IRTKS Regulates MDM2-Mediated p53 Ubiquitination Figure 2. IRTKS directly interacted with p53. (A) Co-localization of IRTKS and p53. HT1080 cells were immunostained with anti-p53 antibody and anti-IRTKS antibody followed by Alexa-488 conjugated anti-mouse antibody and Alexa-546 conjugated anti-rabbit antibody. Images were taken through a confocal microscope. (B) Association of endogenous ITRKS and endogenous p53 in HEK 293 cells. Whole cell lysates (WCL) and immunoprecipitations (IP) with control IgG, p53 antibody, preimmune serum or IRTKS antibody were analyzed by Western blotting. The arrowhead indicates IgG heavy chain. (C) IRTKS interacted with p53 in nucleus. Immunoprecipitations of the nuclear and cytoplasmic extracts from HT1080 cells with p53 antibody were analyzed with IRTKS antibody by Western blotting. (D) Direct interaction between p53 and IRTKS revealed by GST-pulldown assays. Full-length or truncated GST-IRTKS proteins were incubated with in vitro translated 35S-labelled p53 in NETN buffer, and then precipitated with glutathione-Sepharose beads. Bound proteins were resolved by 10% SDS-PAGE followed by autoradiography. GST-IRTKS proteins were visualized by Coomassie blue staining. (E) Interaction site mapping on p53. GST-pulldown assays were performed with recombinant His-IRTKS and full-length or truncated GST-p53. The pulldown samples were subjected to Western blotting with anti-IRTKS and anti-GST antibodies. doi:10.1371/journal.pone.0023571.g002 primary binding site to MDM2 [23,30,31]. This primary interaction has been documented to be essential, but not sufficient, to target p53 degradation. The second binding site of p53 to MDM2 has been identified in its DNA-binding domain [32,33]. A conformational change of MDM2 caused by the primary interaction enables binding of the central region in MDM2 to the DNA-binding domain of p53 [34]. The secondary interaction is required for efficient p53 polyubiquitination and proteasomal degradation [35]. p53 mutants which contain single point mutations in p53 DNA binding domain have increased levels of MDM2-dependent ubiquitination, but not degradation of p53 in vivo [36]. Similarly, IRTKS slightly stabilized p53 protein while increased p53 ubiquitination (Figure 3B), implying that the binding of IRTKS and p53 affected the secondary interaction of p53 and MDM2. Several partners of MDM2, including ARF, L5, L11, and L23, have been reported to bind to the central region of MDM2 [37– 41]. Most of them inhibit MDM2-mediated p53 ubiquitination and degradation, probably by blocking the secondary interaction between MDM2 and p53. Our data suggest that IRTKS bound to both the central domain of MDM2 and the DNA binding domain of p53, and recruited MDM2 to p53 in unstressed cells. IRTKS stabilized the p53-MDM2 interaction at low levels of MDM2, but it could also block the secondary interaction sites of p53-MDM2 interaction. Therefore, IRTKS enhanced p53 ubiquitination but not degradation in unstressed cells. Figure 3. IRTKS enhanced the ubiquitination and cytoplasmic localization of p53. (A) In vivo ubiquitination assay revealed IRTKS-induced p53 ubiquitination. U2OS cells were co-transfected with a 6XHis-Ub expression vector and IRTKS (lane 3). The cell lysates were loaded on nickel (Ni+)- NTA columns. p53 ubiquitination was analyzed with Western blotting by using anti-p53 antibody (DO-1). MDM2 was used as a positive control for p53 ubiquitination (lane1). Note that the IRTKS-induced p53 ubiquitination was in lower molecular weights (,130 kD). (B) IRTKS overexpression resulted in a moderate stabilization of endogenous p53 protein. Half-life of p53 protein was measured in cycloheximide (CHX) treated U2OS cells transfected with FLAG-IRTKS and control plasmid. Protein extracts, prepared at indicated time points after CHX addition, were analyzed by Western blotting. (C) IRTKS increased the cytoplasmic localization of p53. U2OS cells transfected with GFP-p53 and FLAG-IRTKS were stained for IRTKS. The nuclei were counterstained with DAPI and images were taken through a confocal microscope. One hundred cells were counted for each treatment in two separate experiments. The cells with higher levels of GFP-p53 in the cytoplasm versus the nucleus (dark gray bars) and higher levels of p53 in the nucleus versus the cytoplasm (gray bars) were scored. doi:10.1371/journal.pone.0023571.g003 PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23571