XPC promotes MDM 2-mediated degradation of the p 53 tumor suppressor

Although ubiquitin receptor Rad23 has been implicated in bringing ubiquitylated p53 to the proteasome, how Rad23 recognizes p53 remains unclear. We demonstrate that XPC, a Rad23-binding protein, regulates p53 turnover. p53 protein in XPC-deficient cells remains ubiquitylated, but its association with the proteasome is drastically reduced, indicating that XPC regulates a postubiquitylation event. Furthermore, we found that XPC participates in the MDM2-mediated p53 degradation pathway via direct interaction with MDM2. XPC W690S pathogenic mutant is specifically defective for MDM2 binding and p53 degradation. p53 is known to become stabilized following UV irradiation but can be rendered unstable by XPC overexpression, underscoring a critical role of XPC in p53 regulation. Elucidation of the proteolytic role of XPC in cancer cells will help to unravel the detailed mechanisms underlying the coordination of DNA repair and proteolysis. INTRODUCTION One early major discovery that propelled the ubiquitin (Ub)/proteasome system to the forefront in biological research is its tight control on the cellular concentration of p53 tumor suppressor, a master regulator of cell survival and death (Vousden and Prives, 2009; Wade et al., 2010; Brooks and Gu, 2011). Over the years, many laboratories have elucidated the mechanisms of Ub ligase–mediated p53 ubiquitylation under various conditions. One pivotal regulatory mechanism is MDM2-mediated p53 degradation. Specifically, the MDM2 Ub ligase E3 binds to p53 and then covalently attaches Ub onto p53 with the help of E1 and E2 enzymes. Thus far, the studies on the MDM2-p53 circuit concentrated on the mechanisms underlying the MDM2-p53 interaction and MDM2-catalyzed Ub conjugation onto p53 (Wade et al., 2010; Brooks and Gu, 2011). How the Ub-tagged p53 is then transferred to the proteasome remains unclear. Rad23 can directly bind Ub and the proteasome and has been shown to act as an adaptor that shuttles the ubiquitylated proteins to the proteasome (Finley, 2009). Rad23 is required for the degradation of a subset of substrates, including p53 (Glockzin et al., 2003; Dantuma et al., 2009). Because multiple Rad23-like adaptor molecules (e.g., Dsk2, Ddi1) exist and have distinct substrate specificity in vivo, an important unresolved issue is how Rad23 recognizes specific targets (Finley, 2009; Liu and Walters, 2010). One possible helper of Rad23 is its binding protein XPC, a key factor for a conserved DNA repair pathway termed nucleotide excision repair (NER; Friedberg et al., 2006; Sugasawa, 2008). Mutations in XPC can cause xeroderma pigmentosum (XP), a genetic disease that often leads to skin cancer, and are also associated with increased risks for Hodgkin’s disease and other malignancies, including cancer of the lung, bladder, prostate, and pancreas (Hollander et al., 2005; Friedberg et al., 2006; Hirata et al., 2007; El-Zein et al., 2009). Not all the phenotypes associated with XPC patients can be easily explained by DNA repair defects, suggesting that XPC has an important non-NER activity. Consistent with this conjecture, the Lys939Gln allele of XPC is associated with a high risk of cancers, yet wild-type NER activity (Khan et al., 2000; Friedberg et al., 2006; Hirata et al., 2007; El-Zein et al., 2009). Monitoring Editor Thomas Sommer Max Delbrück Center for Molecular Medicine Received: Jun 3, 2013 Revised: Nov 7, 2013 Accepted: Nov 12, 2013 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E13-05-0293) on November 20, 2013. *These authors contributed equally to this work. Address correspondence to: Keke Huo (kkhuo@fudan.edu.cn) or Hai Rao (raoh@ uthscsa.edu). © 2014 Krzeszinski et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. Abbreviations used: FBS, fetal bovine serum; GFP, green fluorescent protein; GST, glutathione S-transferase; NER, nucleotide excision repair; RNAi, RNA interference; Ub, ubiquitin; XP, xeroderma pigmentosum. http://www.molbiolcell.org/content/suppl/2013/11/18/mbc.E13-05-0293v1.DC1 Supplemental Material can be found at: