probed with an anti-His antibody to control for loading. (C) Summary of quantitative Western blot analysis. SUMOylation of the PPARγ K365R mutant in the absence or presence of rosiglitazone or GW1929 was analyzed by an independent quantitative Western blot analysis using fluorescence-labeled secondary antibodies. The values obtained for SUMOylated PPARγ K365R relative to the input signal in the absence of ligands were arbitrarily set to 1.

Figure 5. Transcriptional activity of PPARγ mutants.

(A) HeLa (top) and RAW264.7 (bottom) cells were transfected with the Aox-tk luciferase reporter construct along with the indicated PPARγ1 lysine mutants. Twenty-four hours after transfection, cells were treated with 1 µM rosiglitazone (+) or the vehicle (-), and incubated for additional 24 hours. The reporter activity in the absence of PPARγ was arbitrarily set to 1. Error bars are mean +/− SD. Statistical significance of activation by PPARγ mutants compared to wild type PPARγ in the absence (*) or presence (⁺) of rosiglitazone was calculated using the Student´s t-test. * and ⁺, p<0.05; ** and ⁺⁺, p<0.005. (B) HEK293 cells were transfected with a 5×UAS-driven luciferase reporter along with expression constructs for Gal4 or Gal4-PPARγ-LBD fusions as indicated. Twenty-four hours after transfection, cells were treated with 1 µM rosiglitazone (Rosi) or 1 µM GW1929 for additional 24 hours. The reporter activity in the absence of Gal4 fusions was arbitrarily set to 1. Error bars are mean +/− SD. Statistical significance of activation by Gal4-LBD and Gal4-LBD-K365R compared to Gal4 was calculated by the Student´s t-test. *, p<0.05; **, p<0.005.

Figure 6. Transrepression activity of PPARγ mutants.

(A) RAW264.7 macrophages were transfected with the iNOS luciferase reporter plasmid along with PPARγ mutants. Forty-two hours after transfection, cells were treated for 6 hours with 1 µg/ml LPS and 1 µM rosiglitazone (Rosi) as indicated. The reporter activities in the presence of LPS were set to 100% promoter activity. (B) Hela cells were transfected with the 3xNF-κB luciferase reporter plasmid along with PPARγ mutants. Twenty-four hours after transfection, cells were treated with 1 µM rosiglitazone (Rosi). Four hours prior lysis, 10 ng/ml interleukin-1β (IL-1ß) was added as indicated. The reporter activities obtained by interleukin-1ß stimulation were set to 100% promoter activity. Error bars are mean +/− SD. Statistics was performed using Student´s t-test. *, p<0.05; **, p<0.005.