Account for the observed inhibition of p53 ubiquitylation by MAGE-A2. Another possible explanation for the reduced levels of ubiquitylation is that MAGE-A2 itself is a substrate for MDM2 and can therefore act as a competitive inhibitor of other substrates under our assay conditions. However, we were unable to detect any ubiquitylation of MAGE-A2 by MDM2 under buy T0901317 conditions where MDM2 is able to ubiquitylate p53 and MDM2 itself, qhw.v5i4.5120 thereby ruling out this possibility. In addition to regulating p53 levels, MDM2 interacts with, and mediates the ubiquitylation of, many other cellular proteins including MDM4. To determine whether MAGE-A2 could affect the ubiquitylation of MDM4 by MDM2, MDM4 was introduced into the ubiquitylation assay. The data (Fig 4C) indicate that, as with p53 and MDM2, increasing levels of MAGE-A2 led to a detectable decrease in the ubiquitylation of MDM4 by MDM2. (The reduction in MDM4 ubiquitylation, while evident, appears less robust than with the p53 or MDM2 [panels A and B]. We cannot, therefore, rule out the possibility that other cellular E3 ligases or de-ubiquitylases may have an impact on the magnitude of this response.) Strikingly, however, the presence of even low levels of MAGE-A2 led to a small but significant elevation of MDM4 protein levels. These data suggest that, although MAGE-A2 can inhibit the general ubiquitylation function of MDM2, there is a selective outcome in terms of promoting degradation.PLOS ONE | DOI:10.1371/journal.pone.0127713 May 22,11 /MAGE-A Inhibits MDM2 and Increases MDM4 LevelsMAGE-A governs the levels of endogenous MDMWe and others have previously shown that MAGE-A proteins can directly regulate p53 pnas.1408988111 transcription function but show little influence on regulating wild type p53 protein levels [7,12,13]. Contrary to these observations, other researchers have found that MAGE proteins can influence p53 levels independently of MDM2 by regulating the activity of other p53-targeted ubiquitin ligases [8,17]. Upon re-examination, we confirmed that silencing of MAGE-A expression in U2OS cells, in which p53 levels are governed mainly through the action of MDM2, leads to increased levels of the order HS-173 p53-transcriptional target, p21, yet shows at best only a marginal effect p53 protein levels (S5 Fig). These data support the idea that MAGE-A proteins can acutely down-regulate p53 function without necessarily affecting p53 levels and are consistent with the unaltered p53 levels seen in the ubiquitylation experiment (Fig 4). In contrast to the unaltered p53 levels, the data in Fig 4 indicate that elevated expression of MAGE-A2 leads to increased levels of MDM4. To determine whether the ability of MAGE-A to elevate MDM4 observed in the ubiquitylation assay holds true for endogenous proteins, MDM4 levels were examined following silencing of MAGE-A expression in U2OS cells. The data (Fig 5A) show that decreasing the levels of MAGE-A leads to a significant reduction in MDM4 levels. Consistent with a mechanism involving the ubiquitylation and turnover of MDM4, the reduction on MDM4 levels could be partially rescued by the presence of the proteasomal inhibitor, bortezomid (Fig 5B); (bortezomid, like MG132, is a potent inhibitor ofFig 5. Silencing of MAGE-A expression decreases the levels of MDM4 via the proteasome and increase MDM2/MDM4 association. MAGE-A expression in U2OS cells was silenced by RNAi as previously described or treated with a non-silencing siRNA oligonucleotide as control [12]. The various proteins.Account for the observed inhibition of p53 ubiquitylation by MAGE-A2. Another possible explanation for the reduced levels of ubiquitylation is that MAGE-A2 itself is a substrate for MDM2 and can therefore act as a competitive inhibitor of other substrates under our assay conditions. However, we were unable to detect any ubiquitylation of MAGE-A2 by MDM2 under conditions where MDM2 is able to ubiquitylate p53 and MDM2 itself, qhw.v5i4.5120 thereby ruling out this possibility. In addition to regulating p53 levels, MDM2 interacts with, and mediates the ubiquitylation of, many other cellular proteins including MDM4. To determine whether MAGE-A2 could affect the ubiquitylation of MDM4 by MDM2, MDM4 was introduced into the ubiquitylation assay. The data (Fig 4C) indicate that, as with p53 and MDM2, increasing levels of MAGE-A2 led to a detectable decrease in the ubiquitylation of MDM4 by MDM2. (The reduction in MDM4 ubiquitylation, while evident, appears less robust than with the p53 or MDM2 [panels A and B]. We cannot, therefore, rule out the possibility that other cellular E3 ligases or de-ubiquitylases may have an impact on the magnitude of this response.) Strikingly, however, the presence of even low levels of MAGE-A2 led to a small but significant elevation of MDM4 protein levels. These data suggest that, although MAGE-A2 can inhibit the general ubiquitylation function of MDM2, there is a selective outcome in terms of promoting degradation.PLOS ONE | DOI:10.1371/journal.pone.0127713 May 22,11 /MAGE-A Inhibits MDM2 and Increases MDM4 LevelsMAGE-A governs the levels of endogenous MDMWe and others have previously shown that MAGE-A proteins can directly regulate p53 pnas.1408988111 transcription function but show little influence on regulating wild type p53 protein levels [7,12,13]. Contrary to these observations, other researchers have found that MAGE proteins can influence p53 levels independently of MDM2 by regulating the activity of other p53-targeted ubiquitin ligases [8,17]. Upon re-examination, we confirmed that silencing of MAGE-A expression in U2OS cells, in which p53 levels are governed mainly through the action of MDM2, leads to increased levels of the p53-transcriptional target, p21, yet shows at best only a marginal effect p53 protein levels (S5 Fig). These data support the idea that MAGE-A proteins can acutely down-regulate p53 function without necessarily affecting p53 levels and are consistent with the unaltered p53 levels seen in the ubiquitylation experiment (Fig 4). In contrast to the unaltered p53 levels, the data in Fig 4 indicate that elevated expression of MAGE-A2 leads to increased levels of MDM4. To determine whether the ability of MAGE-A to elevate MDM4 observed in the ubiquitylation assay holds true for endogenous proteins, MDM4 levels were examined following silencing of MAGE-A expression in U2OS cells. The data (Fig 5A) show that decreasing the levels of MAGE-A leads to a significant reduction in MDM4 levels. Consistent with a mechanism involving the ubiquitylation and turnover of MDM4, the reduction on MDM4 levels could be partially rescued by the presence of the proteasomal inhibitor, bortezomid (Fig 5B); (bortezomid, like MG132, is a potent inhibitor ofFig 5. Silencing of MAGE-A expression decreases the levels of MDM4 via the proteasome and increase MDM2/MDM4 association. MAGE-A expression in U2OS cells was silenced by RNAi as previously described or treated with a non-silencing siRNA oligonucleotide as control [12]. The various proteins.