Loss of HAUSP-Mediated Deubiquitination Contributes to DNA Damage-Induced Destabilization of Hdmx and Hdm2

Molecular Cell, Vol. 18, 565–576, May 27, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.04.024

Loss of HAUSP-Mediated Deubiquitination Contributes to DNA Damage-Induced Destabilization of Hdmx and Hdm2 Erik Meulmeester,1,5 Madelon M. Maurice,2,5 Chris Boutell,3 Amina F.A.S. Teunisse,1 Huib Ovaa,4 Tsion E. Abraham,1 Roeland W. Dirks,1 and Aart G. Jochemsen1,* 1 Department of Molecular and Cell Biology Leiden University Medical Center P.O. Box 9503 2300 RA Leiden The Netherlands 2 Hubrecht Laboratory Center for Biomedical Genetics Uppsalalaan 8 3584 CT Utrecht The Netherlands 3 MRC Virology Unit Church Street Glasgow G11 5JR Scotland United Kingdom 4 The Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam The Netherlands

Summary The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitinproteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2. Introduction The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In response to a variety of stresses, p53 is stabilized and activated, resulting in a controlled activation of genes involved in cell cycle arrest, DNA repair, and/or apoptosis. Be*Correspondence: [email protected] 5 These authors contributed equally to this work.

cause p53 plays a pivotal role in the cell’s fate, its activity in normal unstressed cells is tightly regulated. The main regulator of p53 is the E3 ubiquitin ligase Hdm2, which targets p53 for ubiquitin-dependent proteasomal degradation (Kubbutat et al., 1997; Haupt et al., 1997). At the same time, the hdm2 gene is a transcriptional target of p53, thereby forming a negative feedback loop (Prives, 1998; Michael and Oren, 2002). Hdmx is a structural homolog of Hdm2 and binds p53 with analogous requirements as does Hdm2 (Shvarts et al., 1996; Bottger et al., 1999). Although Hdmx lacks detectable E3 ubiquitin ligase activity in cells and is unable to degrade p53, Hdmx can inhibit transcription activation by p53 (Jackson and Berberich, 2000; Stad et al., 2001). The embryonic lethality of mdmx-knockout mice, which is rescued in a p53 null background, underscores the importance of p53 regulation by Mdmx (Parant et al., 2001; Finch et al., 2002; Migliorini et al., 2002). Because mdm2-knockout mice are also embryonic lethal in a p53-dependent manner (Montes de Oca Luna et al., 1995; Jones et al., 1995), it suggests that in early embryonic life, Mdmx and Mdm2 cannot substitute for one another. Recently, we have shown that the hdmx gene is amplified in a set of breast tumors and functions as an oncogene (Danovi et al., 2004). Because Hdm2 levels were not increased and no mutations in p53 were found in tumors containing amplified hdmx, these results provide evidence for an important role of Hdmx in tumor formation. The Hdmx protein is degraded upon treatment of cells with DNA-damaging agents in an Hdm2-dependent but p53-independent manner (Pan and Chen, 2003; Kawai et al., 2003a). Hdm2 induces degradation of Hdmx via the ubiquitin-dependent proteasomal pathway (Pan and Chen, 2003; de Graaf et al., 2003; Kawai et al., 2003a). Although Hdm2 can induce the degradation of both Hdmx and p53, the protein domains required to mediate their ubiquitination/degradation are different. The RING finger domain of Hdm2 is sufficient to ubiquitinate Hdmx, whereas the ubiquitination of p53 also requires the p53 binding and acidic domain of Hdm2 (Kawai et al., 2003b; de Graaf et al., 2003; Meulmeester et al., 2003). The stability of p53 is not only determined by active ubiquitination but also by deubiquitination. p53 has been shown to interact with HAUSP (also known as USP7), which can lead to its deubiquitination and stabilization (Li et al., 2002). However, HAUSP also indirectly affects p53 stability and activity by associating with and deubiquitinating Hdm2, leading to Hdm2 stabilization. Total ablation of HAUSP expression results in such a strong decrease in Hdm2 expression that p53 levels increase, resulting in p53dependent G1 arrest (Li et al., 2004; Cummins et al., 2004). Because Hdm2 also targets Hdmx for ubiquitindependent proteasomal degradation, we wondered whether HAUSP would affect Hdmx stability as well. Here, we show that Hdmx is in a complex with HAUSP, leading to deubiquitination and stabilization of Hdmx. In addition, we provide evidence that the deubiquitination activity of HAUSP toward Hdmx and Hdm2 is im-

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Figure 1. HAUSP Stabilizes p53, Hdm2, and Hdmx (A) U2OS cells (6-well plates) were transfected with expression vectors for HA-p53 (200 ng), with or without Hdm2 (500 ng), in absence or presence of 2 ␮g HAUSP. Western blot was performed on total lysates of the cells harvested 40 hr after transfection. Hdm2 and p53 were detected with the anti-HA antibody; HAUSP was detected with the mouse monoclonal antibody 1G7. Tubulin expression was analyzed as loading control. (B and C) U2OS, H1299 cells were transfected as indicated by using 500 ng HA-Hdmx, 200 ng HA-Hdm2, 1 ␮g or 3 ␮g HAUSP, and 3 ␮g HAUSP (C223S) expression vectors. Hdm2 and HAUSP were detected as in (A); Hdmx was detected with the anti-HA antibody. (D) LS89 cells were incubated with doxycycline (1 ␮g/ml) for the indicated time points. HAUSP was detected with the monoclonal anti-HAUSP antibody 1G7, Hdm2 with 4B2, Hdmx with 6B1A, and p53 with DO-1. Tubulin was analyzed as a loading control. (E) LS89 cells were treated with doxycycline as in (D). RT-PCR was performed on RNA isolated at the indicated time points.

paired after DNA damage, although the general activity of HAUSP is not affected. These results provide a possible mechanism for the temporary instability of Hdm2 and degradation of Hdmx after DNA damage. Results HAUSP Rescues the Degradation of Hdmx by Hdm2 It has recently been shown that HAUSP regulates the stability of p53 and Hdm2, an activity that is dependent on its ubiquitin protease activity (Li et al., 2004; Li et al., 2002; Cummins et al., 2004). Indeed, we also found that coexpression of HAUSP can prevent the Hdm2-mediated degradation of p53, whereas at the same time, Hdm2 levels are increased by ectopic HAUSP expression (Figure 1A). Because Hdmx, like p53, is an important target for the ubiquitin ligase activity of Hdm2, we investigated in a parallel experiment whether HAUSP could also affect the degradation of Hdmx by Hdm2. Hdmx is efficiently degraded by Hdm2 (Figure 1B), but coexpression of HAUSP counteracts the Hdm2-mediated degradation of Hdmx in a dose-dependent manner, whereas the levels of Hdm2 are simultaneously in-

creased by HAUSP. Ectopic Hdmx levels were also slightly elevated upon coexpression of HAUSP in the absence of exogenous Hdm2 (Figure S1 available in the Supplemental Data with this article online). The catalytic mutant of HAUSP (C223S) is inactive in these assays, implying that the rescue of Hdmx degradation by HAUSP is dependent on its function as a deubiquitinating enzyme. Similar results were found in MCF-7 cells (data not shown). To examine whether the increase of Hdmx protein levels by HAUSP is dependent on the presence of p53, similar transfections were performed into the p53-negative H1299 cells. Again, the decrease of Hdmx by Hdm2 is counteracted by HAUSP, implying that the effect of HAUSP is independent of p53 (Figure 1C). Because these experiments were performed with overexpressed Hdmx and Hdm2, we investigated whether endogenous protein levels of Hdmx can also be influenced by HAUSP. For this purpose, we created a derivative from the LS174T colorectal cancer cell line containing a doxycycline-inducible expression vector of Myc-HAUSP (LS89 cell line). The levels of HAUSP increased in time after addition of doxycycline (Figure 1D), resulting in an accumulation of endogenous p53, Hdm2, and also of Hdmx. To exclude the possibility of

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Figure 2. HAUSP Expression Is Essential for Maintenance of Hdmx Protein Level (A) U2OS cells (6 cm dishes) were transfected with 5 ␮g of the indicated pSUPER plasmids. Cells were harvested 72 hr after transfection. The expression of HAUSP, Hdm2, Hdmx, and p53 and tubulin as a loading control were analyzed by Western blot as in Figure 1D. (B) HAUSP RNAi-inducible cells (LS88) were incubated with doxycycline (1 ␮g/ml) for the indicated time points. Total cell extracts were analyzed for the expression of the indicated proteins as described in Figure 1D. (C) HAUSP RNAi-inducible cells (LS88) were either incubated with doxycycline (1 ␮g/ml) for 48 hr or mock treated; subsequently, the cells were incubated for the indicated time points with cycloheximide (50 ␮g/␮l). (D) Total cell extracts of control HCT116 cells and HCT116 HAUSP−/− cells were analyzed as in Figure 1D. (E) U2OS cells were transfected with HA-Hdmx (200 ng) or HA-p53 (50 ng) expression vectors, without or with increasing amounts of HAHdm2 (100 ng, 200 ng, and 400 ng) expression plasmid. The expression of HA-Hdmx, HA-Hdm2, and HA-p53 proteins was analyzed by Western blotting with the use of anti-HA.

transcriptional control of the hdmx gene by HAUSP, RTPCR analysis was performed on RNA isolated from treated LS89 cells, which showed no increase in the levels of hdmx mRNA (Figure 1E) or p53 mRNA (data not shown). These results indicate that HAUSP rescues Hdmx from degradation by Hdm2 and that HAUSP increases the endogenous protein levels of Hdmx. HAUSP Is Critical to Maintain Normal Hdmx Protein Levels To investigate a putative function for endogenous HAUSP in the regulation of Hdmx protein stability, we employed RNA interference to reduce HAUSP expression. U2OS cells were transiently transfected with pSUPER constructs expressing siRNA’s targeting HAUSP. pSUPERMdmx was transfected as control for a-specific effects. RNAi-mediated reduction of HAUSP resulted in a decrease of Hdm2, p53, and of Hdmx expression levels (Figure 2A). Previous reports indicated that a limited re-

duction of HAUSP, as observed in Figure 2A, leads to a decrease in p53 levels, whereas a further reduction or complete ablation of HAUSP expression results in enhanced p53 levels (Li et al., 2004; Cummins et al., 2004). In the latter situation, the increase in p53 levels is attributed to the fact that the absence of HAUSP results in such a dramatic reduction of Hdm2 levels, leaving Hdm2 insufficient to degrade p53. To investigate whether a similar, indirect regulation also occurs on Hdmx, we made use of two cell systems. First, three different doxycycline-inducible HAUSP siRNA expression constructs were stably integrated into a derivative of the colorectal cancer cell line LS174 expressing the tetracycline repressor (Van de Wetering et al., 2002). These three different RNAi constructs did not induce an interferon response on five different targets (ISG20, ISG F3, OAS L, OAS 1, and OAS 3) (data not shown). Incubation of these cell lines (LS88, LS125, and LS126) with doxycycline resulted in a significant reduction of HAUSP ex-

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pression over time (Figure 2B and Figure S2). The levels of Hdm2 significantly decreased by 24 hr after doxycycline treatment, whereas the levels of p53 slightly increased by 24 hr and noticeably increased after 48 and 72 hr of treatment. Interestingly, the levels of Hdmx continued to decrease over time, with similar kinetics to the reduction in HAUSP expression. It is important to note that the parental cells (LS174TR1) did not show a decrease in HAUSP, Hdmx, Hdm2, and p53 levels upon doxycycline addition. To investigate whether the reduction of Hdmx protein level is caused by a decreased protein half-life, a cycloheximide chase was performed with the LS88 cells. Although Hdmx is very stable in the mock-treated cells (Figure 2C, top panels), reducing HAUSP levels by incubation with doxycycline results in a decrease of Hdmx protein half-life (Figure 2C, bottom panels). This result indicates that HAUSP affects Hdmx protein levels by increasing its stability. Because p53 and Hdmx are both targets for Hdm2-mediated ubiquitination and HAUSP-controlled deubiquitination, the continuing decrease in Hdmx levels upon HAUSP depletion, in contrast to the increase in p53 levels (Figure 2B), was unexpected. Even though HAUSP levels are strongly reduced in the LS88 cell line upon doxycycline induction, still some HAUSP is remaining. Therefore, we also made use of HCT116 cells in which the HAUSP gene was homozygously disrupted (Cummins et al., 2004). Confirming previous results, we find that p53 expression is strongly increased in the HCT116/HAUSP−/− cells compared to control HCT116 cells, whereas Hdm2 levels are decreased (Figure 2D). Strikingly, Hdmx levels are dramatically decreased to a virtually undetectable level in the HCT116/HAUSP−/− cells (Figure 2D), confirming the results obtained with the LS88 cells after induction of HAUSP siRNA (Figure 2B). These results suggest that the strongly reduced level of Hdm2 upon HAUSP reduction is sufficient to ubiquitinate and degrade Hdmx but inadequate for p53 degradation. To investigate whether Hdmx is indeed more susceptible to Hdm2-mediated degradation than p53, U2OS cells were transfected with HA-Hdmx or HA-p53 expression vectors with increasing amounts of HA-Hdm2 expression vector. We found that Hdm2 was more efficient at degrading Hdmx compared to p53 (Figure 2E; compare lanes 1 and 3 with lanes 5 and 8; in lanes 3 and 8, Hdm2 expression is comparable). With the same amount of transfected plasmid, the Hdm2 expression is higher when Hdmx is coexpressed compared to coexpression with p53, because Hdmx is stabilizing Hdm2, as we reported before (Stad et al., 2001). These results indicate that HAUSP expression is essential for correct regulation of Hdmx protein levels.

HAUSP Interacts with Hdmx To examine whether regulation of Hdmx protein levels by HAUSP was the result of an interaction between HAUSP and Hdmx, we expressed HA-Hdmx and MycHAUSP in U2OS cells and immunoprecipitated protein complexes with anti-Myc or with anti-Hdmx antibodies. Both HAUSP and Hdmx were deteceted in the appropriate immunoprecipitates, indicating that HAUSP and Hdmx can be found in the same protein complex (Fig-

ure 3A). To obtain more insight into the interaction between HAUSP and Hdmx, GST-Hdmx pull-downs were carried out on 35S-labeled, in vitro-translated HAUSP (Figure 3B). The results that HAUSP binds to both GSTHdmx and to GST-Mdm2, but not GST only, suggest a direct interaction. To determine which domains of Hdmx are required for binding to HAUSP, we made use of several alternative splicing variants of Hdmx, which are schematically depicted in Figure 3C. These splicing variants, which are able to bind p53 with the exception of Hdmx-G (de Graaf et al., 2003 and data not shown), were coexpressed with Myc-HAUSP in U2OS cells, and immunoprecipitations were performed with anti-HAUSP (Figure 3D). These results indicate that the N-terminal, p53 binding domain of Hdmx, expressed by the Hdmx-E form, is sufficient for complex formation with HAUSP. To demonstrate that the interaction between HAUSP and Hdmx is independent of p53, the same experiment was performed with a point mutant of Mdmx (G57A), which is unable to bind p53 (Danovi et al., 2004). Indeed, HAUSP can be found in a complex with Mdmx where p53 is not, again indicating that Mdmx stabilization by HAUSP is independent of p53 (Figure S3). Furthermore, because the Hdmx-G protein, which lacks the p53 binding domain, still interacts with HAUSP, at least one other binding site for HAUSP must be present, analogous to Hdm2, which also contains at least two HAUSP binding sites (Li et al., 2004). The association of endogenously expressed HAUSP and Hdmx proteins was investigated by immunoprecipitations on extracts of three cell lines (MCF-7, C33A, and U2OS). Importantly, HAUSP can be found in the anti-Hdmx immunoprecipitate and vice versa (Figure 3E). These data provide evidence that Hdmx associates with HAUSP in vitro and in vivo, independent of p53. Hdmx Is a Substrate for the Ubiquitin-Specific Protease HAUSP The observation that the catalytic inactive mutant of HAUSP cannot rescue Hdmx from Hdm2-mediated degradation suggested that HAUSP stabilizes Hdmx by deubiquitination. To investigate whether Hdmx can be directly deubiquitinated by HAUSP, we used an in vitro system in which deubiquitination of p53 by HAUSP was shown (Canning et al., 2004). In vitro-translated Hdmx was ubiquitinated by GST-Hdm2, which results in a high molecular weight smear of polyubiquitinated Hdmx species (Figure 4A and Figure S4). The effect of HAUSP was investigated with two conditions. First, different concentrations of HAUSP were added during the ubiquitination incubation, resulting in the inhibition of the formation of high molecular weight Ub-Hdmx species in a dose-dependent manner (see Figure S4). The active site mutant (C223S) of HAUSP did not prevent the formation of Ub-Hdmx species. Because this result does not prove that HAUSP actively deubiquitinates Hdmx, the experiment was also performed post-Hdmx conjugation, i.e., by adding HAUSP after the ubiquitination reaction. To that end, the ubiquitination reaction was stopped by the addition of EDTA, and increasing amounts of purified HAUSP or HAUSP (C223S) were subsequently added. Again, incubation with HAUSP almost completely removed the ubiquitin molecules from

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Figure 3. HAUSP Interacts with Hdmx (A) U2OS cells (9 cm dishes) were transfected with 5 ␮g HA-Hdmx and/or 5 ␮g Myc-HAUSP. The Hdmx proteins were immunoprecipitated with a combination of the anti-Hdmx antibodies p55/p56, and the immunoprecipitates were analyzed by Western blot, with use of the antiMyc antibody to detect HAUSP. Myc-tagged HAUSP was immunoprecipitated with anti-Myc antibody, and HA-Hdmx proteins were detected with the anti-HA antibody. (B) GST-Hdmx, GST-Mdm2, or GST only were incubated with 35S-methionine-labeled, in vitro-translated HAUSP; 10% of the input was loaded as a control. (C) Schematic representations of different splicing variants of Hdmx. (D) Transfection of U2OS cells with 5 ␮g Myc-HAUSP and/or 5 ␮g of the HA-Hdmx splicing variant expression vectors. Immunoprecipitations were performed with the anti-Myc antibody, after which the immunoprecipitates were analyzed with anti-HA. To be able to detect full length Hdmx and Hdmx-E simultaneously, samples were separated on a 15% SDS polyacrylamide gel. (E) Cell extracts from C33A, MCF-7, and U2OS cells were analyzed by immunoprecipitation with the use of a rabbit polyclonal anti-HAUSP, a combination of anti-Hdmx antibodies p55/p56, and a nonimmune antibody as negative control. Hdmx and HAUSP proteins in immunoprecipitates and extracts were detected with anti-HAUSP monoclonal antibody 1G7 and anti-Hdmx monoclonal antibody 6B1A.

Hdmx, whereas the HAUSP (C223S) mutant is inactive (Figure 4A). These data demonstrate that HAUSP is capable of deubiquitinating Hdmx in vitro. To determine whether HAUSP can also deubiquitinate Hdmx in vivo, U2OS cells were transfected with different combinations of HA-Hdmx, Hdm2, and HAUSP expression vectors. Proteasome inhibitor MG132 was added 6 hr prior to harvesting the cells to accumulate ubiquitinated Hdmx species. Hdm2 strongly increased the amount of ubiquitinated Hdmx proteins (Figure 4B, compare lanes 1 and 2). Coexpression of HAUSP results in a major decrease in Ub-Hdmx species, whereas the active site mutant C223S has no effect. The same blot was reprobed with anti-Hdm2, which showed that HAUSP, but not HAUSP (C223S), also deubiquitinates Hdm2 in vivo (Figure 4B). This latter observation confirmed an earlier report showing in vitro deubiquitination of Hdm2 by HAUSP (Li et al., 2004). It should be noted that the HAUSP (C223S) mutant interacted with Hdmx comparable to wild-type (wt) HAUSP (data not shown). A nonrelated deubiquitinating enzyme CYLD was unable to deubiquitinate Hdmx or Hdm2, showing specificity for Hdmx deubiquitination with these experimental

conditions (Figure 4C and data not shown). In addition, CYLD did not affect Hdm2 stability or the Hdm2-mediated degradation of Hdmx (Figure 4D). These results demonstrate that HAUSP can specifically deubiquitinate Hdmx both in vitro and in vivo and provide a potential mechanism for the stabilization effect of HAUSP upon Hdmx. DNA Damage-Induced Degradation of Hdmx Is Not Rescued by HAUSP Previous studies have shown that the Hdmx protein is degraded upon treatment of cells with DNA damaging agents in an Hdm2- and proteasome-dependent manner (Pan and Chen, 2003; Kawai et al., 2003a). Because HAUSP rescues Hdm2-mediated degradation of Hdmx, we wondered whether an increase in HAUSP expression would also prevent the degradation of Hdmx after DNA damage. For this purpose, we made use of the LS89 cell line in which Myc-HAUSP expression can be induced by doxycycline, leading to an increase in Hdmx protein levels (see Figure 1D). These cells were either mock treated or treated with doxycycline (48 hr) and subsequently exposed to the radiomimetic agent neo-

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Figure 4. HAUSP Directly Deubiquitinates Hdmx (A) In vitro deubiquitination of Hdmx by HAUSP. In vitro-translated Hdmx was ubiquitinated by GST-Mdm2 in vitro. After stopping the ubiquitination reaction with EDTA, purified wild-type HAUSP or the HAUSP (C223S) mutant was added and incubation resumed. (B) U2OS cells (6 cm dishes) were transfected with 500 ng of HA-Hdmx, without or with Hdm2 (200 ng), HAUSP (2 ␮g), or HAUSP (C223S) (2 ␮g), all in the presence of 1 ␮g of a His6-tagged ubiquitin expression construct. 24 hr later, cells were treated with MG132 (20 ␮M) for 6 hr prior to harvesting; the His-purified fraction was analyzed for ubiquitinated Hdmx (anti-HA) and ubiquitinated Hdm2 (4B2), whereas the total lysates were used to detect total levels of Hdmx (anti-HA), Hdm2 (4B2), and HAUSP (1G7). (C) U2OS cells were transfected as in (B) with indicated constructs and the use 3 ␮g FLAG-CYLD vector. (D) U2OS cells were transfected as indicated by using 500 ng HA-Hdmx, 200 ng HA-Hdm2, 3 ␮g Myc-HAUSP, or 3 ␮g FLAG-CYLD. Western blot was performed on total lysates. Hdmx and Hdm2 were detected with the anti-HA antibody, HAUSP with anti-Myc antibody, and CYLD with anti-FLAG.

carzinostatin (NCS) for the indicated time periods. Doxycycline treatment increased HAUSP expression levels, resulting in increased p53, Hdm2, and Hdmx protein levels (Figure 5A). Subsequent treatment of these doxycycline-treated cells with NCS did not result in any further increase in p53 levels, whereas induction of p53 by NCS was observed in the mock-treated cells. The appearance of serine15-phosphorylated p53 demonstrates a proper DNA damage response. HAUSP expression was not influenced by DNA damage. Hdm2 levels initially decreased after DNA damage but were restored after 2 hr of NCS treatment, confirming an earlier report (Stommel and Wahl, 2004). Notably, we find a reduction in Hdmx protein levels upon DNA dam-

age, independent of HAUSP expression levels. Similarly, the temporary destabilization of Hdm2 after DNA damage was not rescued by increased HAUSP expression. Similar results were obtained by using etoposide as DNA damaging agent (data not shown). These findings were unexpected, because ectopic HAUSP expression was shown to stabilize both Hdmx and Hdm2 (Figure 4) (Li et al., 2004). These results suggest that DNA damage prevents the stabilization of Hdmx and Hdm2 by HAUSP. To investigate this possibility, LS89 cells were either mock treated or doxycycline treated for 48 hr to induce HAUSP expression. Subsequently, cells were incubated with NCS or mock treated, and 2 hr post NCS treatment, cycloheximide was added,

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Figure 5. DNA Damage Prevents Stabilization of Hdmx by HAUSP (A) LS89 cells were incubated with doxycycline (1 ␮g/ml) for 48 hr to induce HAUSP expression. The cells were subsequently mock treated or treated with 500 ng/ml NCS for the indicated time points. Total cell extracts were analyzed as described in Figure 1D. In addition, the antiphospho-Serine15-p53 antibody was used to monitor the DNA damage response. (B and C) LS89 cells were incubated with doxycycline (1 ␮g/ml) for 48 hr to induce HAUSP expression or mock treated. Subsequently, the cells were treated for 2 hr with NCS (500 ng/ml) or mock treated and then incubated with cycloheximide for the indicated time points. Although the levels of Hdm2 and Hdmx were increased by HAUSP induction, exposures were chosen such that the signals at t = 0 are similar to facilitate comparison of the stability.

and the stability of Hdm2 and Hdmx was analyzed over the appropriate time scale. Because the half-life of Hdm2 is much shorter than that of Hdmx, cells were harvested at shorter intervals to analyze Hdm2 (Figure 5B) compared to Hdmx (Figure 5C). As depicted in Figure 5B, the stability of Hdm2 increased upon HAUSP induction in the absence of DNA damage. However, increased HAUSP expression does not rescue the transient decrease in Hdm2 stability upon DNA damage. Similarly, increased HAUSP expression does not prevent the DNA damage-induced destabilization of Hdmx (Figure 5C). These results suggest that the balance between deubiquitination and ubiquitination of Hdmx and Hdm2 is disturbed after DNA damage. One potential explanation could be that HAUSP cannot deubiquitinate either Hdmx or Hdm2 after DNA damage. To examine this possibility, we performed the in vivo ubiquitination/deubiquitination assay after NCS treatment (Figure 6). As previously shown in Figure 4, in the absence of NCS treatment, HAUSP effectively inhibits Hdm2-mediated ubiquitination of Hdmx and Hdm2 self-ubiquitination (Figure 6A, compare lanes 3 and 5). NCS treatment slightly increases the amount of polyubiquitinated

Hdm2 and Hdmx species, observed as a small increase in the fraction of high molecular weight ubiquitinated proteins (Figure 6A, compare lanes 3 and 4). Importantly, upon DNA damage, HAUSP is less efficient in the deubiquitination of Hdmx and Hdm2 (Figure 6A, compare lanes 5 and 6). We next questioned the mechanism by which the deubiquitination of Hdmx/Hdm2 by HAUSP could be impaired. One possibility would be that DNA damage inactivates the enzymatic activity of HAUSP. To investigate this option, we made use of an HA-tagged ubiquitin (HA-Ub) probe fused to a C-terminal electrophilic trap to measure the general activity of HAUSP. Such a probe has been used to investigate the activity of HAUSP and other ubiquitin proteases in cells (Ovaa et al., 2004). Because we specifically wanted to investigate HAUSP activity and show the specificity for active HAUSP, MCF-7 cells were transfected with HAUSP, catalytic inactive HAUSP (C223S), or mock transfected. After 24 hr, HAUSP was immunoprecipitated from NCS- or mock-treated cells and labeled with the HA-Ub probe. The amount of HAUSP reacting with the HA-Ub probe, indicating the activity of HAUSP, was not attenuated upon DNA damage (Fig-

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Figure 6. DNA Damage Inhibits the Interaction between HAUSP-Hdmx and HAUSP-Hdm2 (A) Transfections were performed into U2OS cells, and cell extracts were analyzed as described for Figure 4B. Cells were treated with MG132 (20 ␮M) for 5 hr and, where indicated, with 500 ng/ml NCS prior to harvesting. (B) Immunoprecipitated HAUSP was incubated with the HA-Ub probe and resolved on SDS-PAGE. Active HAUSP was visualized with antiHA monoclonal antibody, whereas the total amount of immunoprecipitated HAUSP was detected with anti-HAUSP monoclonal antibody. (C) MCF7 cells were treated with NCS (1000 ng/ml) for 3 hr or mock treated. Immunoprecipitations were performed with the anti-rabbit polyclonal HAUSP antibody, after which the immunoprecipitates and total lysates were analyzed as in Figure 1D. (D) Hdmx was immunoprecipitated from mock-treated or NCS-treated (1000 ng/ml) cells. Where indicated, the anti-Hdmx immunoprecipitate was treated with alkaline phosphatase in the presence or absence of phosphatase inhibitors (PI). Each immunoprecipitate was subsequently divided and incubated with in vitro-translated p53 (20%) or in vitro-translated HAUSP (70%), after which the bound proteins were analyzed by gel electrophoresis and autoradiography. 10% of each immunoprecipitation was analyzed by immunoblotting to control for the amount of Hdmx immunoprecipitated.

ure 6B). Transfection of the catalytic inactive mutant did not increase anti-HA signal above mock-transfected cells, indicating the specificity of the HA-Ub probe for the active enzyme. This result indicates that the enzymatic activity of HAUSP is not affected by treatment of the cells with double-strand DNA breaks-inducing agents. To investigate whether changes in subcellular localization of HAUSP and/or Hdmx/Hdm2 upon DNA damage could explain the inability of HAUSP to deubiquitinate Hdmx/Hdm2, we performed immunofluorescence studies. Normal growing MCF-7 cells show both a cytoplasmic and nuclear localization of Hdmx, which is almost exclusively shifted toward nuclear staining after NCS treatment (Figure S5), confirming earlier reports (Pan and Chen, 2003). HAUSP is mainly found in the nucleoplasm, with a minority of the protein

found in PML nuclear bodies, as reported previously (Everett et al., 1997). Upon DNA damage, the localization of HAUSP is not altered and still colocalizes with the PML bodies. Hdm2 is found in the nucleus and increases upon NCS treatment. These results do not explain the inability of HAUSP to deubiquitinate and stabilize Hdmx and Hdm2 upon DNA damage. Because it is very likely that HAUSP needs to interact with Hdmx and Hdm2 to deubiquitinate these proteins, the effect of DNA damage upon these interactions was investigated by immunoprecipitation experiments. To prevent the degradation of Hdmx/Hdm2 after DNA damage, which would complicate the interpretation of the results, the proteasome inhibitor MG132 was added to all cells prior to NCS treatment. Incubation with MG132 alone does not affect the interaction between HAUSP and Hdmx/Hdm2

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Figure 7. A Model for HAUSP Function in the Hdmx, Hdm2, p53 Pathway (A) In normal growing cells, Hdmx stabilizes Hdm2 by inhibition of autoubiquitination, whereas Hdm2 degrades both p53 and Hdmx. The levels of Hdmx, Hdm2, and p53 are regulated by a balance between ubiquitination by Hdm2 and deubiquitination by HAUSP. Therefore, HAUSP impinges directly on p53 but also indirectly via the p53’s negative regulators Hdm2 and Hdmx. (B) Upon DNA damage, the interactions between HAUSP and Hdmx/Hdm2 are impaired, leading to a decrease of Hdmx/Hdm2 levels. This reduction of Hdmx and Hdm2 likely contributes to activation of p53 after DNA damage.

(data not shown). It was found that the amount of immunoprecipitated HAUSP is comparable with or without NCS treatments, as is the amount of coprecipitated p53. However, upon DNA damage, the amount of Hdmx/Hdm2 that is coprecipitated with HAUSP is significantly reduced, whereas the levels of Hdmx/Hdm2 in the cell extracts are not affected by NCS in the presence of MG132 (Figure 6C). We have recently shown that DNA damage-induced ubiquitination of Hdmx is at least partly ATM dependent and can be inhibited by an ATM inhibitor (Pereg et al., 2005). Therefore, we investigated whether the DNA damage-induced reduction of Hdmx/HAUSP interaction could be prevented by caffeine, a well-known inhibitor of ATM/ATR activity. Indeed, we find that the interaction between HAUSP and Hdmx is not significantly impaired after DNA damage in the presence of caffeine (Figure S6), indicating that ATM/ATR-dependent phosphorylation is important for the regulation of the Hdmx/HAUSP interaction. To provide further evidence for a role of Hdmx phosphorylation in the reduction of interaction between Hdmx and HAUSP, Hdmx was immunoprecipitated from mocktreated or NCS-treated cells. The immunoprecipitates were subsequently incubated with in vitro-translated HAUSP or p53 (Figure 6D). As found with the endogenous interaction, Hdmx from NCS-treated cells has less affinity for HAUSP. However, treatment of the immunoprecipitated Hdmx from NCS-treated cells with alkaline phosphatase largely restored the interaction. These results indicate that upon DNA damage, the interaction between HAUSP and Hdm2/Hdmx is weakened, most likely caused by ATM/ATR-dependent phosphorylation of Hdmx, resulting in impaired deubiquitination and thus destabilization of Hdmx and Hdm2. Discussion The results described in this study demonstrate an important role of the HAUSP ubiquitin protease in the control of Hdmx stability. HAUSP interacts with and deubiquitinates Hdmx, thereby counteracting the Hdm2mediated ubiquitination and degradation of Hdmx. Because Hdm2 levels are concomitantly increased with Hdmx upon HAUSP overexpression, HAUSP apparently has a dominant role in protecting Hdmx from Hdm2mediated degradation. A similar situation is observed

in the rescue of Hdm2-induced degradation of p53 by HAUSP (Li et al., 2004). Previous studies on the regulation of p53 and Hdm2 by HAUSP reported a dual effect of HAUSP on p53 stability. HAUSP interacted with and deubiquitinated p53, leading to increased p53 levels. On the other hand, HAUSP expression was found to be necessary to maintain normal Hdm2 levels. Ablation of HAUSP expression strongly destabilized Hdm2, leaving insufficient Hdm2 to ubiquitinate p53, leading to increased p53 levels (Li et al., 2004). Our results add another twist to this story, on the basis of which we propose a model in which HAUSP activity plays an important role in the regulation of not only the half-life of p53 and Hdm2 but also of Hdmx during normal growth conditions and after DNA damage (Figure 7). With the use of similar techniques (RNAi in LS174T, U2OS cells, and MCF-7 cells) and the same cell lines (HCT116 HAUSP−/− and control HCT116 cells) as used in the studies on Hdm2 and p53, we demonstrate that HAUSP is essential for maintaining Hdmx protein levels under normal growth conditions. It implies that two targets for the Hdm2 ubiquitin ligase activity are distinctly regulated. Cells that lack HAUSP have still sufficient levels of Hdm2 to degrade Hdmx, but not p53. This result can, at least partly, be explained by our observation that Hdm2 degrades Hdmx more efficiently than p53. Alternatively, one could argue that, in addition to Hdm2, mammalian cells express another ubiquitin ligase that can stimulate the ubiquitination and degradation of Hdmx, the expression of which is not decreased upon knockdown of HAUSP. However, several lines of evidence indicate an important role of Hdm2 in the regulation of Hdmx protein levels. First, it has been shown that decreasing the endogenous Hdm2 expression by RNAi results in an increase of Hdmx (Linares et al., 2003). Second, a side-by-side comparison of Mdmx expression in wt MEFs, p53−/− MEFs, and p53/mdm2−/− MEFs showed an increased Mdmx expression in the latter cells (Figure S7). Lastly, treatment of cells with DNA damaging agents results in an Hdm2-dependent degradation of Hdmx (Pan and Chen, 2003; Kawai et al., 2003a). Remarkably, we find that ectopically expressed HAUSP is unable to rescue DNA damagemediated degradation of Hdmx. It could be hypothesized that not the decrease in HAUSP activity triggers the degradation of Hdmx but rather an increase in Hdm2 activity. However, Kawai et al. (2003a) have

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shown that an increase in Hdm2 levels is not necessary for DNA damage-induced Hdmx degradation. Furthermore, we recently found that the interaction between Hdmx and Hdm2, as assessed by immunoprecipitations, is not detectably altered after DNA damage (Pereg et al., 2005). Third, we show that at early time points after DNA damage, Hdm2 levels temporarily decrease, confirming the results of Stommel and Wahl (2004). At that time, Hdmx degradation has already started (Figure 5A). All these results argue against an increase in Hdm2 activity directed toward Hdmx leading to its DNA damage-induced degradation. It is important to notice that the transient decrease of Hdm2 is observed at early time points (1 hr after NCS; Figure 5A) and also after the induction of HAUSP expression. Thus, HAUSP is unable to inhibit the autoubiquitination and destabilization of Hdm2 upon DNA damage. This inability to stabilize Hdm2 and Hdmx appears to be caused by reduction in the affinity of HAUSP for Hdmx and Hdm2 and not by a decrease in HAUSP enzymatic activity. Intriguingly, the temporary destabilization of Hdm2 after DNA damage was shown to depend on the activity of DNA damage-induced kinases, ATM, ATR, and/or DNA-PK (Stommel and Wahl, 2004). Moreover, we have recently shown that NCS-induced DNA damage results in multiple phosphorylations of Hdmx, of which one site is a direct target of ATM (S403). Each of these sites is important for Hdmx-mediated ubiquitination and degradation after DNA damage (Pereg et al., 2005). This completely fits with our findings presented here that an ATM/ATR kinase activity is involved in the dissociation of Hdmx and HAUSP (Figure 6C) but that a phospho-mimicking mutation of the direct ATM target in Hdmx is not sufficient to reduce the interaction. These results suggest that multiple phosphorylations may play a role in the loss of Hdmx/HAUSP interaction after DNA damage. In conclusion, our results indicate that HAUSP has an important role in the DNA damageinduced destabilization of Hdmx and Hdm2. Because both Hdmx degradation and Hdm2 destabilization are essential for a proper p53 response after DNA damage (Kawai et al., 2003a; Stommel and Wahl, 2004), the regulation by HAUSP activity clearly plays an essential and intriguing role in the cellular DNA damage stress response. Experimental Procedures Plasmids pSUPER-HAUSP plasmids were a gift from R. Bernards (Brummelkamp et al., 2003), pSUPER-Hdmx has been described elsewhere (Danovi et al., 2004). pSUPER-Mdmx construct contains the target sequence 5#-GAATCTTGTTACATCAGCT-3# and efficiently knocks down mouse Mdmx expression, but not human Hdmx (A.G.J. and E.M., unpublished data). The expression vectors for HA-p53, HAHdmx, HA-Hdmx-G, HA-Hdmx-A, and HA-Hdmx-E have been described previously (de Graaf et al., 2003; Danovi et al., 2004). The inducible RNAi constructs targeting the hausp mRNA were generated as described previously (Van de Wetering et al., 2003). The sequence of the oligonucleotides cloned into the pTER plasmids to generate the LS88, LS125, and LS126 cell lines are available upon request. Plasmids pCL-HAUSP and pCL-HAUSP (C223S) have been described before (Canning et al., 2004; Everett et al., 1997). Full-length, Myc-tagged HAUSP expression construct was generated by cloning HAUSP cDNA into a Myc tag-expressing pcDNA3 plasmid (pGloMyc; Roose et al., 1998) by standard sub-

cloning and PCR-based methods using blunted EcoRI and NotI sites. To generate an inducible version of full-length Myc-HAUSP, Myc-HAUSP cDNA was transferred into a pcDNA4/TO plasmid by using the HindIII and NotI sites. Cell Lines, Cell Culture, Transfections, and In Vivo Ubiquitination HCT116 and H1299 cells were cultured in RPMI medium with 10% fetal bovine serum (FBS). U2OS cells, C33A cells, and MCF7 cells were maintained in DMEM 10% FBS. H1299, U2OS, and MCF7 cells were transfected with the Fugene reagent (Roche Molecular Diagnostics) per the manufacturer’s protocol. LS174TR1 cells, expressing the Tet-repressor, were cultured in RPMI with 10% FBS, blasticidin (10 ␮g/ml; Van de Wetering et al., 2003). The LS174TR1 cells were transfected with pTER-HAUSP or the Tet-inducible MycHAUSP vector. Zeocin-resistant clones were tested by immunoblotting for their ability to downregulate (LS88, LS125, and LS126) or upregulate (LS89) HAUSP expression, respectively, after addition of doxycyline (1 ␮g/ml) to the medium. LS88, LS125, LS126 (inducible RNAi for HAUSP), and LS89 cells (inducible MycHAUSP) were grown on RPMI with 10% FBS under selective pressure (blasticidin [10 ␮g/ml] and zeocin [400 ␮g/ml]). The parental LS174TR1 cells were used as a negative control in all experiments. Analysis of the effects of HAUSP on the in vivo ubiquitination of Hdmx by Hdm2 was performed as described before (Meulmeester et al., 2003). Protein half-life studies were performed by incubating the cells with cycloheximide (CHX; 50 ␮g/ml) for the indicated time points. RNA Isolation and RT-PCR Total RNA was isolated from LS89 and LS174TR1 cells by using the Promega SV RNA Isolation Kit (Promega). cDNA was generated from 2 ␮g total RNA with random hexamers and the Superscript First-Strand Synthesis System (Gibco-BRL). PCR was carried out for 32 cycles for Hdmx with an annealing temperature of 52°C; for GAPDH, the annealing temperature was 60°C with 26 cycles. PCR products were analyzed on ethidium bromide-stained agarose gels. Sequences of primers are available upon request. In Vitro Deubiquitination of Hdmx 35 S-methionine-labeled, in vitro-translated Hdmx was prepared in a T7 polymerase-driven rabbit reticulocyte lysate transcription and translation system (Promega). In vitro ubiquitin ligase and deubiquitination assays were performed as described previously (Canning et al., 2004; Boutell and Everett, 2003). Activity Assay for HAUSP To measure the activity of HAUSP, an HA-tagged ubiquitin with a C-terminal electrophilic trap (vinyl methyl ester) was used, synthesized via an intein-based method as described (Ovaa et al., 2004). HAUSP was immunoprecipitated from MCF-7 cells either mock treated or treated with NCS (500 ng/ml) for 4 hr. Subsequently, the immunoprecipated HAUSP was incubated with the HA-tagged ubiquitin probe for 1 hr at 37°C in Giordano (150) buffer, and the reaction was stopped by adding 3× sample buffer. Samples were analyzed by immunoblotting. Antibodies, Immunoblotting, and Immunoprecipitation The primary antibodies used were as follows: mouse monoclonal antibodies anti-HA (HA.11; Covance Research Products), anti-p53 (DO-1), and anti-Hdm2 (SMP14) from Santa Cruz Biotechnology; anti-myc (9E10, Roche Molecular Diagnostics); anti-FLAG (M2) and antiα-Tubulin (Sigma-Aldrich); anti-lacZ (D19-2F3-2; Roche Molecular Diagnostics); anti-Hdm2 (4B2; gift from Dr. A. Levine); anti-HAUSP (1G7 and 7G9, raised against, respectively, the N terminus and C terminus of HAUSP; M.M.M, unpublished data); and anti-Hdmx (6B1A; Ramos et al., 2001). Furthermore, goat-anti-Hdmx antibody D19 (Santa Cruz Biotechnology), rabbit anti-Hdmx polyclonal antibodies p55 and p56 (Ramos et al., 2001), anti-phospho-p53 (Ser15) rabbit polyclonal antibodies (Cell-Signaling Technology), rabbit polyclonal anti-p53 (FL393, Santa Cruz), anti-PML antibody (5E-10) (Stuurman et al., 1992), rabbit-anti-HAUSP polyclonal antibody (Bethyl Laboratories), and monoclonal anti-phospho H2AX (Upstate Biotechnology Inc.) were used. Caffeine was obtained from Sigma-

HAUSP Critically Regulates Hdmx Protein Levels 575

Aldrich. For immunoprecipitation experiments, cells were lysed in Giordano (150) buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% Triton X-100, and 5 mM EDTA,) with 10% glycerol and protease inhibitors. Immunoprecipitation of Hdmx was performed with a mixture of the rabbit anti-Hdmx polyclonal sera p55/p56. Immunoprecipitation of HAUSP was performed with anti-Myc antibody for transfected Myc-HAUSP or with rabbit polyclonal anti-HAUSP for endogenous HAUSP. Immunoblotting was performed as described (Meulmeester et al., 2003). In Vitro Interaction Assays 35 S-methionine-labeled, in vitro-translated HAUSP was prepared in a T7 polymerase-driven wheat germ-coupled transcription and translation system (Promega). An aliquot was mixed with 5 ␮g of bacterially expressed GST-Hdmx, GST-Mdm2, or GST only and tumbled for 2 hr at 4°C in Giordano (150) buffer and precipitated with glutathione beads. Immunoprecipitated Hdmx from mock-treated or NCS-treated cells was incubated with alkaline phosphatase (CIP, 2 U, Biolabs) for 20 min at 30°C in the presence or absence of phosphatase inhibitors. The immunoprecipitates were incubated with 35S-methioninelabeled, in vitro-translated HAUSP or p53 and tumbled O/N at 4°C in Giordano (150) buffer. Complexes were washed four times in Giordano (150) buffer, resolved by SDS-PAGE, and detected by autoradiography.

Supplemental Data Supplemental Data include Supplemental Experimental Procedures, Supplemental References, and seven figures and are available with this article online at http://www.molecule.org/cgi/ content/full/18/5/565/DC1/.

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Acknowledgments −/−

We’d like to thank B. Vogelstein for providing the HCT116 HAUSP cells; A. Levine, D. Lane, and M. Oren for antibodies and expression plasmids; R. Bernards for pSUPER-HAUSP and CYLD expression plasmids; R. Everett for pCL-HAUSP expression plasmids; K. Vousden for GST-Mdm2 expression plasmid; and Y. Shiloh for providing NCS. Furthermore, financial support by H. Clevers and R. Everett is gratefully acknowledged. We thank J.-C. Marine and P. de Graaf for critically reading the manuscript and W. Helvensteyn, M. Groenewoud, and M. Melis for practical assistance. E.M. is supported by a grant from the Dutch Cancer Society and M.M.M. is supported by a Zon-MW fellowship. Received: December 8, 2004 Revised: March 22, 2005 Accepted: April 28, 2005 Published: May 26, 2005 References Bottger, V., Bottger, A., Garcia-Echeverria, C., Ramos, Y.F., van der Eb, A.J., Jochemsen, A.G., and Lane, D.P. (1999). Comparative study of the p53-mdm2 and p53-MDMX interfaces. Oncogene 18, 189–199.

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