IRTKS Regulates MDM2-Mediated p53 Ubiquitination Figure 4. IRTKS interacted with MDM2. (A) Colocalization of IRTKS and MDM2. HT1080 cells were transfected with MDM2 and immunostained with anti-MDM2 antibody and anti-IRTKS antibody. (B) Interaction of endogenous IRTKS and endogenous MDM2 in HT1080 cells. Whole cell lysate (WCL) and immunoprecipitations (IP) with preimmune serum or IRTKS antibody, control IgG, MDM2 antibody were analyzed by Western blotting. (C) Mapping of IRTKS domain for MDM2 binding. GST-pulldown assay was performed with recombinant GST-IRTKS variations and His-MDM2 to reveal the direct interaction between IRTKS and MDM2, and map the interaction site of IRTKS. (D) Interaction site mapping on MDM2 for IRTKS. doi:10.1371/journal.pone.0023571.g004 The effects of MDM2-mediated ubiquitination on p53 are in a delicate balance to determine the cell fate during normal homeostasis and stress response. The negative feedback loop between MDM2 and p53 ensures that p53 is maintained at low levels under normal conditions or after stress resolved [4]. Interestingly, IRTKS could be also involved in the negative feedback regulation. Here we showed that p53 dissociated with IRTKS after DNA damage. IRTKS polyubiquitination and degradation by high levels of MDM2 might increase the direct interaction of the secondary sites of p53 and MDM2 for p53 polyubiquitination and degradation. Together, these data led us to propose a novel mechanism for maintaining the balance of MDM2-mediated ubiquitination on p53 during normal homeostasis and stress response. In unstressed cells with low levels of MDM2, IRTKS stabilized the MDM2-p53 interaction and enhanced MDM2-mediated p53 ubiquitination, thereby inhibiting p53 transcriptional activity. In response to DNA damage, the stabilized MDM2-IRTKS dissociated from p53. MDM2 may possess E3 ligase for self-degradation and IRTKS ubiquitination. Late after DNA damage, MDM2, as a p53 target, was up-regulated and high levels of MDM2 mediated the polyubiquitination and degradation of both IRTKS and p53. Several proteins have been proved to act as a regulator and substrate of MDM2 in the negative feedback regulation loop between p53 and MDM2. MDM2 can downregulate p53 protein synthesis by inducing the polyubiquitination of L26, a ribosomal protein which stimulates translation of p53 mRNA after DNA damage [42]. Another ribosomal protein S7 interacts with MDM2, inhibiting the E3 ligase activity of MDM2 and stabilizing both MDM2 and p53. S7 itself is ubiqutinated by MDM2 and ubiquitinated S7 selectively destabilizes MDM2, enhancing the p53 stress response [43]. Herein, we demonstrated IRTKS can regulate p53 by modulating the p53-MDM2 interaction as a scaffold protein and substrate of MDM2. Previous studies suggest that IRSp53-like proteins, including IRTKS, contribute to actin dynamics and membrane protrusion. This function is mainly achieved by the IMD which is the most conserved domain of these proteins [12]. The IMD, also known as an I-BAR (inverse Bin-amphiphysin-Rvs167) domain, binds F- actin, RAC, and lipid, triggering protrusive membrane deforma- tion [10]. Here we show that the IMD of IRTKS was responsible for its association with p53 and MDM2, indicating the IMD- contain proteins might have combination functions of cell mobility and apoptosis. This finding suggests that the IRSp53-like proteins PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23571