IRTKS Regulates MDM2-Mediated p53 Ubiquitination Figure 5. IRTKS promoted MDM2-mediated p53 ubiqutination. (A) IRTKS only enhanced p53 ubiquitination mediated by low levels of MDM2. In vitro ubiquitination assays were performed with recombinant His-p53, His-IRTKS and varying amounts of His-MDM2, and analyzed with p53-specific monoclonal antibody (DO-1) by Western blotting. (B) IMD was essential for IRTKS to promote MDM2-mediated p53 ubiquitination. In vitro ubiquitination assays were performed with p53, low levels of MDM2 and varying amounts of IRTKS (lane 1–3) or IMD deletion mutant IRTKS (lane 4–6). (C) IRTKS promoted low levels of MDM2-mediated ubiquitination of p53 in vivo. H1299 cells were cotransfected with the plasmids for expression HA-ubiquitin, p53, varying amounts of MDM2 and IRTKS or control plasmid. Cells were treated with 25 mM MG-132 for 4 h and the lysates were immunoprecipitated with anti-HA antibody. p53 ubiquitination was analyzed by western blot with anti-p53 antibody(DO-1). (D) IRTKS could not affect MDM2-induced p53 neddylation. H1299 cells were cotransfected with the indicated plasmids and treated with 25 mM MG-132 for 4 h. The cell lysates were immunoprecipitated with anti-HA antibody. p53 neddylation was analyzed by western blot with anti-p53 antibody(DO-1). doi:10.1371/journal.pone.0023571.g005 may have diverse functions in cytoplasm and nucleus. While the middle region of IRSp53 is a partial CRIB domain which binds to active CDC42, IRTKS can not interact with CDC42 and its middle region provided a second binding site for IRTKS-p53 interaction. Therefore, it remains to determine whether the other IRSp53-like proteins can affect p53 ubiquitination. As a tumor suppressor, p53 is presumed as a protein that acts negatively to prevent motility and tumor metastasis [44]. Actin polymerization and membrane deformation are important pro- cesses for cell mobility [45]. Recently, a p53 cofactor JMY, has been identified as an actin nucleation factor which is involved in to cell motility [46]. Under DNA damage, JMY augments in nucleus and drives the p53 transcription, its influence on cell motility is reduced [47]. As IRTKS was involved in both actin dynamic and p53 modification, it remains to investigate whether IRTKS is another protein for coordinating cell mobility with the p53 response. And further studies need to address the behavior of IRTKS in tumor as it has diverse functions in inhibiting p53 activation in nucleus and promoting cell mobility in cytoplasm. Materials and Methods Plasmids and antibodies The entire open reading frame of human IRTKS was amplified from a human fetal liver cDNA library (Clontech) and subcloned into mammalian cell expression vectors pFLAG-CMV 2 (Sigma) for mammalian expression. Plasmids for expressing p53 and MDM2 in mammalian cells were constructed in pcDNA 3.0. For in vitro experiment, IRTKS, p53 and MDM2 including their truncations were constructed by PCR, and subcloned into pGEX-5X-1 and PET-28a. For immunoblotting and immunoprecipitation detection of endogenous IRTKS, a rabbit polyclonal antibody was raised against GST-IRTKS. p53 antibody DO-1 and FL393, MDM2 antibody H-221 (Santa Cruz) were used for immunoprecipitation. p53 antibody 1C12 (Cell Signaling Technology) and MDM2 antibody H-221 were used for immunofluorescence staining. p53 antibody DO-1 and FL393, MDM2 antibody H-221, cleaved PARP (Asp214) antibody (Cell Signaling Technology ) and PIG3 antibody A-5 (Santa Cruz) were used for Western blotting. PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23571