Otub1 stabilizes MDMX and promotes its proapoptotic function at the mitochondria

Otub1 regulates p53 stability and activity via non-canonical inhibition of UbcH5, the MDM2 cognate ubiquitin-conjugating enzyme (E2). However, whether Otub1 regulates MDMX stability and activity is not clear. Here we report that Otub1 also suppresses MDM2-mediated MDMX ubiquitination in cells and in vitro, independently of its deubiquitinating enzyme activity. Consequently, overexpression of Otub1 markedly stabilized MDMX and increased its levels, whereas knockdown of Otub1 reduced the levels of MDMX. Interestingly, MDMX induced by Otub1 can localize to mitochondria in addition to the cytosol, enhance p53 phosphorylation at S46 (p53S46P) and promote mitochondria-mediated apoptotic pathway. Knockdown of MDMX reduced Otub1-induced p53S46P, which was shown to be critical for p53's mitochondrial function and apoptotic activity. Furthermore, Otub1 promotes UV-irradiation-induced p53S46P and apoptosis, which can be significantly inhibited by MDMX depletion. Together, these results suggest that Otub1 stabilizes MDMX and promotes p53S46P and mitochondria-mediated apoptosis, providing an alternative mechanism of Otub1's role in apoptosis.


INTRODUCTION
The tumor suppressor protein p53 inhibits cell growth and proliferation in response to stress by transcriptionally activating or suppressing myriad target genes, whose protein products induce cell cycle arrest, cell death, senescence and other outcomes [1][2][3]. p53 can also directly trigger mitochondria-mediated apoptosis and necrosis through transcription-independent mechanisms [4][5][6][7][8]. p53 directly interacts with anti-apoptotic Bcl-2 family proteins such as Bcl-xL and Bcl-2 at mitochondria to neutralize their inhibition of proapoptotic proteins, leading to Bax or Bak oligomerization [7]. p53 can directly interact with Bax, leading to Bax oligomerization, mitochondria outer membrane permeabilization (MOMP) and cytochrome C release [4]. It can also bind to Bak and disrupt the Bak-MCL1 interaction, causing the oligomerization of Bak and release of cytochrome C as well [9]. A recent study showed that p53 also triggers mitochondria-dependent necrosis in response to oxidative stress by binding to the mitochondrial permeability transition pores (PTPs) [8]. Thus the cytoplasmic p53 plays a key role in negatively regulating cell growth by inducing cell death pathways.
Here we report that Otub1 suppresses MDM2mediated MDMX ubiquitination and degradation. Overexpression of Otub1 markedly stabilized MDMX and induced its levels, whereas knockdown of Otub1 reduced the levels of MDMX. Similar to its regulation of p53, Otub1 stabilizes MDMX independently of its Dub activity. We show that both wild-type Otub1 and its catalyticallyinactive mutant (C91S), suppresses MDM2-mediated MDMX ubiquitination in cells and in vitro. Interestingly, the stabilized MDMX can localize into mitochondria and mediate mitochondrial apoptotic pathway. Knockdown of MDMX reduced Otub1-induced p53 phosphorylation at S46, which was shown to be critical for p53's mitochondria function, and apoptotic activity. Also, Otub1 promotes UV-irradiation-induced apoptosis, which can be inhibited by MDMX depletion. Together, these results suggest that Otub1 also induces mitochondrial-mediated apoptosis by stabilizing MDMX and relocalizing it to mitochondria to induce mitochondria-mediated apoptosis.

Otub1 stabilizes MDMX and increases its levels
To test whether Otub1 regulates MDMX levels, we first performed transient transfection followed by IB analysis in H1299 cells. As shown in Figure 1A and as expected [32,33], overexpression of MDM2 markedly reduced the levels of MDMX (compare lane 3 to lane 2). Further expression of Otub1 (lane 4), but not Otub2 (lane 5), completely abolished this reduction ( Figure 1A). Doxycycline-induced expression of Otub1 in T-Rex-U2OS-Flag-Otub1 cells also drastically induced the levels of MDMX, as well as p53 and its target MDM2 in a timedependent manner ( Figure 1B). This induction of MDMX is due to protein stabilization, as the levels of MDMX mRNA did not change compared to the marked increase of p21 and MDM2 mRNA levels when Otub1 expression is induced ( Figure 1C). Also, induced overexpression of Otub1 drastically prolonged the half-life of MDMX as determined by half-life assays ( Figure 1D). These results suggest that overexpression of Otub1 alleviates MDM2-mediated MDMX degradation. The stabilization of MDMX by Otub1 does not depend on Otub1's deubiquitinating enzyme activity, as both wild-type (wt) and the catalytic-inactive C91S mutant are equally able to induce the levels of MDMX ( Figure 1E). By contrast, the D88A mutant, which is unable to bind to and suppress UbcH5 and unable to induce p53 [39,40], also failed to induce MDMX levels ( Figure 1E), suggesting that this mutant may cause overall structural changes in the OTU domain that abolish Otub1's activity to suppress E2. Consistently, knockdown of endogenous Otub1 by two different siRNAs significantly reduced the levels of MDMX in cells ( Figure 1F). Thus endogenous Otub1 also regulates the MDMX levels.

Otub1 directly suppresses MDM2-mediated MDMX ubiquitination in cells and in vitro
To understand how Otub1 stabilizes MDMX, we tested whether Otub1 suppresses MDM2-mediated MDMX ubiquitination, similar to the suppression of p53 ubiquitination [39,40]. We first performed in vivo ubiquitination assays in H1299 cells using Ni 2+ -NTA purification method. As shown in Figure 2A

Otub1-stabilized MDMX localizes to the mitochondria
As MDMX assists MDM2 to degrade p53, thus inhibiting p53 function [26][27][28][29][30][31] while Otub1 expression stabilizes p53 [39], the Otub1-stabilized MDMX may be excluded from suppressing p53. We therefore examined the cellular localization of MDMX upon Otub1 expression. As shown in Figure 3A, cell fractionation assays revealed that Otub1 expression not only significantly induced the nuclear levels of p53 and MDMX, but also induced the levels of cytoplasmic MDMX and p53 (lane 4, Figure 3A). Of note, unlike p53, most of which was accumulated in the nucleus, the majority of MDMX was accumulated in the cytoplasm. These results suggest that Otub1 increases the cytoplasmic levels of both MDMX and p53. As p53 can directly induce mitochondrial apoptosis pathway [4][5][6][7] and it has recently been shown that MDMX also possesses pro-apoptotic function in mitochondria [41], we then asked whether MDMX relocalizes to the mitochondria upon Otub1 overexpression. Indeed, mitochondria fractionation assays showed that MDMX (together with p53) was accumulated in the mitochondria fraction ( Figure 3B). Thus, Otub1 stabilizes MDMX in the cytosol and promotes MDMX's re-localization to the mitochondria.
Otub1-stabilized MDMX contributes to p53 S46 phosphorylation and p53-mediated apoptosis As described in our previous work, Otub1 stabilizes and activates p53 and can induce apoptosis [39]. It also has been shown that MDMX can promote p53 phosphorylation at S46 (p53-S46P) [41,42] and this phosphorylation promotes p53 function to induce apoptosis [43,44]. Indeed, we found that overexpression of Otub1 in U2OS cells induced the levels of p53-S46P  Figure 4A) and caused concomitant induction of cleaved PARP and caspase-9 ( Figure 4A), indicative of intrinsic apoptosis. Interestingly, knockdown of MDMX by siRNA significantly attenuated the induction of p53-S46P, cleaved PARP and cleaved caspase-9 by Otub1 overexpression ( Figure 4B). Thus, Otub1-stabilized MDMX may be critical for p53 phosphorylation at S46 and play a proapoptotic role in the context of Otub1 overexpression.

Otub1 promotes UV-induced apoptosis that involves MDMX stabilization
We have previously shown that Otub1 plays a critical role in p53 induction in response to DNA damage such as UV irradiation [39]. Consistent with this notion, induced overexpression of Otub1 significantly promoted UV irradiation-induced apoptosis, as indicated by   Figure 5A) and dose-( Figure 5B) dependent manner. Also, Otub1 expression promoted UV irradiationinduced p53-S46P ( Figure 5C). Furthermore, knockdown of MDMX significantly attenuated the induction of p53-S46P, cleaved PARP and Caspase-9 in response to UV irradiation ( Figure 5D). Thus, our results support that elevated MDMX can relocalize into the mitochondria, promote p53-S46P, and induce intrinsic apoptosis.

DISCUSSION
In this study, we show that Otub1 inhibits MDM2-mediated MDMX ubiquitination, leading to the stabilization of MDMX, its accumulation in mitochondria and the cytosol, p53 S46 phosphorylation, and mitochondria-mediated apoptosis. Knockdown of MDMX impaired both the p53-S46P and the induction of apoptosis induced by Otub1 expression as well as in response to UV irradiation, revealing an important role for MDMX in Otub1-induced and mitochondria-mediated apoptosis ( Figure 6). Our finding is also consistent with a previously suggested proapoptotic function of MDMX in mitochondria via promoting p53-S46P and neutralizing anti-apoptotic Bcl-2 family proteins.
As an oncoprotein, the main function of MDMX is to suppress p53 activity and assist MDM2 to ubiquitinate and degrade p53. This inhibitory role is indispensable as deleting p53 rescues the lethal phenotype of the MDMX knockout mice [45][46][47], similar to that in MDM2 −/− p53 −/− double knockout mice [48,49]. However, additional evidence suggests that MDMX also possesses intrinsic anti-tumorigenic activity via p53-dependent and p53independent mechanisms in certain conditions. For example, deleting MDMX increases tumorigenesis in MDM2 transgenic mice, indicating that MDMX can inhibit tumorigenesis when MDM2 is overexpressed [50]. Unlike MDM2 −/− p53 −/− double knockout cells, MDMX −/− p53 −/− double knockout cells induce multipolar mitosis resulting in increased cell proliferation and spontaneous transformation in MEF cells and increased spontaneous tumorigenesis in mice [51]. Additionally, it has been shown that cisplatin caused cell death requires MDMX-mediated mitochondrial intrinsic apoptosis pathway [52]. Also, overexpression of MDMX has been shown to stabilize p53 and increase p53-dependent cell death in response to stress [53]. Mechanistically, this proapoptotic function occurs in the cytoplasm, as MDMX has been shown to localize into the mitochondria and promote p53-dependent intrinsic apoptotic pathway by facilitating p53 phosphorylation at S46 and the binding of p53 to and neutralizing Bcl-2 [41,42]. More recently, it has been shown that in response to severe DNA damage, MDMX can dissociate from MDM2 and bind to and stabilize serine-threonine kinase HIPK2 to stimulate p53 phosphorylation at Ser46 [42]. Also, overexpression of MDMX is associated with an increased likelihood of survival in glioma patients [54]. In addition, when highly overexpressed, MDMX can inhibit MDM2-mediated p53 ubiquitination and degradation possibly by competing with MDM2 for binding to p53 and directly suppressing MDM2 autoubiquitination activity [23,25,26,33]. Together, these studies suggest that MDMX, when highly overexpressed, could stabilize p53 and increase p53 cytoplasmic function to mediate intrinsic apoptotic function.
Our observation here supports the proapoptotic function of MDMX when its expression is highly elevated, in this case, by Otub1 overexpression, as MDMX was localized in the mitochondria with concomitant increase of p53-S46P when Otub1 was induced and knockdown of MDMX alleviated the p53-S46P and intrinsic apoptosis. Our finding also extends  The cells were assayed by IB using indicated antibodies. (C) Otub1 promotes p53-S46P following UV irradiation. T-Rex-U2OS-Flag-Otub1 cells were cultured without or with 2 ug/ml Dox for 24 hours, followed by UV irradiation. The cells were assayed by IB using the indicated antibodies. (D) Knockdown of MDMX attenuates p53-S46P and induction of apoptosis in response to UV irradiation. T-Rex-U2OS-Flag-Otub1 cells infected with scrambled or MDMX shRNA were cultured without or with 2 ug/ml Dox for 24 hours, followed by UV irradiation (40 J/m 2 ) for 6 hours. The cells were then assayed by IB to detect the expression of indicated proteins. can also stabilize MDMX, which accumulates in the mitochondria and promotes p53 phosphorylation at S46, resulting in mitochondriamediated apoptosis.
Oncotarget 11059 www.impactjournals.com/oncotarget the function of Otub1 in inducing apoptosis via a novel MDMX-mediated mechanism in the cytoplasm. Thus, it is likely that Otub1 stabilizes p53 and promotes p53′s transcriptional-dependent and transcriptionalindependent function with the later mechanism involving MDMX localization in the mitochondria and its role in promoting p53-S46P. The p53-S46P kinase HIPK2 can be activated by UV irradiation-induced DNA damage [43,44]. p53, once phosphorylated at S46P, can induce key target genes involved in mitochondria-mediated apoptotic pathway. MDMX is also specifically localized in the cytoplasm following UV irradiation [55]. Indeed, we previously showed that Otub1 plays a critical role in p53 stabilization and activation following UV irradiation [56] and in this study we further showed that Otub1 markedly promotes UV-induced p53 activation. Knockdown of MDMX reduced UV-induced p53-S46P and apoptosis upon Otub1 expression. Thus, Otub1 may interplay with MDMX in the cytoplasm to mediate UVirradiation-induced apoptosis. It is interesting in future studies to test how Otub1 is activated by UV damage to suppress MDM2-mediated MDMX ubiquitination and degradation.
In summary, we have found that Otub1 promotes p53 function by stabilizing MDMX, which in turn contributes to the p53-induced mitochondria-mediated apoptosis pathway. Like the stabilization of p53, the MDMX stabilization also results from the non-canonical role of Otub1 via suppressing ubiquitin E2.

In vivo ubiquitination assay
In vivo ubiquitination assay under denaturing conditions was conducted in H1299 cells as previously described [39,56]. Briefly, cells transfected with indicated plasmids were treated with 40 μM MG132 for 6 h before harvesting. The cells were harvested at 48 h after transfection, and 20% of the cells were used for direct IB and the rest of cells were used for ubiquitination assays under denaturing conditions using Ni 2+ -NTA pulldown. After washing, the bead-bound proteins were analyzed by IB.

Reverse transcriptase-Quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA was isolated from cells using Trizol ® reagent following manufacturer's protocol (Invitrogen). Reverse transcriptions were performed as described [39]. Quantitative real-time PCR was performed on an ABI 7300 real-time PCR system (Applied Biosystems) using iTaq ™ Universal SYBR Green Supermix (Bio-Rad) as described previously [39,58]. All reactions were carried out in triplicate. The relative gene expression was calculated using the ΔCτ method following the manufacturer's protocol. The primers for p21, mdm2 and GAPDH were described [39]. The primers for MDMX are 5′-GCCTTGAGGAAGGATTGGTA-3′ and 5′-TCGACAATCAGGGACATCAT-3′.

Cell fractionation
T-Rex-U2OS-Flag-Otub1 cells cultured in the presence or absence of doxycycline (2 µg/ml) were resuspended in a hypotonic buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT) and homogenized. After centrifugation, the supernatant was collected as the cytoplasmic fraction. The pellets were resuspended in buffer C (20 mM HEPES pH 7.9, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT, 25% Glycerol) and sonicated. The nuclear fraction (supernatant) was collected by centrifugation. For isolating mitochondria, cells were trypsinized and washed twice in phosphate-buffered saline (PBS) and resuspended in ice-cold IB cells -1 buffer (225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA, 30 mM Tris-HCl pH7.4, 1 mM DTT and protease inhibitors). After hypotonic swelling on ice, the cells were homogenized in dounce tissue grinder until 80-90% of cell damage was attained. The homogenate was centrifuged twice at 600 g for 5 min at 4°C. The supernatant was collected and centrifuged at 7000 g for 10 min at 4°C. The supernatant was saved as the cytoplasmic fraction, and the mitochondrial pellet was resuspended in IB cells -2 buffer (225 mM mannitol, 75 mM sucrose, 30 mM Tris-HCl pH7.4, 1 mM DTT and protease inhibitors), followed by a 10 min centrifugation at 10,000 g. The mitochondrial pellet was lysed in mitochondria lysis buffer (0.5% TritonX-100, 30 mM Tris-HCl pH7.4, 200 mM KCl, 5 mM EDTA, 1 mM DTT and protease inhibitors) and assayed by IB.