Oxidative stress-induced p53 activity is enhanced by a redox-sensitive TP53INP1 SUMOylation

Abstract

Tumor Protein p53-Induced Nuclear Protein 1 (TP53INP1) is a tumor suppressor that modulates the p53 response to stress. TP53INP1 is one of the key mediators of p53 antioxidant function by promoting the p53 transcriptional activity on its target genes. TP53INP1 expression is deregulated in many types of cancers including pancreatic ductal adenocarcinoma in which its decrease occurs early during the preneoplastic development. In this work, we report that redox-dependent induction of p53 transcriptional activity is enhanced by the oxidative stress-induced SUMOylation of TP53INP1 at lysine 113. This SUMOylation is mediated by PIAS3 and CBX4, two SUMO ligases especially related to the p53 activation upon DNA damage. Importantly, this modification is reversed by three SUMO1-specific proteases SENP1, 2 and 6. Moreover, TP53INP1 SUMOylation induces its binding to p53 in the nucleus under oxidative stress conditions. TP53INP1 mutation at lysine 113 prevents the pro-apoptotic, antiproliferative and antioxidant effects of TP53INP1 by impairing the p53 response on its target genes p21, Bax and PUMA. We conclude that TP53INP1 SUMOylation is essential for the regulation of p53 activity induced by oxidative stress.

Figure 1

TP53INP1 is conjugated to SUMO1 on lysine 113. (a) Schematic representation of the two isoforms of TP53INP1. Positions of lysines in the sequence are indicated by red bars. (b) U2OS cells were transfected with GFP-TP53INP1α, GFP-TP53INP1β and 6HF-SUMO1 expressing constructs. Twenty-four hours post transfection, 80% of cells were lysed in Guanidine-HCl containing buffer and 6HF-SUMO1 conjugates were isolated on Ni²⁺-NTA agarose beads. The remaining 20% were lysed in non-denaturing buffer to detect non-modified TP53INP1, as described under Materials and methods. SUMOylated proteins and input sample were resolved by SDS-PAGE, and TP53INP1 was revealed by western blotting using anti-GFP antibodies. (c) U2OS cells were transfected with the wild type (WT) or the K113R mutant TP53INP1α together with the 6HF-SUMO1 construct. 6HF-SUMO1 conjugates were isolated under strong denaturing conditions as in (b), and TP53INP1 was detected using the monoclonal anti-TP53INP1 F8 antibody. (d) U2OS cells were transfected with 6HF-SUMO1, 6HF-Ubiquitin and 6HF-Nedd8 constructs, together with the WT or K113R mutant TP53INP1α. 6HF-modified proteins were isolated as in (b), and TP53INP1 was revealed using the F8 antibody

Figure 2

TP53INP1 SUMOylation depends on oxidative stress and participates in the proapoptotic activity of TP53INP1. (a) U2OS cells stably overexpressing GFP-TP53INP1α were transfected with the 6HF-SUMO1 construct. Eight hours later, cells were treated with 10 mM of NAC during 16 h and 6HF-SUMO1 modified proteins were isolated as described previously. TP53INP1 was revealed using the F8 antibody. (b) TP53INP1α was induced by ponasterone A (PonA) in U2OS cells, which were transfected with the 6HF-SUMO1 construct. Twenty-four hours later, cells were treated with the indicated amounts of H₂O₂ during 15 min. 6HF-SUMO1 modified proteins were isolated and TP53INP1 was revealed using the F8 antibody. (c) U2OS cells were transfected with the WT or K113R mutant GFP-TP53INP1α, or with an empty vector as a control. Transfection efficiency was normalized by GFP epifluorescence, and clonogenicity assays were performed for 2 weeks. The graph represents the number of colonies observed after crystal violet staining at the end of the experiment. Results are presented as the percent of the number of colonies observed with the empty vector ±S.D. Graphs represent data from three independent experiments. *P<0.05. (d) U2OS cells were transfected with the WT or K113R mutant TP53INP1α, or with an empty vector as a control. Sixteen hours later, cells were exposed to 1 mM of H₂O₂ for 1 h, and incubated for another 24-h period. Caspase 3/7 activity was measured using a pro-fluorescent caspase-3/7 consensus substrate. Results are expressed in fold change compared with the control ±S.D. Graphs represent data from three independent experiments.*P<0.05. (e) U2OS cells stably expressing WT or K113R mutant TP53INP1 were seeded on coverslips, and exposed to 1 mM of H₂O₂ for 1 h the day after. Cells were fixed 24 h post treatment, and apoptotic cells were stained using a colorimetric TUNEL assay. The × 20 magnification was used to count the number of TUNEL-positive cells. Data presented are the mean of five fields counting (*P⩽0.05) from two independent experiments (n=2)

Figure 3

SUMOylation is necessary for TP53INP1-mediated activation of p53 transcriptionnal activity. (a) U2OS cells were co-transfected with p53 and a luciferase reporter gene construct p53-TA-luc. Where indicated, the WT or K113R mutant TP53INP1α was co-transfected with the previous constructs. Sixteen hours later, cells were exposed to H₂O₂ 1 mM for 1 h, and incubated for an additional 24-h period, luciferase activity was measured in cell lysates. The graph represents luciferase activity normalized by a transfection efficiency control as described in Materials and Methods; results are expressed as fold changes compared with the control ±S.D. The assay was performed three times in triplicate with comparable results (*P<0.05). (b) U2OS cells were transfected with the WT or K113R mutant TP53INP1, or with an empty vector as a control and 16 h later, cells were exposed to 1 mM of H₂O₂ for 1 h. After incubation for another 12-h period total RNA was extracted using TRIzol (Invitrogen) and mRNA levels of p21, Bax and PUMA were analyzed by RT-qPCR. Results are expressed as fold changes compared with the control ±S.D. The assay was performed three times in triplicate with comparable results (*P<0.05)

Figure 4

SUMOylation is crucial for TP53INP1 interaction with p53 in cellulo under oxidative stress. (a) U2OS cells stably expressing WT TP53INP1 were transfected with p53 expression construct, seeded on coverslips 24 h post transfection and treated with H₂O₂ the day after. Cells were then fixed and stained for PLA immunofluorescence experiments. A positive signal in PLA experiments is observed as red dots and requires the spatial proximity between two proteins (<40 nm). Nuclei are stained by blue-fluorescent DAPI. (b) Quantification of TP53INP1-p53 interaction. Quantification of PLA dots was performed in a cell-by-cell manner using the NIS element AR software. A minimum of 15 cells for each condition has been used. PLAs were performed at least three times with comparable results. Graphs represent data from three independent experiments ±S.D. (*P<0.05). (c) U2OS cells stably expressing wild type or K113R mutant GFP-TP53INP1α were transfected with a p53 expressing construct. TP53INP1 was precipitated with anti-GFP antibodies, immunoprecipitated proteins were fractioned on SDS-PAGE, and p53 was detected by western blotting. Amount of precipitated TP53INP1 and expression levels in input samples were controlled

Figure 5

SUMOylation is not involved in TP53INP1-mediated autophagy. (a) U2OS stably expressing the WT or K113R mutant TP53INP1 were seeded on coverslips, and treated with Bafilomycin A1 100 nM for 6 h to block the autophagic flux. Cells were then fixed and immunostaining was performed using anti-GFP and anti-LC3 antibodies. Green staining represents TP53INP1 and red staining endogenous LC3. Merged images are shown in yellow. (b) U2OS cells were seeded on coverslips and transfected with the WT or K113R mutant TP53INP1, or with an empty vector as a control. Twenty-four hours later, cells were incubated either with rapamycin 10 μM for 6 h followed by EBSS treatment for 30 min, or with Bafilomycin A1 100 nM for 6 h. Cells were then fixed and immunostained using an anti-LC3 antibody. Autophagy was measured by quantification of LC3-positive dots per cell (n=50), data represent LC3 puncta by cell ±S.D. (*P<0.05). (c) U2OS cells were transfected with WT or K113R mutant TP53INP1, or with an empty vector as a control. Cell lysates were prepared and proteins were resolved by SDS-PAGE. Total levels of p62, LC3-I and II, TP53INP1 and β-Tubulin were monitored by western blotting. Right panel shows a densitometric quantification of bands. All assays were performed at least three times with comparable results (*P<0.05). NS, non significant differences

Figure 6

SUMO1 conjugation to TP53INP1 is mediated by Cbx4 and PIAS3, and deconjugation by SENP1, SENP2 and SENP6. (a) U2OS cells expressing TP53INP1α were transfected with 20 nM of siRNA targeting Cbx4 and PIAS3 alone or together with an siRNA targeting the Ubc9. Eight hours later, cells were transfected with the 6HF-SUMO1 construct, and 6HF-SUMO1 modified proteins were isolated by Ni²⁺ pull down 48 h later as indicated in the legend of Figure 1. TP53INP1 was revealed by western blotting using the F8 antibody. (b) U2OS cells stably expressing TP53INP1α wild type or the K113R mutant were transfected with the 6HF-SUMO1 construct, alone or in combination with a 3 × Flag-PIAS3 plasmid. 6HF-SUMO1 modified proteins were isolated by Ni²⁺ pull down, and TP53INP1 was revealed by western blotting using anti-GFP antibody. (c) U2OS cells were transfected with WT TP53INP1α GFP tagged, or with an empty vector as a control. TP53INP1 was precipitated using anti-GFP antibodies, immunoprecipitated proteins were fractioned on SDS-PAGE, and endogenous Cbx4 and PIAS3 was detected by western blotting. Amount of precipitated TP53INP1 and expression levels in input samples were controlled. (d) U2OS cells expressing TP5INP1α were transfected with siRNA targeting SENP1, SENP2 or SENP6 and 8 h later, cells were transfected with the 6HF-SUMO1 construct. The day after, cells were treated with NAC for an additional 24-h period, and 6HF-SUMO1 conjugates were isolated as described above. TP53INP1 was revealed using the anti-GFP antibody. Bottom panel shows a densitometric quantification of bands from the Ni²⁺ pull down assay

Figure 7

Oxidative stress-induced p53 activity by a redox-sensitive TP53INP1 SUMOylation. During oxidative stress, TP53INP1 expression is induced by p53. If stress induces autophagy, then TP53INP1 interacts with LC3 in the cytoplasm, goes to the autophagososmes to displace p62 triggering autophagic-associated cell death. If stress induces oxidation, then TP53INP1 is SUMOylated, interacting with p53 in the nucleus and favoring p53 transcriptional activity on pro-apoptotic genes