serum depletion-induced cell death (Figure 1A), implying that IRTKS may possess anti-apoptotic activity. HT1080 cells express wild type p53, which mediates the apoptotic response to DNA damage [15]. We found that IRTKS overexpression reduced the number of apoptotic cells upon UV exposure (Figure 1B and Figure S1A). By contrast, IRTKS depletion by RNA interference (RNAi) enhanced UV irradiation-induced apoptosis (Figure 1C and Figure S1B) and the apoptosis-related cleavage of poly(ADP- ribose) polymerase (PARP) (Figure S1C). We also infected p53-null SOAS-2 cells with adenoviruses expressing p53 and IRTKS, and found that IRTKS overexpression inhibited apoptosis mediated by ectopic p53(Figure 1D and Figure S1D) while IRTKS knockdown increased p53-induced apoptosis in SOAS-2 cells (Figure 1E and Figure S1E). These observations demonstrated that IRTKS negatively modulated p53-induced apoptosis. p53-induced apoptosis is largely dependent on its transactivation of such proapoptotic genes as BAX, PUMA and PIG3 [16–18]. We sought to determine the effect of IRTKS induction on the expression of p53 target genes. We found that IRTKS overexpres- sion slightly decreased the expression of PIG3 and MDM2 in SOAS- 2 cells (Figure 1D, right panel). By contrast, deletion of IRTKS increased the expression of PIG3 and MDM2 in SOAS-2 cells (Figure 1E, right panel). Induction of IRTKS overexpression in HT1080 cells upon doxycycline withdrawal also reduced the levels of PIG3, PUMA and MDM2 with no apparent effect on the expression of p53 (Figure 1F). Furthermore, in SOAS-2 cells, using a luciferase reporter system under the control of the BAX promoter, we found that IRTKS overexpression dose-dependently inhibited p53-induced luciferase activity (Figure 1G). These data showed that IRTKS attenuated the transactivation of p53 on its downstream genes, suggesting that IRTKS may exert its anti-apoptotic effect through modulating p53 transcriptional activity. IRTKS interacted directly with p53 in nucleus We were interested in determining whether IRTKS exerted its effects on p53 through its physical association with the protein. We defined the subcellular localization of both proteins by immuno- fluorescence assays, which showed that both IRTKS and p53 were co-localized in the nucleus (Figure 2A), suggesting that IRTKS could physically associate with p53. Reciprocal co-immunopre- cipitations (Co-IPs) using antibodies against IRTKS and p53 confirmed their association in HEK 293 and HT1080cells (Figure 2B, Figure S2A). Co-IPs with nuclear and cytoplasmic extracts from HT1080 cells further revealed that, consistent with the data from immunofluorescence assays (Figure 2A), endogenous IRTKS interacted with p53 exclusively in the nucleus (Figure 2C). Direct interaction of IRTKS with p53 was further demonstrated in vitro by GST-pulldown assays (Figure 2D). Additionally, two regions of IRTKS (N-terminal 1–250 and 250–350 amino acid residues), which contain the IMD domain and an uncharacterized domain, respectively, were found to interact with p53 by GST- pulldown experiments with a series of deletion variants of IRTKS (Figure 2D). Consistently, two regions of p53 were involved in the direct interaction of IRTKS and p53. While the p53 deletion variants containing its C-terminal regulatory domain (CRD) strongly bound to IRTKS, the DNA binding domain (DBD) of p53 also interacted with IRTKS (Figure 2E and Figure S2B). These findings indicated that IRTKS physically associated with p53 in the nucleus via their functional domains. IRTKS increases the ubiquitination and the cytoplasmic localization of p53 p53 can be negatively regulated at the transcriptional, translational and post-translational levels [1]. As IRTKS did not IRTKS Regulates MDM2-Mediated p53 Ubiquitination alter the abundance of p53 mRNA and protein levels (Figure 1F and Figure S3), we proposed that IRTKS negatively modulated p53 activity at the post-transcriptional level. We found that IRTKS overexpression failed to induce changes in phosphoryla- tion and acetylation of certain amino acid residues of p53 under stress or unstress conditions, including serine 15, 20, 37, 46, 392, and lysine 382 (Figure S3). However, p53 ubiquitination with lower molecular weights (,130 Kd) was significantly increased in HT1080 cells overexpressing IRTKS (Figure S4A). By contrast, IRTKS knockdown reduced the ubiquitination of endogenous p53 (Figure S4B). In vivo ubiquitination assay in U2OS cells further confirmed that, distinct from the p53 ubiquitination pattern by ectopic MDM2, IRTKS indeed mediated p53 ubiquitination with increasing lower molecular weight modifications (Figure 3A). Previous reports have demonstrated that p53 polyubiquitination is associated with its proteasomal degradation while p53 mono- ubiquitination induces its nuclear export. Our data here showed that IRTKS did not affect the protein levels of endogenous and ectopic p53 (Figure S3 and S4; Figure 1D and 1E). On the contrary, IRTKS overexpression slightly stabilized endogenous p53 in U2OS cells treated with cycloheximide (Figure 3B). Our immunofluorescence assay revealed that IRTKS overexpression increased the cytoplasmic localization of p53 (Figure 3C). These data indicated that IRTKS-induced p53 ubiquitination affects the subcellular localization of p53. IRTKS interacts with MDM2 IRTKS was previously considered as a scaffold protein without enzymatic activity [13,19]. IRTKS alone also failed to demon- strate any catalytic activity on p53 ubiquitination in our in vitro ubiquitination assays (Figure S6B, lane 1–4). Therefore, IRTKS might function as an adaptor for the ubiquitin E3 ligases to mediate p53 ubiquitination. As known, MDM2 is essential for regulating p53 function and catalyzes p53 ubiqitination for degradation and nuclear export dose-dependently [7,20,21]. In MDM2 cells, IRTKS only increased p53 ubiquitination when MDM2 was co-transfected to the cells (Figure S5), implying that IRTKS affected MDM2-mediated p53 ubiquitination (Figure S5). To assess the possibility that IRTKS could serve as an adaptor of MDM2, we performed MDM2 and IRTKS co-localization assays, which disclosed an almost identical subcellular localization of IRTKS and MDM2, especially in the nucleus (Figure 4A). Co-IP analysis additionally revealed that endogenous IRTKS and MDM2 were reciprocally immunoprecipitated (Figure 4B), sug- gesting that IRTKS indeed physically associated with MDM2. In vitro GST-pulldown assays with purified recombinant proteins confirmed their physical interaction, where both the IMD domain of IRTKS and 150–300 residues of MDM2 were required for their interaction (Figure 4C and 4D). These data implied that IRTKS could recruit both MDM2 and its substrate p53 through the IMD domain and serve as a cofactor of MDM2. IRTKS promoted p53 ubiquitination induced by low levels of MDM2 We further explored the role of IRTKS in MDM2-mediated p53 ubiquitination by assessing the activity of MDM2 for p53 ubiquitination using in vitro ubiquitination assays with recombi- nant MDM2 and p53. Consistent with the previous study [7], MDM2 induced p53 monoubiquitination and polyubiquitination dose-dependently (Figure S6A). Using this ubiquitination system, we found that IRTKS only enhanced low molecular weight of p53 ubiquitination (,130 Kd) mediated by low levels of MDM2, but had no effect on p53 ubiquitination induced by high levels of MDM2 (Figure 5A). Moreover, IRTKS variant with IMD deletion PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23571