IRTKS Regulates MDM2-Mediated p53 Ubiquitination were collected and analysed by FACS after staining with propidium iodide (PI) at the indicated time. The sub-G1 population was counted as the percentage of cell death. The inducible expression of flag-tagged IRTKS was assessed by Western blotting with anti-FLAG and anti-IRTKS antibodies (right panel). (B, C) IRTKS inhibited UV-induced apoptosis. HT1080 cells with overexpression (B) or knockdown (C) of IRTKS were exposed to UV irradiation and analyzed for apoptosis by Annexin V-FITC/PI (propidium iodide) staining. Representative results of four independent experiments were shown. P,0.005. The expression of IRTKS was assessed by Western blotting (right panel). (D) Overexpression of IRTKS inhibited p53-induced apoptosis. SAOS-2 cells infected with adenoviruses for IRTKS or/and p53 overexpression were analyzed with PI staining by FACS. Data were shown as mean 6 s.d. (n = 3), p,0.01. The expression of p53, IRTKS and p53 inducible gene products (PIG3 and MDM2) were analyzed by Western blotting (right panel). (E) Knockdown of IRTKS enhanced p53-induced apoptosis. SAOS-2 cells were treated with IRTKS-siRNA for 24 h, then infected with Ad- p53 or control virus Ad-GFP, and analyzed for apoptosis. Data were shown as mean 6 s.d. (n = 3), p,0.01. The expression of p53, IRTKS and p53 inducible gene products (PIG3 and MDM2) were analyzed by Western blotting (right panel). (F) IRTKS inhibited the expression of p53-inducible genes. Total RNA from the IRTKS Tet-off HT1080 cells in the presence (10 ng/ml) or absence of doxycycline was subjected to real-time quantitative PCR (qPCR) analysis. Data were expressed as mean 6 s.d. (n = 3). p,0.01. (G) IRTKS decreased the transactivation of p53. p53 transactivation activity on the BAX promoter was measured by luciferase reporter gene assay in SAOS-2 cells. doi:10.1371/journal.pone.0023571.g001 failed to enhance MDM2-induced p53 ubiquitination (Figure 5B), indicating that the IMD region which was required for the physical association of IRTKS with both MDM2 and p53, was crucial for IRTKS-induced p53 ubiquitination. In vivo ubiquitination assays using H1299 cells also revealed that IRTKS promoted p53 ubiquitination only with low levels of MDM2 (Figure 5C, lane 4 compared with lane 2). It has been reported that MDM2 mediates both ubiquitination and neddyla- tion of p53. MDM2-mediated NEDD8 modification inhibits p53 transcription activity [22]. Herein, in vivo neddylation assay showed that IRTKS overexpression could not alter MDM2- mediated neddylation of p53 (Figure 5D). Therefore, as a cofactor of MDM2, IRTKS only enhanced p53 ubiquitination induced by low level MDM2. IRTKS enhanced the p53-MDM2 interaction at low levels of MDM2 Previous studies showed that the N terminal regions of both MDM2 and p53 provide the primary site for their physical interactions [23], which are distinct from the domain that mediated their binding to IRTKS shown here. Our data indicated that the IMD domain was required for IRTKS binding to p53 and MDM2 and for its effect on MDM2-mediated p53 ubiquitination. The crystal structure of IRTKS indicates that IMD could self- associate into an 180 A˚-long zeppelin-shaped dimer [11,24]. Moreover, an uncharacterized domain of the middle region of IRTKS could provide a secondary site for interaction with p53. These observations raised the intriguing possibility that IRTKS could affect the p53-MDM2 interaction in vivo. Co-IP assays revealed that endogenous p53, MDM2 and IRTKS were all immunoprecipitated with antibodies against p53 or MDM2 (Figure 6A), suggesting that they were present in the same protein complex. We then evaluated the effect of IRTKS on the interaction between MDM2 and p53. Interestingly, a cotransfec- tion assay in H1299 cells showed that the interaction between MDM2 and p53 was significantly enhanced by ectopic IRTKS with low levels of MDM2 (Figure 6B, lane 4 compared with lane 3), whereas no effect on the association of high levels of MDM2 with p53 was detected (Figure 6B, lanes 5 and 6). Additionally, consistent with our findings in in vitro and in vivo ubiquitination assays, p53 ubiquitination at low molecular (,130 kD) was increased at low levels of MDM2 with IRTKS overexpression (Figure 6B, lane 4 compared with lane 3). In contrast, IRTKS knockdown attenuated the p53-MDM2 interaction and p53 ubiquitination at low levels, not high levels, of MDM2 (Figure 6C). These data together indicated that IRTKS induced p53 ubiquitination through enhancing the interaction between p53 and low levels of MDM2 in unstressed cells. MDM2 is an E3 ubiquitin ligase for IRTKS degradation The protein levels of IRTKS were significantly decreased after DNA damage in HT1080 cells (Figure S1B and Figure 7A) but not in p53-null SAOS-2 cells (Figure S7A), suggesting that IRTKS could be decrease by DNA damage induced p53 activation. This finding promoted us to assess the dynamics of IRTKS expression. Interestingly, MDM2 overexpression reduced the protein levels of IRTKS (Figure 7B), suggesting that MDM2 may mediate IRTKS degradation. Both in vitro and in vivo ubiquitination assays demonstrated that MDM2 mediated IRTKS ubiquitination in a dose-dependent manner (Figure 7C and 7D). These results strongly indicated that MDM2 as an E3 ubiquitin ligase affected IRTKS stability at high activity levels via ubiquitination and proteasomal degradation. We then investigated the effect of DNA damage on the interaction of IRTKS, p53 and MDM2. UV irradiation in HT1080 cells significantly attenuated the interaction between endogenous IRTKS and p53 (Figure S7B). Moreover, the DNA- damaging agent etoposide abolished the IRTKS-p53 interaction and stabilized the IRTKS-MDM2 interaction in U2OS cells when all three proteins were stabilized by MG132 (Figure 7E). These data suggested that DNA damages affected the IRTKS-p53 and IRTKS-MDM2 interactions differently and specifically. Discussion P53-induced transcriptional alteration causes a variety of cell fate changes, including apoptosis, growth arrest, senescence, DNA repair. For maintenance the homeostasis of cell, various processes are equipped to modulate p53 activity. Adjusting the protein levels and transcriptional activity of p53 are two essential processes for cells to maintain the physiological state [25]. Although poly- ubiquitination of p53 targets its proteasomal degradation, several studies have demonstrated that p53 ubiquitination also affects its activation. WWP1-mediated p53 ubiqitination causes stabilization of p53 but inactivates p53 transactivation [26]. Ubc13 elicits K63- dependent ubiquitination of p53, increases p53 localization to the cytoplasm and p53 stability, and decreases its transcriptional activity [27]. CUL7 causes only mono- or di-ubiquitination of p53, and promotes cell growth through antagonizing the function of p53 [28]. p53 ubiquitination could be important for keeping p53 inactive. De-ubiquitination of p53 is essential for p53-mediated mitochondrial apoptosis and for recycling of p53 from cytoplasm to nucleus [21,29]. In this study, our data showed that IRTKS decreased p53 activity and increased low levels of MDM2- mediated p53 ubiquitination via binding to both p53 and MDM2. How, then, IRTKS promoted MDM2-mediated p53 ubiquiti- nation but not degradation via direct interaction? N-terminal transactivation domain of p53 has been characterized as the PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23571