Here we show that activation of the Caspase-2-PIDDosome promotes cleavage of Mdm2. Caspase-2 directly cleaves Mdm2 in a conserved DVPD367 site, leading to separation of the p53 binding domain and the RING finger responsible for p53 ubiquitination. As a result, Mdm2 cleavage inhibits its E3 ubiquitin ligase activity, leading to increased p53 levels and activity. We show that Mdm2 cleavage in human lung cancer cells promotes p53-dependent cell cycle arrest and subsequent drug resistance. Furthermore, Mdm2 cleavage by the Caspase-2-PIDDosome occurs in response to DNA damage and contributes to the maintenance of p53 levels. Together, p53 activation of the Caspase-2-PIDDosome establishes a positive feedback loop that promotes p53 stability and activity following DNA damage ().
Previous studies showed that Mdm2 is cleaved by a caspase-3-like activity under apoptotic and non-apoptoptic, growth arrest conditions (Chen et al., 1997
; Erhardt et al., 1997
; Pochampally et al., 1999
; Pochampally et al., 1998
). The caspase responsible for Mdm2 cleavage was p53-inducible (Pochampally et al., 1999
) and distinct from Caspases 1, 3, 6, 7 and 8 (Erhardt et al., 1997
; Pochampally et al., 1999
; Pochampally et al., 1998
). Thus, the identity of the caspase that directly cleaved Mdm2 and its mechanism of induction by p53 remained unknown. Our studies resolve these issues by demonstrating that 1) Caspase-2 directly cleaves Mdm2 and 2) the mechanism of Caspase-2 activation is due to p53-mediated induction of PIDD.
Very few Caspase-2 cleavage targets are known, making its biological role elusive. Interestingly, the Caspase-2 cleavage site identified here in Mdm2 (DVPD) is highly conserved among vertebrates and similar to the known Caspase-2 cleavage site, ESPD, of Golgin-160 (Mancini et al., 2000
). Although studied here in the context of DNA damage and p53 activation, we speculate that this pathway may be important for p53 regulation more broadly. Recent studies by Ho et al. have implicated Caspase-2 as a tumor suppressor, and the authors noted that p53 activity is reduced in caspase-2
null MEFs. This phenotype is consistent with our model in which Caspase-2 depletion reduces Mdm2 cleavage, thus permitting increased expression of full-length Mdm2 and enhanced degradation of p53. Consistent with Ho et al., we observe that Caspase-2 knockdown accelerates cell growth and results in reduced p53 and p21 levels (data not shown).
In contrast to our findings, previous studies implicated Caspase-2 in heat shock-induced apoptosis and failed to detect Caspase-2 activation upon other types of genotoxic stress (Tu et al., 2006
). Because these studies were performed in T cells and splenocytes, it remains possible that cell-type specific differences explain this discrepancy. We initially tested the effects of PIDD in response to cisplatin in lung cancer cells, but began to use U2OS cells for their ease of manipulation for siRNA and cDNA transfections. We tested doxorubicin on U2OS cells, as it is a comment therapeutic agent for osteosarcoma and subsequently confirmed that doxorubicin and neocarzinostatin, a double-strand break inducer, also affect Mdm2 cleavage in lung cancer cells ( and Supp Fig S5C
Mdm2 contains other potential caspase cleavage sites located N-terminal to the DVPD site. Because Caspase-3 can cleave Mdm2 at high levels, it is possible that Caspase-2 initially cleaves Mdm2 under low levels of DNA damage, while excessive damage activates Caspase-3, leading to complete degradation of Mdm2 via additional cleavage sites. Recent studies have identified a mechanism of Mdm2 degradation involving phosphorylation by casein kinase 1 and subsequent β-TRCP-mediated destruction (Inuzuka et al., 2010
). The levels of DNA damage used in our experiments promote growth arrest and did not lead to complete degradation of Mdm2. However, upon higher levels of damage, we observed a reduction in full-length and cleaved Mdm2 (Supp Fig S5A
). Therefore, we hypothesize that the mode of Mdm2 regulation identified here is particularly important under reparable levels of DNA damage.
Mdm2 interaction with p53 has been shown to inhibit p53-transcriptional activity in vitro, but genetically-engineered mouse models have led to uncertainty about the in vivo relevance of these findings (Itahana et al., 2007
). Itahana et al. demonstrated that Mdm2
C462A mutant mice, in which Mdm2 lacks ubiquitin-conjugation function but retains p53-binding capacity, are embryonic lethal, similar to Mdm2
null mice. Embryonic lethality of Mdm2
C462A mice is rescued by p53 loss indicating that Mdm2 binding to p53 is not sufficient to inhibit p53 in vivo (Itahana et al., 2007
). Therefore, it may not be surprising that in the presence of Mdm2 p60 we find stabilized p53 that appears to be transcriptionally active and capable of inducing p21
and other p53 target genes (data not shown). How Mdm2 cleavage ultimately impacts the transcriptional program induced by p53 is an interesting avenue of further investigation.
Mdm2 cleavage in human lung cancer cells led to p21 induction and cell cycle arrest. We hypothesize that this mechanism of transient Mdm2 inhibition could serve to protect p53
wild-type tumors treated with chemotherapy, if enhanced p53 preferentially induces cell cycle arrest and/or DNA damage repair. Consistent with this hypothesis, we recently observed that Pidd
is more highly induced in cisplatin-resistant tumors compared to naïve tumors in response to chemotherapy in vivo (Oliver et al., 2010
), suggesting that p53 may promote arrest or repair in this context. Given that context-specific post-translational modifications of p53 may also regulate its activity, it remains possible that p53 stability may promote functions other than growth arrest such as apoptosis, senescence or autophagy under different cellular conditions.
Mdm2 can bind a number of important proteins via its N-terminal and central acidic domains, such as Rb and ARF, and can regulate the activity of other proteins by ubiquitination (Coutts et al., 2009
). Therefore, Mdm2 cleavage by Caspase-2 may have important consequences for the regulation of a variety of p53-dependent and -independent processes. Importantly, Mdm2 binds the Mdm2 homolog, MdmX, through their C-terminal RING domains (Tanimura et al., 1999
). Thus, Mdm2 cleavage is predicted to abolish interaction with MdmX. The DVPD cleavage site in Mdm2 is also conserved in MdmX (Pochampally et al., 1998
), but we do not detect MdmX cleavage under these conditions (Supp Fig S5D
). MdmX is detected largely in the nucleus of human lung cancer cell lines, which may preclude efficient interaction with Caspase-2 in the cytoplasm (Supp Fig S5D
). In either case, cleavage of MdmX and/or Mdm2 at DVPD would be expected to abolish Mdm2/MdmX heterodimer formation (Wade et al., 2010
) and prevent Mdm2-mediated ubiquitination of p53. While our studies did not preclude that Mdm2 also inhibits the sumoylation and neddylation of p53, ubiquitination of p53 is known to control its levels, whereas sumoylation and neddylation have not been implicated in p53 stability, suggesting these modifications are most likely ubiquitin (Dei and Gu, Trends in Mol Med, 2010). Mdm2 has been shown to degrade MdmX upon DNA damage (de Graaf et al., 2003
; Pan and Chen, 2003
), suggesting that cleaved Mdm2 p60 could enhance MdmX levels and counter p53 activity. Inhibition of the Caspase-2-PIDDosome upon DNA damage did not appear to enhance MdmX degradation in the assays used here (data not shown).
Many p53 autoregulatory loops impinge on Mdm2 (Harris and Levine, 2005
; Lu, 2010
) and regulate Mdm2 by a variety of mechanisms, including interaction with MdmX, auto-ubiquitination, phosphorylation, and here, cleavage (Wade et al., 2010
). We observe that inhibition of the Caspase-2-PIDDosome does not prevent p53 induction upon DNA damage, but impacts the dynamics of p53 levels over time. During the past decade, it has become appreciated that p53 exhibits pulsatile dynamics in response to DNA damage (Batchelor et al., 2008
; Lahav et al., 2004
; Loewer et al., 2010
). We speculate that the p53-Caspase-2-PIDDosome-Mdm2 pathway identified here may contribute to p53 pulses during DNA damage, by shutting down Mdm2 activity and re-establishing p53 activity. This positive feedback loop initiated by p53-induction of PIDD
may thus serve as an important counterbalance to p53-induction of Mdm2
. Together, these findings highlight the complexity of the feedback loops dictating p53 response, and undoubtedly contribute to the cell's ability to control p53 with exquisite sensitivity.