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Modification of MDMX by sumoylation
BBRC Biochemical and Biophysical Research Communications 332 (2005) 702–709 www.elsevier.com/locate/ybbrc
Modification of MDMX by sumoylation Yu Pan, Jiandong Chen * Molecular Oncology Program, H. Lee Moffitt Comprehensive Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA Received 19 April 2005 Available online 10 May 2005
Abstract MDMX is a homolog of MDM2 and is critical for regulating p53 function during mouse development. MDMX level is regulated by MDM2-mediated poly-ubiquitination, which results in its accelerated degradation after DNA damage or expression of ARF. In this report, we demonstrate that MDMX can be modified by conjugation to SUMO-1 both in vivo and in vitro. We found that double mutation of two lysine residues, K254 and K379, abrogated MDMX sumoylation in vivo. Experiments using the sumoylation-deficient MDMX mutant showed that it undergoes normal ubiquitination and degradation by MDM2, normal nuclear translocation and degradation after DNA damage, and inhibits p53 with wild type efficiency. Therefore, sumoylation is not required for several activities of MDMX under our assay conditions. Ó 2005 Elsevier Inc. All rights reserved. Keywords: MDMX; MDM2; p53; ARF; Sumoylation; Ubiquitination; Degradation
The p53 pathway is regulated by multiple mechanisms, which is critical for its ability to respond to stress and suppress tumor formation. P53 turnover is regulated by MDM2, which functions as a ubiquitin E3 ligase to promote p53 ubiquitination and degradation by the proteasome [1]. Stress signals such as DNA damage induce p53 accumulation by phosphorylation of p53 and MDM2 [2]. Mitogenic signals activate p53 by induction of the ARF tumor suppressor encoded by an alternative open reading frame in the p16INK4a locus, which inhibits the E3 ligase function of MDM2 [1,3]. MDM2 also regulates the acetylation of p53 by preventing the binding of p300/CBP or recruitment of HDAC1 [4–6]. Recent studies revealed that p53 is also regulated by the MDM2 homolog MDMX [7]. MDMX shares strong homology to MDM2 at the amino acid sequence level and can bind to p53 and inhibit its transcription function in transient transfection assays. MDMX alone does not promote p53 ubiquitination or degradation in vivo *
Corresponding author. Fax: +1 813 903 6817. E-mail address: jchen@moffitt.usf.edu (J. Chen).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.05.012
[8]. However, formation of MDM2–MDMX heterodimer stimulates the ubiquitin E3 ligase activity of MDM2 for itself and p53 [9,10], suggesting that MDMX may function as a regulator or cofactor of MDM2. Knockout of MDM2 in mice results in embryonic lethality due to hyper-activation of p53 [11]. Several studies showed that MDMX-null mice also die in utero in a p53-dependent fashion, which can be rescued by crossing into the p53-null background [12–14]. Therefore, MDMX is also an important regulator of p53 during embryonic development. MDMX overexpression can lead to transformation in cell culture; MDMX gene amplification and overexpression have been observed in certain tumors with wild type p53, suggesting that it may contribute to p53 inactivation in cancer [15,16]. Recently, p53 and MDM2 have been found to be modified by SUMO-1 conjugation (small ubiquitin-like modifier) [17–20]. Sumoylation of p53 and MDM2 in vitro is carried out by a universal SUMO activating E1 enzyme (SEA1/SEA2 heterodimer) and SUMO conjugating enzyme E2 (ubc9) [21]. SUMO is activated in an ATP-dependent manner by E1, transferred to E2, and subsequently attached to the e-amino group
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of lysines on target proteins [21]. The sumoylated lysine residue in many substrates often locates within a consensus sequence of wKxE where w is an aliphatic residue [22]. Lysine 386 is the major sumoylation site for p53 [17,18], whereas the sumoylation site on MDM2 has not been positively identified [20,23]. The functional role of sumoylation is diverse among different substrates. It has been proposed to be important for regulating the localization, activity, and degradation of target proteins [21]. Initial studies using p53 386 lysine-to-arginine mutant resistant to sumoylation suggested that sumoylation moderately enhances p53 transcription activity [17,18]. Muller et al. [19] found that the p53 386K mutant has slightly impaired apoptotic activity. Other studies did not observe such an effect [24,25], possibly due to different assay conditions. MDM2 sumoylation has been shown to be stimulated by ARF through nucleolar targeting of MDM2 [20,23]. MDM2 and ARF cooperatively stimulate p53 sumoylation [23], suggesting a potential mechanism that contributes to ARF activation of p53. Despite efforts by several laboratories, the sumoylation site on MDM2 has not been determined, possibly due to the presence of multiple alternative sites. The lack of sumoylation-deficient MDM2 mutants prevents further investigation of the function of this modification. Recent work from several laboratories showed that MDMX is regulated by MDM2-mediated ubiquitination and degradation [10,26–28]. Ubiquitination of MDMX leads to increased degradation after DNA damage and expression of ARF [26,27], suggesting that ubiquitination is an important modification on MDMX. In this report, we present evidence that similar to p53 and MDM2, MDMX is also modified by sumoylation. Two lysine residues critical for MDMX sumoylation have been identified. MDMX point mutants that cannot be modified by sumoylation have similar properties as wild type MDMX in several functional assays, suggesting that sumoylation is not required for these MDMX functions.
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carded. Cell lysate (10–50 lg protein) was fractionated by SDS–PAGE and transferred to Immobilon P filters (Millipore). The filter was blocked for 1 h with phosphate-buffered saline (PBS) containing 5% non-fat dried milk, 0.1% Tween 20. MDMX was detected by monoclonal antibody 8C6 [34]; MDM2 was detected by monoclonal antibody 3G9 [32]; and p53 was detected by monoclonal antibody DO-1 (Pharmingen). The filter was developed using ECL-plus reagent (Amersham). In vivo sumoylation and ubiquitination assays. H1299 cells in 9 cm plates were transfected with 5 lg each of His6-SUMO-1 or His6ubiquitin and 5 lg of human MDMX expression plasmids using conventional calcium phosphate precipitation method. Thirty-two hours after transfection, cells from each plate were collected into two aliquots. One aliquot was used for conventional Western blot to confirm the expression of transfected proteins. The second aliquot was used for purification of His6-tagged proteins by Ni2+–NTA beads. The cell pellet was lysed in buffer A (6 M guanidinium–HCl, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris–Cl, pH 8.0, 5 mM imidazole, and 10 mM b-mercaptoethanol) and incubated with Ni2+–NTA beads (Qiagen) for 4 h at room temperature. The beads were washed with buffers A, B (8 M urea, 0.1 M Na2PO4/NaH2PO4, 0.01 M Tris– Cl, pH 8.0, and 10 mM b-mercaptoethanol), C (8 M urea, 0.1 M Na2PO4/NaH2PO4, 0.01 M Tris–Cl, pH 6.3, and 10 mM b-mercaptoethanol), and bound proteins were eluted with buffer D (200 mM imidazole, 0.15 M Tris–Cl, pH 6.7, 30% glycerol, 0.72 M b-mercaptoethanol, and 5% SDS). The eluted proteins were analyzed by Western blot for the presence of MDMX by 8C6. In vitro sumoylation assay. Human MDMX was translated in vitro using the TNT coupled rabbit reticulocyte lysate system (Promega) in the presence of [35S]methionine. Two microliters of in vitro translation product was used in a 20 ll reaction containing 25 mM Tris–Cl (pH 7.5), 4 mM MgCl2, 1 mM DTT, 3 mM ATP, 500 ng purified UbcH9, 5 lg purified SUMO-1, and 16 lg HeLa Fraction II (A.G. Scientific). The mixture was incubated for 2 h at 30 °C, boiled in SDS sample buffer, and fractionated by SDS–PAGE. Radiolabeled proteins were detected by autofluorography. Immunofluorescence staining. Cells cultured on chamber slides were transfected with indicated MDMX expression plasmids using Lipofectamine-Plus reagents (Life Technologies). Twenty-four hours after transfection, cells were fixed with acetone–methanol (1:1) for 3 min at room temperature, blocked with PBS + 10% normal goat serum (NGS) for 20 min, and incubated with 8C6 diluted in PBS + 10% NGS for 2 h. The slides were washed with PBS + 0.1% Triton X-100, incubated with FITC-goat-anti-rabbit IgG in PBS + 10% NGS for 1 h, washed with PBS + 0.1% Triton X-100, and mounted.
Results Materials and methods Cell lines and plasmids. H1299 (non-small cell lung carcinoma, p53null) and U2OS (osteosarcoma, p53 wild type, ARF-deficient) cells were maintained in DMEM with 10% fetal bovine serum. Plasmids expressing His6-ubiquitin, and His6-SUMO-1 and HA-SUMO-1 were kindly provided by Drs. David Lane, Ronald Hay, and Honggang Wang [18,29]. ARF deletion and point mutants were kind gifts from Dr. Yue Xiong [30]. SENP1 was kindly provided by Dr. Edward Yeh [31]. MDM2 mutations were described previously [32,33]. Point mutants of p53 and MDM2 were created using the Quick Change kit from Stratagene. All p53, MDM2, and ARF constructs used in this study were of human origin. Western blot. Cells were lysed in lysis buffer [50 mM Tris–HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.5% NP40, and 1 mM PMSF], centrifuged for 5 min at 10,000g, and the insoluble debris were dis-
MDMX is post-translationally modified by SUMO-1 MDM2 has been demonstrated to be modified by SUMO-1. Since MDMX and MDM2 share significant sequence homology, we speculated that MDMX might also be sumoylated in vivo. To detect MDMX sumoylation, we coexpressed MDMX and His6-SUMO-1 by transient transfection into H1299 cells. MDMX conjugated to His6-SUMO-1 was purified by Ni2+–NTA beads under denaturing conditions and detected by Western blot with MDMX-specific monoclonal antibody 8C6 using a previously described assay [23,34]. The result showed that coexpression of MDMX and His6-SUMO-1 resulted in the appearance of MDMX
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Fig. 1. Sumoylation of MDMX in vivo and in vitro. (A) MDMX was coexpressed with His6-SUMO-1 in H1299 cells by transient transfection. His6sumoylated proteins were purified, and MDMX was detected by Western blotting with 8C6 antibody. The lower panels are control Western blots of whole cell lysate (10% of the amount used for Ni2+–NTA purification). SENP1 is a desumoylating protease and MDM2DRING is a 1–440 fragment of MDM2. Transfection efficiency was verified by expression of cotransfected GFP plasmid. (B) In vitro sumoylation of MDMX. Human MDMX and ARF were translated in vitro in the presence of [35S]methionine. Two microliters of in vitro translation product was incubated with ATP, purified UbcH9, purified SUMO-1, and HeLa E1 fraction. Samples were analyzed by SDS–PAGE and autoradiography.
species at 100 kDa molecular weight, suggesting that MDMX was sumoylated in vivo (Fig. 1A). Expression of the desumoylating enzyme SENP1 completely eliminated the 100 kDa MDMX band [31], consistent with it being a sumoylated form of MDMX. It has been demonstrated that sumoylation of MDM2 can be significantly enhanced by ARF [20,23]. To test if ARF also stimulates the sumoylation of MDMX, ARF was cotransfected together with MDMX and His6-SUMO-1. Similar to MDM2 sumoylation, MDMX sumoylation was also moderately enhanced by the overexpression of ARF (Fig. 1A). We have recently reported that MDM2 functions as an E3 ligase for MDMX poly-ubiquitination [26]. Furthermore, MDM2 expression also stimulates p53 sumoylation [23]. Therefore, we tested whether MDM2 and p53 regulate MDMX sumoylation. The results showed that expression of MDM2 led to degradation of MDMX in a RING finger-dependent fashion as expected. The level of MDMX sumoylation was also reduced accordingly, suggesting that MDM2 does not stimulate MDMX sumoylation (Fig. 1A). Coexpression of wild type p53 significantly reduced transfection efficiency and MDMX expression level due to squelching effects (Fig. 1A). However, the corresponding reduction of sumoylated MDMX level also ruled out a stimulatory effect. Next, we tested whether MDMX sumoylation can be recapitulated in cell-free conditions. In vitro translated MDMX was incubated with HeLa cell fraction containing SUMO E1, purified E2, and ATP using a previously described procedure [23], significant modification of MDMX was observed after addition of purified SUMO-1 (Fig. 1B). Addition of in vitro translated ARF had no effect on MDMX sumoylation level in this
assay, possibly due to insufficient amount and poor binding to MDMX under the assay condition, or that the in vivo stimulatory effect involved additional factors. This result showed that similar to many other sumoylation substrates, MDMX sumoylation can occur efficiently in vitro in the presence of E1 and E2, without an obvious requirement for E3. Identification of MDMX sumoylation sites The consensus sequence for sumoylation has been identified as wKXE, where w represents a large hydrophobic amino acid and K stands for the SUMO conjugating lysine residue. However, despite the presence of such consensus sequences, previous studies were not able to definitively identify the sumoylation site on MDM2 [20,23]. To determine the region containing sumoylation sites on MDMX, a panel of MDMX deletion mutants was generated with N or C terminal truncations (Fig. 2A). The MDMX deletion mutants were coexpressed with His6-SUMO-1 by transient transfection into H1299 cells, purified by Ni2+–NTA beads under denaturing conditions, and detected by Western blot with MDMX polyclonal antibody. The result suggested that major sumoylation sites are contained within the region 250–490 (Fig. 2B). The MDMX sequence contains three sites that conform to the wKXE consensus, lysine 254 (IKVE), 379 (IKKE), and 478 (CKKE) (Fig. 2D). To directly test whether these sites are required for sumoylation, single or combined site-directed mutagenesis of these three lysines was performed. The mutants were tested for their sumoylation efficiency by transient transfection of H1299 cells. MDMX sumoylation was not affected by
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Fig. 2. Mapping of sumoylation sites on MDMX. (A) A panel of MDMX deletion mutants was generated and tested for their sumoylation level. MDMX deletion mutants were coexpressed with His6-SUMO-1 in H1299 cells by transient transfection. His6-sumoylated proteins were purified, and MDMX deletion mutants were detected by Western blotting with a polyclonal rabbit-anti-MDMX antibody. The lower panel is Western blot of whole cell lysate showing the expression of the MDMX deletion mutants (10% of the amount used for Ni2+–NTA purification). (B) Diagram of MDMX mutants and summary of results. Major MDMX sumoylation sites are contained within the 250–490 region. (C) MDMX mutants containing single or compound lysine-to-arginine mutations in residue 254, 379, and 478 were coexpressed with His6-SUMO-1 in H1299 cells by transient transfection. His6-sumoylated proteins were purified, and MDMX was detected by Western blotting with 8C6 antibody. The lower panel is control Western blot of whole cell lysate (10% of the amount used for Ni2+–NTA purification) to confirm expression of MDMX point mutants. (D) Diagram of MDMX mutants with the important lysine residues in bold. Double mutations of lysine 254 and 379 are required for preventing sumoylation of MDMX.
the mutation of any single lysines. However, double mutation of K254 and K379 to arginine abrogated the sumoylation of MDMX (Fig. 2C). Therefore, K254 and K379 are likely to be the major sumoylation sites, which is consistent with the deletion mutant analysis. MDMX sumoylation does not affect its stability and regulation by DNA damage MDMX forms a heterodimer with MDM2, and is poly-ubiquitinated and degraded by MDM2. MDMX is also modified by mono-ubiquitination in the absence of MDM2 expression through an unknown mechanism [26]. To test if blocking MDMX sumoylation affected its ubiquitination, we coexpressed MDMX and His6ubiquitin by transient transfection into H1299 cells. MDMX conjugated to His6-ub was purified by Ni2+–NTA beads under denaturing conditions and detected by Western blot with MDMX-specific monoclo-
nal antibody 8C6. The result showed that K254 and K379 single or double mutations had no significant effect on MDMX mono-ubiquitination levels (Fig. 3A). Interestingly, mutating K478 to arginine led to a significantly higher mono-ubiquitination level either alone or in the triple mutant background. Since K478 is located immediately after the last cysteine residue of the RING finger, its mutation to arginine may have stimulated the weak self-ubiquitination of MDMX [9]. Next, the MDMX sumoylation-deficient double or triple-point mutants were cotransfected with MDM2 in H1299 cells. Poly-ubiquitination of the MDMX mutants by MDM2 was similar to wild type MDMX (Fig. 3A), suggesting that lack of sumoylation did not affect MDMX ubiquitination by MDM2. Surprisingly, the K478R mutant was also poly-ubiquitinated and degraded at similar efficiency compared to wild type MDMX, suggesting that increased basal mono-ubiquitination did not affect poly-ubiquitination and
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Fig. 3. Ubiquitination and degradation of MDMX sumoylation mutants. (A) MDMX point mutants and His6-ubiquitin were coexpressed by transient transfection into H1299 cells. MDMX conjugated to His6-ub was purified by Ni2+–NTA beads under denaturing conditions and detected by Western blot with MDMX antibody 8C6. The lower panels are control Western blots of whole cell lysate to verify uniform expression of MDMX mutants and similar transfection efficiency. Basal mono-ubiquitination of MDMX in the absence of MDM2 cotransfection was elevated in the K478R mutant. Poly-ubiquitination of MDMX in the presence of MDM2 cotransfection was not affected by the mutations. (B) Degradation of MDMX sumoylation mutant. MDMX sumoylation-deficient 254/379R double mutant (1 lg) was cotransfected with increasing amounts of MDM2 plasmid (0.5–2.5 lg) into H1299 cells. The level of MDMX was detected by Western blot with 8C6 antibody.
degradation by MDM2. To further compare the sensitivity of MDMX sumoylation mutants to MDM2-mediated degradation, a titration experiment was performed with a fixed amount of the MDMX plasmid and increasing amounts of MDM2 plasmid in the transfection. The dose-dependent degradation by MDM2 was also similar between wild type and mutant MDMX (Fig. 3B). Previous studies showed that MDMX is destabilized upon DNA damage in an MDM2-dependent fashion [26,27]. Recent work in our laboratory suggested that DNA damage-induced phosphorylation of MDMX may play a role in accelerating MDMX degradation (manuscript in preparation). To test if blocking MDMX sumoylation affected its regulation by DNA damage, U2OS cells were stably transfected with control vector, wild type MDMX, and MDMX sumoylation-deficient mutants. Stable colonies were pooled, treated with 10 Gy gamma radiation, and collected at different time points. We found that both wild type and sumoylation-deficient MDMX were degraded at similar efficiency after DNA damage (Fig. 4A). MDMX is mainly localized in the cytoplasm of cells when expressed at high levels and undergoes nuclear
translocation after DNA damage through both MDM2-mediated and MDM2-independent mechanisms [34]. When compared to wild type MDMX, the sumoylation-deficient MDMX mutants were also localized in the cytoplasm of transfected cells and efficiently translocated into the nucleus after ionizing radiation (Fig. 4B). Therefore, we conclude that sumoylation is not required for the regulation of MDMX localization and stability after DNA damage. In further experiments, we compared the ability of MDMX sumoylation mutants to inhibit p53 transcriptional function. The MDMX mutants were cotransfected with p53 expression plasmid and p53-responsive luciferase reporter into p53-null H1299 cells, additionally, MDMX mutants were cotransfected with p53-luciferase reporter into p53-wild type U2OS cells to determine the effect on endogenous p53 transcriptional activity. In both the tests, the MDMX sumoylation mutants exhibited similar ability to inhibit p53 function compared to wild type. Coimmunoprecipitation experiments with MDM2 and p53 also did not reveal changes in the ability of the sumoylation mutants to bind MDM2 or p53 (data not shown). Therefore, we
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Fig. 4. Degradation and nuclear translocation of MDMX sumoylation mutants after DNA damage. (A) U2OS cells were stably transfected with control vector, wild type MDMX, and MDMX sumoylation-deficient mutants. Pooled stable transfectants were treated with ionizing radiation and collected at different time points. An example at the 6 h time point is shown. The levels of MDMX, MDM2, p53, and actin were detected by Western blot. (B) MDMX mutants were stably transfected into U2OS cells and treated with gamma radiation. Cells were fixed 18 h after irradiation and MDMX localization was determined by staining with 8C6 antibody and FITC-anti-mouse IgG. MDMX sumoylation mutants translocated into the nucleus similar to wild type MDMX. An example of the 254/379R double mutant is shown.
conclude that sumoylation of MDMX is not required for suppression of p53 activity under our experimental conditions.
Discussion The results described above showed that similar to p53 and MDM2, MDMX also undergoes modification by sumoylation. Conjugation to SUMO-1 was detected both in vivo and in vitro. Similar to other sumoylation substrates, MDMX sumoylation in vitro is highly efficient in the presence of E1 and E2 enzymes, suggesting that specific E3 is not required for this reaction. Previous studies failed to identify the lysine residue important for sumoylation on MDM2, possibly due to the presence of multiple alternative sites [20,23]. Through deletion and point mutagenesis, we were able to determine that K254 and K379 are important for MDMX sumoylation. Single mutations on each site had limited effect on MDMX sumoylation, but the K254R/K379R double mutant largely abrogated the modification. Sequence comparison showed that these sites are not conserved on MDM2, suggesting that MDM2 sumoylation involves other lysine residues. Furthermore, mutation of
K254 and K379 did not affect MDMX ubiquitination, eliminating the possibility that sumoylation and ubiquitination compete for the same lysines on MDMX. An unresolved question is the role of sumoylation in the function and regulation of the p53 pathway. Previous studies showed that sumoylation of p53 had very moderate positive effects on its transcription activation function [35]. Inability to identify the sumoylation site on MDM2 hampered investigation of the role of this modification on MDM2 and p53 function. Generation of MDMX sumoylation-deficient mutants in this study enabled us to test whether sumoylation of MDMX is involved in several aspects of MDMX function and regulation. At present, the results suggest that MDMX sumoylation is not required for MDMX ubiquitination and degradation by MDM2. Furthermore, regulation of MDMX degradation and nuclear translocation by DNA damage are not affected by lack of sumoylation. The regions of MDMX that contain the sumoylation sites are not directly involved in binding to p53, therefore it is not surprising that p53 binding and regulation by the MDMX mutants are similar to wild type. Comparison between the level of total MDMX expression in the transfected cells and the amount of sumoylated MDMX recovered by His6-SUMO-1
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purification indicated that only a small fraction of MDMX (<5%) is conjugated to SUMO-1 at any given time. Therefore, it is possible that the modification regulates a specific sub-population of MDMX. Therefore, the impact of this modification on MDMX function may not be evident by the overexpression assays we employed. Although the functional analyses so far have not revealed a requirement for sumoylation in several aspects of MDMX regulation, we cannot rule out a role for sumoylation in regulating other yet to be identified MDMX functions. The findings and reagents described in this report should facilitate future study of the role of sumoylation in regulating MDMX and the p53 pathway.
Acknowledgments We thank the Moffitt Molecular Biology Core for DNA sequence analysis. Plasmids expressing His6-ubiquitin and His6-SUMO-1 were kindly provided by Drs. David Lane and Ronald Hay. SUMO-specific isopeptidase SENP1 was kindly provided by Dr. Edward Yeh. This work was supported by grants from the American Cancer Society and National Institutes of Health to J. Chen.
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Modification of MDMX by sumoylation Yu Pan, Jiandong Chen * Molecular Oncology Program, H. Lee Moffitt Comprehensive Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA Received 19 April 2005 Available online 10 May 2005
Abstract MDMX is a homolog of MDM2 and is critical for regulating p53 function during mouse development. MDMX level is regulated by MDM2-mediated poly-ubiquitination, which results in its accelerated degradation after DNA damage or expression of ARF. In this report, we demonstrate that MDMX can be modified by conjugation to SUMO-1 both in vivo and in vitro. We found that double mutation of two lysine residues, K254 and K379, abrogated MDMX sumoylation in vivo. Experiments using the sumoylation-deficient MDMX mutant showed that it undergoes normal ubiquitination and degradation by MDM2, normal nuclear translocation and degradation after DNA damage, and inhibits p53 with wild type efficiency. Therefore, sumoylation is not required for several activities of MDMX under our assay conditions. Ó 2005 Elsevier Inc. All rights reserved. Keywords: MDMX; MDM2; p53; ARF; Sumoylation; Ubiquitination; Degradation
The p53 pathway is regulated by multiple mechanisms, which is critical for its ability to respond to stress and suppress tumor formation. P53 turnover is regulated by MDM2, which functions as a ubiquitin E3 ligase to promote p53 ubiquitination and degradation by the proteasome [1]. Stress signals such as DNA damage induce p53 accumulation by phosphorylation of p53 and MDM2 [2]. Mitogenic signals activate p53 by induction of the ARF tumor suppressor encoded by an alternative open reading frame in the p16INK4a locus, which inhibits the E3 ligase function of MDM2 [1,3]. MDM2 also regulates the acetylation of p53 by preventing the binding of p300/CBP or recruitment of HDAC1 [4–6]. Recent studies revealed that p53 is also regulated by the MDM2 homolog MDMX [7]. MDMX shares strong homology to MDM2 at the amino acid sequence level and can bind to p53 and inhibit its transcription function in transient transfection assays. MDMX alone does not promote p53 ubiquitination or degradation in vivo *
Corresponding author. Fax: +1 813 903 6817. E-mail address: jchen@moffitt.usf.edu (J. Chen).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.05.012
[8]. However, formation of MDM2–MDMX heterodimer stimulates the ubiquitin E3 ligase activity of MDM2 for itself and p53 [9,10], suggesting that MDMX may function as a regulator or cofactor of MDM2. Knockout of MDM2 in mice results in embryonic lethality due to hyper-activation of p53 [11]. Several studies showed that MDMX-null mice also die in utero in a p53-dependent fashion, which can be rescued by crossing into the p53-null background [12–14]. Therefore, MDMX is also an important regulator of p53 during embryonic development. MDMX overexpression can lead to transformation in cell culture; MDMX gene amplification and overexpression have been observed in certain tumors with wild type p53, suggesting that it may contribute to p53 inactivation in cancer [15,16]. Recently, p53 and MDM2 have been found to be modified by SUMO-1 conjugation (small ubiquitin-like modifier) [17–20]. Sumoylation of p53 and MDM2 in vitro is carried out by a universal SUMO activating E1 enzyme (SEA1/SEA2 heterodimer) and SUMO conjugating enzyme E2 (ubc9) [21]. SUMO is activated in an ATP-dependent manner by E1, transferred to E2, and subsequently attached to the e-amino group
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of lysines on target proteins [21]. The sumoylated lysine residue in many substrates often locates within a consensus sequence of wKxE where w is an aliphatic residue [22]. Lysine 386 is the major sumoylation site for p53 [17,18], whereas the sumoylation site on MDM2 has not been positively identified [20,23]. The functional role of sumoylation is diverse among different substrates. It has been proposed to be important for regulating the localization, activity, and degradation of target proteins [21]. Initial studies using p53 386 lysine-to-arginine mutant resistant to sumoylation suggested that sumoylation moderately enhances p53 transcription activity [17,18]. Muller et al. [19] found that the p53 386K mutant has slightly impaired apoptotic activity. Other studies did not observe such an effect [24,25], possibly due to different assay conditions. MDM2 sumoylation has been shown to be stimulated by ARF through nucleolar targeting of MDM2 [20,23]. MDM2 and ARF cooperatively stimulate p53 sumoylation [23], suggesting a potential mechanism that contributes to ARF activation of p53. Despite efforts by several laboratories, the sumoylation site on MDM2 has not been determined, possibly due to the presence of multiple alternative sites. The lack of sumoylation-deficient MDM2 mutants prevents further investigation of the function of this modification. Recent work from several laboratories showed that MDMX is regulated by MDM2-mediated ubiquitination and degradation [10,26–28]. Ubiquitination of MDMX leads to increased degradation after DNA damage and expression of ARF [26,27], suggesting that ubiquitination is an important modification on MDMX. In this report, we present evidence that similar to p53 and MDM2, MDMX is also modified by sumoylation. Two lysine residues critical for MDMX sumoylation have been identified. MDMX point mutants that cannot be modified by sumoylation have similar properties as wild type MDMX in several functional assays, suggesting that sumoylation is not required for these MDMX functions.
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carded. Cell lysate (10–50 lg protein) was fractionated by SDS–PAGE and transferred to Immobilon P filters (Millipore). The filter was blocked for 1 h with phosphate-buffered saline (PBS) containing 5% non-fat dried milk, 0.1% Tween 20. MDMX was detected by monoclonal antibody 8C6 [34]; MDM2 was detected by monoclonal antibody 3G9 [32]; and p53 was detected by monoclonal antibody DO-1 (Pharmingen). The filter was developed using ECL-plus reagent (Amersham). In vivo sumoylation and ubiquitination assays. H1299 cells in 9 cm plates were transfected with 5 lg each of His6-SUMO-1 or His6ubiquitin and 5 lg of human MDMX expression plasmids using conventional calcium phosphate precipitation method. Thirty-two hours after transfection, cells from each plate were collected into two aliquots. One aliquot was used for conventional Western blot to confirm the expression of transfected proteins. The second aliquot was used for purification of His6-tagged proteins by Ni2+–NTA beads. The cell pellet was lysed in buffer A (6 M guanidinium–HCl, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris–Cl, pH 8.0, 5 mM imidazole, and 10 mM b-mercaptoethanol) and incubated with Ni2+–NTA beads (Qiagen) for 4 h at room temperature. The beads were washed with buffers A, B (8 M urea, 0.1 M Na2PO4/NaH2PO4, 0.01 M Tris– Cl, pH 8.0, and 10 mM b-mercaptoethanol), C (8 M urea, 0.1 M Na2PO4/NaH2PO4, 0.01 M Tris–Cl, pH 6.3, and 10 mM b-mercaptoethanol), and bound proteins were eluted with buffer D (200 mM imidazole, 0.15 M Tris–Cl, pH 6.7, 30% glycerol, 0.72 M b-mercaptoethanol, and 5% SDS). The eluted proteins were analyzed by Western blot for the presence of MDMX by 8C6. In vitro sumoylation assay. Human MDMX was translated in vitro using the TNT coupled rabbit reticulocyte lysate system (Promega) in the presence of [35S]methionine. Two microliters of in vitro translation product was used in a 20 ll reaction containing 25 mM Tris–Cl (pH 7.5), 4 mM MgCl2, 1 mM DTT, 3 mM ATP, 500 ng purified UbcH9, 5 lg purified SUMO-1, and 16 lg HeLa Fraction II (A.G. Scientific). The mixture was incubated for 2 h at 30 °C, boiled in SDS sample buffer, and fractionated by SDS–PAGE. Radiolabeled proteins were detected by autofluorography. Immunofluorescence staining. Cells cultured on chamber slides were transfected with indicated MDMX expression plasmids using Lipofectamine-Plus reagents (Life Technologies). Twenty-four hours after transfection, cells were fixed with acetone–methanol (1:1) for 3 min at room temperature, blocked with PBS + 10% normal goat serum (NGS) for 20 min, and incubated with 8C6 diluted in PBS + 10% NGS for 2 h. The slides were washed with PBS + 0.1% Triton X-100, incubated with FITC-goat-anti-rabbit IgG in PBS + 10% NGS for 1 h, washed with PBS + 0.1% Triton X-100, and mounted.
Results Materials and methods Cell lines and plasmids. H1299 (non-small cell lung carcinoma, p53null) and U2OS (osteosarcoma, p53 wild type, ARF-deficient) cells were maintained in DMEM with 10% fetal bovine serum. Plasmids expressing His6-ubiquitin, and His6-SUMO-1 and HA-SUMO-1 were kindly provided by Drs. David Lane, Ronald Hay, and Honggang Wang [18,29]. ARF deletion and point mutants were kind gifts from Dr. Yue Xiong [30]. SENP1 was kindly provided by Dr. Edward Yeh [31]. MDM2 mutations were described previously [32,33]. Point mutants of p53 and MDM2 were created using the Quick Change kit from Stratagene. All p53, MDM2, and ARF constructs used in this study were of human origin. Western blot. Cells were lysed in lysis buffer [50 mM Tris–HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.5% NP40, and 1 mM PMSF], centrifuged for 5 min at 10,000g, and the insoluble debris were dis-
MDMX is post-translationally modified by SUMO-1 MDM2 has been demonstrated to be modified by SUMO-1. Since MDMX and MDM2 share significant sequence homology, we speculated that MDMX might also be sumoylated in vivo. To detect MDMX sumoylation, we coexpressed MDMX and His6-SUMO-1 by transient transfection into H1299 cells. MDMX conjugated to His6-SUMO-1 was purified by Ni2+–NTA beads under denaturing conditions and detected by Western blot with MDMX-specific monoclonal antibody 8C6 using a previously described assay [23,34]. The result showed that coexpression of MDMX and His6-SUMO-1 resulted in the appearance of MDMX
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Fig. 1. Sumoylation of MDMX in vivo and in vitro. (A) MDMX was coexpressed with His6-SUMO-1 in H1299 cells by transient transfection. His6sumoylated proteins were purified, and MDMX was detected by Western blotting with 8C6 antibody. The lower panels are control Western blots of whole cell lysate (10% of the amount used for Ni2+–NTA purification). SENP1 is a desumoylating protease and MDM2DRING is a 1–440 fragment of MDM2. Transfection efficiency was verified by expression of cotransfected GFP plasmid. (B) In vitro sumoylation of MDMX. Human MDMX and ARF were translated in vitro in the presence of [35S]methionine. Two microliters of in vitro translation product was incubated with ATP, purified UbcH9, purified SUMO-1, and HeLa E1 fraction. Samples were analyzed by SDS–PAGE and autoradiography.
species at 100 kDa molecular weight, suggesting that MDMX was sumoylated in vivo (Fig. 1A). Expression of the desumoylating enzyme SENP1 completely eliminated the 100 kDa MDMX band [31], consistent with it being a sumoylated form of MDMX. It has been demonstrated that sumoylation of MDM2 can be significantly enhanced by ARF [20,23]. To test if ARF also stimulates the sumoylation of MDMX, ARF was cotransfected together with MDMX and His6-SUMO-1. Similar to MDM2 sumoylation, MDMX sumoylation was also moderately enhanced by the overexpression of ARF (Fig. 1A). We have recently reported that MDM2 functions as an E3 ligase for MDMX poly-ubiquitination [26]. Furthermore, MDM2 expression also stimulates p53 sumoylation [23]. Therefore, we tested whether MDM2 and p53 regulate MDMX sumoylation. The results showed that expression of MDM2 led to degradation of MDMX in a RING finger-dependent fashion as expected. The level of MDMX sumoylation was also reduced accordingly, suggesting that MDM2 does not stimulate MDMX sumoylation (Fig. 1A). Coexpression of wild type p53 significantly reduced transfection efficiency and MDMX expression level due to squelching effects (Fig. 1A). However, the corresponding reduction of sumoylated MDMX level also ruled out a stimulatory effect. Next, we tested whether MDMX sumoylation can be recapitulated in cell-free conditions. In vitro translated MDMX was incubated with HeLa cell fraction containing SUMO E1, purified E2, and ATP using a previously described procedure [23], significant modification of MDMX was observed after addition of purified SUMO-1 (Fig. 1B). Addition of in vitro translated ARF had no effect on MDMX sumoylation level in this
assay, possibly due to insufficient amount and poor binding to MDMX under the assay condition, or that the in vivo stimulatory effect involved additional factors. This result showed that similar to many other sumoylation substrates, MDMX sumoylation can occur efficiently in vitro in the presence of E1 and E2, without an obvious requirement for E3. Identification of MDMX sumoylation sites The consensus sequence for sumoylation has been identified as wKXE, where w represents a large hydrophobic amino acid and K stands for the SUMO conjugating lysine residue. However, despite the presence of such consensus sequences, previous studies were not able to definitively identify the sumoylation site on MDM2 [20,23]. To determine the region containing sumoylation sites on MDMX, a panel of MDMX deletion mutants was generated with N or C terminal truncations (Fig. 2A). The MDMX deletion mutants were coexpressed with His6-SUMO-1 by transient transfection into H1299 cells, purified by Ni2+–NTA beads under denaturing conditions, and detected by Western blot with MDMX polyclonal antibody. The result suggested that major sumoylation sites are contained within the region 250–490 (Fig. 2B). The MDMX sequence contains three sites that conform to the wKXE consensus, lysine 254 (IKVE), 379 (IKKE), and 478 (CKKE) (Fig. 2D). To directly test whether these sites are required for sumoylation, single or combined site-directed mutagenesis of these three lysines was performed. The mutants were tested for their sumoylation efficiency by transient transfection of H1299 cells. MDMX sumoylation was not affected by
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Fig. 2. Mapping of sumoylation sites on MDMX. (A) A panel of MDMX deletion mutants was generated and tested for their sumoylation level. MDMX deletion mutants were coexpressed with His6-SUMO-1 in H1299 cells by transient transfection. His6-sumoylated proteins were purified, and MDMX deletion mutants were detected by Western blotting with a polyclonal rabbit-anti-MDMX antibody. The lower panel is Western blot of whole cell lysate showing the expression of the MDMX deletion mutants (10% of the amount used for Ni2+–NTA purification). (B) Diagram of MDMX mutants and summary of results. Major MDMX sumoylation sites are contained within the 250–490 region. (C) MDMX mutants containing single or compound lysine-to-arginine mutations in residue 254, 379, and 478 were coexpressed with His6-SUMO-1 in H1299 cells by transient transfection. His6-sumoylated proteins were purified, and MDMX was detected by Western blotting with 8C6 antibody. The lower panel is control Western blot of whole cell lysate (10% of the amount used for Ni2+–NTA purification) to confirm expression of MDMX point mutants. (D) Diagram of MDMX mutants with the important lysine residues in bold. Double mutations of lysine 254 and 379 are required for preventing sumoylation of MDMX.
the mutation of any single lysines. However, double mutation of K254 and K379 to arginine abrogated the sumoylation of MDMX (Fig. 2C). Therefore, K254 and K379 are likely to be the major sumoylation sites, which is consistent with the deletion mutant analysis. MDMX sumoylation does not affect its stability and regulation by DNA damage MDMX forms a heterodimer with MDM2, and is poly-ubiquitinated and degraded by MDM2. MDMX is also modified by mono-ubiquitination in the absence of MDM2 expression through an unknown mechanism [26]. To test if blocking MDMX sumoylation affected its ubiquitination, we coexpressed MDMX and His6ubiquitin by transient transfection into H1299 cells. MDMX conjugated to His6-ub was purified by Ni2+–NTA beads under denaturing conditions and detected by Western blot with MDMX-specific monoclo-
nal antibody 8C6. The result showed that K254 and K379 single or double mutations had no significant effect on MDMX mono-ubiquitination levels (Fig. 3A). Interestingly, mutating K478 to arginine led to a significantly higher mono-ubiquitination level either alone or in the triple mutant background. Since K478 is located immediately after the last cysteine residue of the RING finger, its mutation to arginine may have stimulated the weak self-ubiquitination of MDMX [9]. Next, the MDMX sumoylation-deficient double or triple-point mutants were cotransfected with MDM2 in H1299 cells. Poly-ubiquitination of the MDMX mutants by MDM2 was similar to wild type MDMX (Fig. 3A), suggesting that lack of sumoylation did not affect MDMX ubiquitination by MDM2. Surprisingly, the K478R mutant was also poly-ubiquitinated and degraded at similar efficiency compared to wild type MDMX, suggesting that increased basal mono-ubiquitination did not affect poly-ubiquitination and
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Fig. 3. Ubiquitination and degradation of MDMX sumoylation mutants. (A) MDMX point mutants and His6-ubiquitin were coexpressed by transient transfection into H1299 cells. MDMX conjugated to His6-ub was purified by Ni2+–NTA beads under denaturing conditions and detected by Western blot with MDMX antibody 8C6. The lower panels are control Western blots of whole cell lysate to verify uniform expression of MDMX mutants and similar transfection efficiency. Basal mono-ubiquitination of MDMX in the absence of MDM2 cotransfection was elevated in the K478R mutant. Poly-ubiquitination of MDMX in the presence of MDM2 cotransfection was not affected by the mutations. (B) Degradation of MDMX sumoylation mutant. MDMX sumoylation-deficient 254/379R double mutant (1 lg) was cotransfected with increasing amounts of MDM2 plasmid (0.5–2.5 lg) into H1299 cells. The level of MDMX was detected by Western blot with 8C6 antibody.
degradation by MDM2. To further compare the sensitivity of MDMX sumoylation mutants to MDM2-mediated degradation, a titration experiment was performed with a fixed amount of the MDMX plasmid and increasing amounts of MDM2 plasmid in the transfection. The dose-dependent degradation by MDM2 was also similar between wild type and mutant MDMX (Fig. 3B). Previous studies showed that MDMX is destabilized upon DNA damage in an MDM2-dependent fashion [26,27]. Recent work in our laboratory suggested that DNA damage-induced phosphorylation of MDMX may play a role in accelerating MDMX degradation (manuscript in preparation). To test if blocking MDMX sumoylation affected its regulation by DNA damage, U2OS cells were stably transfected with control vector, wild type MDMX, and MDMX sumoylation-deficient mutants. Stable colonies were pooled, treated with 10 Gy gamma radiation, and collected at different time points. We found that both wild type and sumoylation-deficient MDMX were degraded at similar efficiency after DNA damage (Fig. 4A). MDMX is mainly localized in the cytoplasm of cells when expressed at high levels and undergoes nuclear
translocation after DNA damage through both MDM2-mediated and MDM2-independent mechanisms [34]. When compared to wild type MDMX, the sumoylation-deficient MDMX mutants were also localized in the cytoplasm of transfected cells and efficiently translocated into the nucleus after ionizing radiation (Fig. 4B). Therefore, we conclude that sumoylation is not required for the regulation of MDMX localization and stability after DNA damage. In further experiments, we compared the ability of MDMX sumoylation mutants to inhibit p53 transcriptional function. The MDMX mutants were cotransfected with p53 expression plasmid and p53-responsive luciferase reporter into p53-null H1299 cells, additionally, MDMX mutants were cotransfected with p53-luciferase reporter into p53-wild type U2OS cells to determine the effect on endogenous p53 transcriptional activity. In both the tests, the MDMX sumoylation mutants exhibited similar ability to inhibit p53 function compared to wild type. Coimmunoprecipitation experiments with MDM2 and p53 also did not reveal changes in the ability of the sumoylation mutants to bind MDM2 or p53 (data not shown). Therefore, we
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Fig. 4. Degradation and nuclear translocation of MDMX sumoylation mutants after DNA damage. (A) U2OS cells were stably transfected with control vector, wild type MDMX, and MDMX sumoylation-deficient mutants. Pooled stable transfectants were treated with ionizing radiation and collected at different time points. An example at the 6 h time point is shown. The levels of MDMX, MDM2, p53, and actin were detected by Western blot. (B) MDMX mutants were stably transfected into U2OS cells and treated with gamma radiation. Cells were fixed 18 h after irradiation and MDMX localization was determined by staining with 8C6 antibody and FITC-anti-mouse IgG. MDMX sumoylation mutants translocated into the nucleus similar to wild type MDMX. An example of the 254/379R double mutant is shown.
conclude that sumoylation of MDMX is not required for suppression of p53 activity under our experimental conditions.
Discussion The results described above showed that similar to p53 and MDM2, MDMX also undergoes modification by sumoylation. Conjugation to SUMO-1 was detected both in vivo and in vitro. Similar to other sumoylation substrates, MDMX sumoylation in vitro is highly efficient in the presence of E1 and E2 enzymes, suggesting that specific E3 is not required for this reaction. Previous studies failed to identify the lysine residue important for sumoylation on MDM2, possibly due to the presence of multiple alternative sites [20,23]. Through deletion and point mutagenesis, we were able to determine that K254 and K379 are important for MDMX sumoylation. Single mutations on each site had limited effect on MDMX sumoylation, but the K254R/K379R double mutant largely abrogated the modification. Sequence comparison showed that these sites are not conserved on MDM2, suggesting that MDM2 sumoylation involves other lysine residues. Furthermore, mutation of
K254 and K379 did not affect MDMX ubiquitination, eliminating the possibility that sumoylation and ubiquitination compete for the same lysines on MDMX. An unresolved question is the role of sumoylation in the function and regulation of the p53 pathway. Previous studies showed that sumoylation of p53 had very moderate positive effects on its transcription activation function [35]. Inability to identify the sumoylation site on MDM2 hampered investigation of the role of this modification on MDM2 and p53 function. Generation of MDMX sumoylation-deficient mutants in this study enabled us to test whether sumoylation of MDMX is involved in several aspects of MDMX function and regulation. At present, the results suggest that MDMX sumoylation is not required for MDMX ubiquitination and degradation by MDM2. Furthermore, regulation of MDMX degradation and nuclear translocation by DNA damage are not affected by lack of sumoylation. The regions of MDMX that contain the sumoylation sites are not directly involved in binding to p53, therefore it is not surprising that p53 binding and regulation by the MDMX mutants are similar to wild type. Comparison between the level of total MDMX expression in the transfected cells and the amount of sumoylated MDMX recovered by His6-SUMO-1
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purification indicated that only a small fraction of MDMX (<5%) is conjugated to SUMO-1 at any given time. Therefore, it is possible that the modification regulates a specific sub-population of MDMX. Therefore, the impact of this modification on MDMX function may not be evident by the overexpression assays we employed. Although the functional analyses so far have not revealed a requirement for sumoylation in several aspects of MDMX regulation, we cannot rule out a role for sumoylation in regulating other yet to be identified MDMX functions. The findings and reagents described in this report should facilitate future study of the role of sumoylation in regulating MDMX and the p53 pathway.
Acknowledgments We thank the Moffitt Molecular Biology Core for DNA sequence analysis. Plasmids expressing His6-ubiquitin and His6-SUMO-1 were kindly provided by Drs. David Lane and Ronald Hay. SUMO-specific isopeptidase SENP1 was kindly provided by Dr. Edward Yeh. This work was supported by grants from the American Cancer Society and National Institutes of Health to J. Chen.
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