Abstract
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.
Highlights
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 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]
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)
Summary
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 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]. P53 can directly trigger mitochondria-mediated apoptosis and necrosis through transcription-independent mechanisms [4,5,6,7,8]. Unlike MDM2, MDMX does not possess ubiquitin E3 activity towards p53 [24, 25] It assists MDM2 to inhibit p53 by binding to MDM2 through their RING domains and stabilizes MDM2 [26,27,28,29,30,31], whereas MDM2 in turn ubiquitinates and degrades MDMX in response to DNA damage [32, 33]. The p53-MDM2-MDMX axis is critical for normal control of the levels and activity of p53 and is tightly regulated by various posttranslational modifications in response to stress
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