Insulin Receptor Tyrosine Kinase Substrate Enhances Low Levels of MDM2-Mediated p53 Ubiquitination Ke-Sheng Wang 1 , Gang Chen1,2, Hai-Lian Shen1, Ting-Ting Li1,2, Fei Chen1, Qin-Wan Wang 1 , Zhi-Qin Wang 1 , Ze-Guang Han1,3, Xin Zhang 1 * 1 Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China, 2 School of Bioengineering, East China University of Science and Technology, Shanghai, China, 3 Rui-Jin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Abstract The tumor suppressor p53 controls multiple cellular functions including DNA repair, cell cycle arrest and apoptosis. MDM2- mediated p53 ubiquitination affects both degradation and cytoplasmic localization of p53. Several cofactors are known to modulate MDM2-mediated p53 ubiquitination and proteasomal degradation. Here we show that IRTKS, a novel IRSp53-like protein inhibited p53-induced apoptosis and depressed its transcription activity. IRTKS bound directly to p53 and increased p53 ubiquitination and cytoplasmic localization. Further studies revealed that IRTKS interacted with MDM2 and promoted low levels of MDM2-mediated p53 ubiquitination in vitro and in vivo. In unstressed cells with low levels of MDM2, IRTKS was found to stabilize the interaction of p53 and MDM2. In stressed cells, IRTKS dissociated from p53, and high levels of MDM2 induced by p53 activation mediate IRTKS poly-ubiquitination and subsequent proteasomal degradation. These data suggest that IRTKS is a novel regulator of p53, modulating low level of MDM2-mediated p53 ubiquitination in unstressed cells. Citation: Wang K-S, Chen G, Shen H-L, Li T-T, Chen F, et al. (2011) Insulin Receptor Tyrosine Kinase Substrate Enhances Low Levels of MDM2-Mediated p53 Ubiquitination. PLoS ONE 6(8): e23571. doi:10.1371/journal.pone.0023571 Editor: Deanna Koepp, University of Minnesota, United States of America Received May 9, 2011; Accepted July 20, 2011; Published August 24, 2011 Copyright: 2011 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by Chinese High-Tech Research and Development Program grants (2006AA02A305), the China National Key Projects for Infectious Disease (2008ZX10002-020 and 2008ZX10002-021), and Shanghai Commission for Science and Technology (10JC1411900). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: zhang68@hotmail.com Introduction The activity of p53 is controlled by a complicated network during normal homeostasis and stress response [1]. Multiple post- translational modifications of p53, including phosphorylation, acetylation, methylation, glycosylation, ubiquitination and ubiq- uitin-like modification, have been documented for p53 regulation [2,3]. Among these modifications, ubiquitination plays a pivotal role in controlling p53 activity under different conditions [4]. Ubiquitin conjugates to proteins either in a monomeric manner (monoubiquitination) to cause conformational change to the substrate or in a polyubiquitin chain which signals the substrate to be degraded by the 26S proteasome. While polyubiquitination serves as a signal for proteasomal degradation which requires an at least four-subunit-long multiubiquitin chain per individual lysine residue, monoubiquitination has been shown to be involved in a variety of cellular processes, including protein trafficking, DNA repair and transcriptional regulation [5,6]. MDM2, a key regulator of p53, catalyzes both monoubiquitination and polyubiquitination on p53 in a dose-dependent manner. High levels of MDM2 mediate p53 polyubiquitination and proteasomal degradation, whereas low levels of MDM2 induce the monoubiquitination and nuclear export of p53 [7]. Some cofactors, including L5, L11, L23, p300/CBP, Yin Yang 1, MDMx, UBE4B, have been characterized to regulate MDM2-mediated ubiquitination and degradation [8,9]. But the regulation of low levels of MDM2-mediated p53 monoubiquitina- tion and cytoplasmic translocation has been rarely reported. IRSp53-like proteins contain a conserved IRSp53/MIM homol- ogy domain (IMD) at the N-terminus, and a canonical SH3 domain near the C-terminus [10]. The IMD belongs to the larger family of Bin-amphyipysin-Rvs67 (BAR) domains that can bundle actin filaments and induce membrane protrusions [11]. The IRSp53 family is involved in the formation of filopodia and lamellipodia [12]. Insulin receptor tyrosine kinase substrate (IRTKS), an IRSp53-like protein, has been recognized as a substrate for the insulin receptor tyrosine kinase and its overexpression induces clustering of short actin bundles [13]. However, IRTKS is largely localized in the nucleus wherein its function remains unknown. In this study, we first found that IRTKS suppressed the p53 induced apoptosis and transactivation activity. IRTKS interacted directly with both p53 and MDM2 through its IMD domain, and enhances low level MDM2-mediated p53 ubiquitination in vivo and in vitro. Interestingly, the effect of IRTKS on the p53-MDM2 axis is dependent on the protein levels of MDM2. At normal physiological status, the interaction between p53 and low level MDM2 was enhanced by IRTKS. During stress, p53 dissociated with IRTKS. And high levels of MDM2 induce ubiquitin-mediated degradation of IRTKS. These data together suggested that the scaffold protein, IRTKS may balance the MDM2 activity in mediating p53 ubiqitination. Results IRTKS suppressed p53-induced apoptosis and negatively regulated the transcription activity of p53 To investigate the function of IRTKS, we established a stable HT1080 cell line (HT1080-IRTKS) with expressing ectopic FLAG-tagged IRTKS upon withdrawal of doxycycline [14]. Interestingly, overexpression of IRTKS remarkably suppressed PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23571