SUMO-specific protease 2 in Mdm2-mediated regulation of p53

Abstract

Genetic analysis in mice has revealed a key genetic pathway, SUMO-specific protease 2 (SENP2)-Mdm2-p53, essential for trophoblast development. Targeted disruption of SENP2 impairs the G-S transition required for mitotic and endoreduplication cell cycles during the expansion of trophoblast stem cells and their differentiation into polyploidy cells, respectively. The disruption disturbed the subcellular distribution and SUMO modification of Mdm2, leading to interference with p53 degradation. Here, we further explore the mechanism underlying SENP2-mediated regulation of Mdm2 in p53-induced cellular stress. We identify a specific isoform of SENP2 necessary and sufficient to negatively regulate the p53-dependent transcription and its related stress responses. This isoform-specific effect is attributed to the differential compartmentalization of SENP2. SUMO conjugation of Mdm2 induces its co-localization and association with SENP2 in promyelocytic leukemia bodies. Biochemical studies show that SENP2 catalyzes the desumoylation process of Mdm2. SENP2-dependent regulation of Mdm2 is sensitive to its p53-binding activity. Our findings led us to propose a mechanism underlying the SENP2-mediated regulation of Mdm2 that is critical for genome integrity in p53-dependent stress responses.

Figure 1

Isoform-specific regulation of p53 by SENP2. (a) Immunoblot analysis shows the p53 level elevated by genetic inactivation of SENP2 in TS cells. (b) The loss of SENP2 induces enhancement of the p53-dependent transcription. The protein level of p53 is downregulated by high levels of SENP2 (c), but not SENP2-M (e) and SENP2-S (g) isoforms. Immunoblot analysis of p53 was performed in HCT116 colon cancer cells containing knockout (p53−/−) and wild-type p53 (p53+/+), with (+) or without (−) transfection of MT-SENP2 isoforms as indicated. Overexpression of SENP2 (d), but not SENP2-M (f) or SENP2-S (h), inhibits the p53-mediated transcription. The p53−/− and p53+/+ HCT116 cells were co-transfected with p53-luc (a luciferase reporter under control of p53-response elements) and MT (−) or MT-SENP2 (+) DNA plasmids. RLA shows the activity of p53-dependent transcription (n=3). Actin level is used as a loading control for immunoblot analysis. (i) Diagram illustrating the three isoforms of SENP2. NLS, nuclear localization sequence; NES, nuclear export signal

Figure 2

SENP2 interferes with the DNA damage-induced cell death and the growth factor-deprived stress mediated by p53. (a) Disruption of p53 results in the survival of HCT116 cells treated with doxorubicin. (b) Immunoblot analysis indicates the expression of SENP2 isoforms, SENP2, SENP2-M and SENP2-S, in HCT116 stably transformed variants, HCT116-SENP2, HCT116-SENP2M and HCT116-SENP2S, respectively. Repression of p53 is detected in majority of the HCT116-SENP2 sublines, but not the HCT116-SENP2M and HCT116-SENP2S derivatives. The number indicates the different stably transformed lines. Actin level is also analyzed as a loading control. (c) The doxorubicin-mediated cell survival problem is lost in the HCT116-SENP2, but not in HCT116-SENP2M and HCT116-SENP2S, cell lines. (d) Expression of the myc-tagged (MT) SENP2, but not SENP-M, SENP2-S or MT, has a preventive effect on the percentage of HCT116 cells undergoing DNA damage-induced apoptosis with doxorubicin treatment for 24 h as determined by immunostaining of activated caspase-3 and TUNEL staining. (e) HCT116-SENP2 variants expressing high levels of SENP2 are able to grow under a growth factor-deprived stress condition, reminiscent to the knockout of p53 in HCT116 cells

Figure 3

Differential localizations of SENP2 isoforms. Three different isoforms of the myc-tagged SENP2, SENP2 (a–d and i–p), SENP2-M (e, f and q–t) and SENP2-S (g, h and u–x) are transiently expressed in C57MG mammary epithelial cells. 3D images (a, c, e and g) of immunostained cells as well their shadow views (b, d, f and h) reveal their subcellular distributions (green). Arrowheads indicate the perinuclear staining of SENP2-M (e and f). (i–x) Sectioned views show double labeling of SENP2 isoforms (green) with subcellular markers (red), lamin B (i–l and u–x), PML (m–p) and GS28 (q–t). Immunostained cells were counterstained by DAPI (blue). Three-color merge images (l, p, t and x), SENP2 and DAPI (i, m, q and u), marker and DAPI (j, n, r and v); SENP2 and marker (k, o, s and w) show localization of SENP2 isoforms in the cell

Figure 4

SUMO conjugation affects the subcellular distribution of Mdm2. HCT116 cells transfected with GFP-tagged Mdm2 (a–c), Mdm2-SUMO (d–f) or Mdm2-SUMO^ΔGG (g–m), were analyzed by fluorescent imaging. (j–m) Co-localization analysis reveals that the nuclear dotted staining of Mdm2-SUMO^ΔGG co-localizes with PML in HCT116 cells

Figure 5

Isoform-specific co-localization of SENP2 with SUMO-conjugated Mdm2. HCT116 cells transfected with GFP-tagged Mdm2 (a–l) or Mdm2-SUMO (m–x), and different isoforms of myc-tagged SENP2, were analyzed by fluorescent imaging. (a–l) Double-labeling analysis shows no co-localization of Mdm2 with SENP2 (a–d), SENP2-M (e–h) and SENP2-S (i–l). (m–p) Triple labeling of cells with Mdm2-SUMO, PML and SENP2 isoforms reveals that SENP2 (m–p), but not SENP2-M (q–t) and SENP2-S (u–x), co-localizes with SUMO-conjugated Mdm2 at the PML bodies

Figure 6

SENP2 associates with Mdm2 and Mdm2-SUMO. Co-immunoprecipitation reveals that SENP2 forms protein complexes with Mdm2 and Mdm2-SUMO. (a) HCT116 cells, which transiently express either Mdm2 or Mdm2-SUMO with or without MT-SENP2, were analyzed by immunoprecipitation with anti-Mdm2 antibodies, followed by immunoblot with anti-MT or anti-Mdm2 antibodies. (b) HCT116-SENP2 cells, which were transfected by GFP-tagged Mdm2 or Mdm2-SUMO, were analyzed by immunoprecipitation with anti-MT antibodies, followed by immunoblot with anti-Mdm2 or anti-MT antibodies. Total cell extracts directly analyzed by immunoblot analysis show the protein expression levels (a and b). Actin level is used as a loading control

Figure 7

SENP2 regulates the SUMO modification of Mdm2. SENP2-mediated desumoylation of Mdm2 is analyzed by the in vivo (a and b) and in vitro (c and d) systems. (a and b) The effect of SENP2 expression on the sumoylation status of endogenous Mdm2 is assayed by IP–IB analysis. The SENP2 expression reduces the sumoylated Mdm2 in the HA-SUMO1 transfected HCT116 cells (a) and HCT116-SENP2 cells (b). Total cell extracts directly analyzed by immunoblot analysis show the protein expression levels. Actin level is used as a loading control. (c) In vitro desumoylation assay shows that the addition of purified MT-SENP2 (+), but not MT (−), diminishes the level of SUMO-conjugated Mdm2 while the presence of SUMO protease inhibitor, NEM, prevents the desumoylation activity. Protein extracts, isolated from the HA-SUMO1-transfected HCT116 cells, were treated with purified SENP2 or MT, with or without the presence of NEM, followed by immunoblot analyses. (d) In vitro reconstitution analysis using recombinant enzymes and substrates reveals that SUMO conjugation of Mdm2 is reversed by purified SENP2. GST-tagged Mdm2 was sumoylated by recombinant Ubc9, SAE1/2 and GST-SUMO1 proteins, followed by desumoylation with purified SENP2

Figure 8

SENP2-dependent SUMO modification of Mdm2 modulates the p53 level. (a) Mdm2 is required for SENP2-dependent regulation of p53. Immunoblot analysis shows the levels of p53 and p21 affected by Mdm2 RNA interference (Mdm2 siRNA) in HCT116 and HCT116-SENP2 cells. (b) SENP2-mediated repression of p53 is sensitive to the inhibition of Mdm2 by Nutlin-3. Immunoblot analysis shows the levels of p53 and p21 affected by the SENP2 expression in HCT116 cells in the presence or absence of Nutlin-3. The expression of SENP2 also reduces the level of Mdm2-SUMO independent of Nutlin-3. (c) Effects of Nutlin-3 on the sumoylation of Mdm2 and Mdm2-mediated regulation of p53 and p21. Immunoblot analysis reveals that Nutlin-3 interferes with the p53 and p21 levels by the expression of MT, Mdm2, Mdm2-SUMO and Mdm2-SUMO^ΔGG in HCT116 cells. (d) The ubiquitin E3 ligase activity is maintained in the fusion proteins of Mdm2 and SUMO1. In vitro ubiquitination assay reveals that purified GFP-tagged Mdm2, Mdm2-SUMO and Mdm2-SUMO^ΔGG, but not GFP, are able to modify a recombinant GST-tagged p53 protein with ubiquitin. Actin level is used as a loading control for immunoblot analysis

Figure 9

DNA damage-induced apoptosis promotes the interaction of Mdm2 and SENP2 at the PML bodies. (a) Triple labeling of Mdm2, PML and activated caspase-3 reveals that Mdm2 accumulates in the PML bodies of cells undergoing p53-dependent apoptosis. Mdm2-GFP was expressed in HCT116 cells without or with the addition of doxorubicin for 24 h. Mdm2 co-localized with PML in cells undergoing apoptosis identified by immunostaining of activated caspase-3. (b) Triple labeling of p53, PML and activated caspase-3 shows that the doxorubicin-induced apoptosis does not promote p53 accumulation in the PML bodies. (c) Graph indicating the percentage of Mdm2-GFP positive cells displaying PML body distribution, and undergoing programmed cell death in the course of doxorubicin treatment. (d) Graph showing the percentage of HCT116-SENP2 cells displaying the PML body distribution of SENP2 in the course of doxorubicin treatment (P-value: ^*P<0.035; ^**P<0.021; ^***P<0.017). (e) Immunoblot and immunoprecipitation–immunoblot analyses reveal that the induction of p53 and p21 coincides with the accumulation of sumoylated Mdm2 upon doxorubicin treatment. Actin level is used as a loading control. (f) DNA damage response promotes accumulations of Mdm2 and SUMO1 in the PML bodies. HCT116 cells transfected with GFP-tagged Mdm2 were cultured in the presence or absence of doxorubicin for 24 h, followed by triple labeling analysis

Figure 10

Model for SENP2-mediated modulation of Mdm2 in regulating p53. The diagram illustrates the mechanism underlying the regulation of Mdm2 by the SUMO pathway to control the cellular level of p53. SENP2 regulates p53 through modulation of Mdm2. SENP2 interacts with sumoylated Mdm2 and regulates its SUMO conjugation at the PML body. The SENP2-dependent SUMO modification acts upstream of the binding of Mdm2 and p53 in the regulatory pathway. Desumoylation of Mdm2 permits its binding and ubiquitination of p53, which is then degraded through the proteolysis system