Abstract 1008: IRTKS suppresses p53 activity through promoting MDM2 mediated p53 monoubiquitination
Authors: Xin Zhang, Kesheng Wang, Hailian Sheng, Tingting Li, Gang Chen, Fei Chen, Qinwan Wang, Zhihong Cheng, Zhiqin Wang, Zeguang Han
Journal: Cancer Research (2010)

Abstract The ubiquitination of p53, including poly- and mono- ubiqitination, is important for the regulation of p53 function. The E3 ubiquitin ligase MDM2 mediates both monoubiquitination and polyubiquitination on p53 in a dose-dependent manner. Low levels of MDM2 induce monoubiquitination and nuclear export of p53, while high levels promote its polyubiquitination and degradation. The regulator of MDM2 mediated p53 monoubiquitination has been rarely identified, whereas several cofactors have been defined to regulate MDM2 induced p53 degradation. Insulin Receptor Tyrosine Kinase Substrate (IRTKS), an widespread expressed IRSP53/MIM homology domain(IMD) containing protein, has been reported as a scaffold protein in actin dynamics and membrane protrusions by associating with RAC1. As a protein located ubiquitously in cells, the function of IRTKS in nucleus remains to be elucidated. Here, we showed that ectopic IRTKS reduced and knockdown of IRTKS increased the apoptosis induced by UV irradiation in HT1080 cells in a p53-dependent manner. IRTKS inhibited the apoptosis and the expression of p53 target genes, BAX, PIG3 and MDM2, in p53 null SOAS-2 cells with force expressed p53. We found that IRTKS could directly bind to p53 in cellular nucleus in unstressed cells by using reciprocal co-immunoprecipitation analysis and fluorescence resonance energy transfer (FRET) assay. In vitro binding experiment indicated that IMD and the central region of IRTKS, DNA bind domain and C-terminal regulating domain of p53 were responsible for the interaction. Interestingly, over-expression of IRTKS induced p53 ubiquitination and nuclear export without affecting its degradation. Because IRTKS alone could not mediate p53 ubiquitination in vitro, we examined the association of IRTKS and MDM2 by using the co-immunoprecipitation analysis and in vitro binding experiment. The directly interaction through IMD of IRTKS and the central region of MDM2 has been detected. Both in vitro and in vivo ubiquitin experiment showed that IRTKS only enhanced p53 monoubiquitination mediated by low levels of MDM2, but not affected its polyubiquitination. In H1299 cells cotransfected with IRTKS, p53 and different amount MDM2, the co-immunoprecipitation experiment indicated that the association of MDM2 and p53 was significantly enhanced by ectopic IRTKS at low levels of MDM2. In IRTKS siRNA-treated H1299 cells, the p53-MDM2 interaction was reduced at low levels of MDM2. Interestingly, overexpression of MDM2 reduced the protein levels of IRTKS in HT1080 and H1299 cells. Additional in vivo and in vitro ubiquitin assays unraveled that MDM2 is an E3 ubiquitin ligase of IRTKS and mediated IRTKS polyubiquitination at high levels. Taking together, we demonstrated that IRTKS, as a scaffold protein, may play an important role in fine tune of p53 functions in an MDM2 dependent manner during normal homeostasis of the unstressed cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1008.