Disruption of the gatekeeper p53 tumor suppressor is involved in various virus-associated tumorigeneses, with aberrant ubiquitination as the major cause of p53 abnormalities in virus-associated tumors. Of note, wild-type p53 is accumulated in Epstein-Barr virus (EBV)-associated tumors, especially in nasopharyngeal carcinoma (NPC). We have previously identified that p53 is accumulated and phosphorylated by EBV oncoprotein latent membrane protein 1 (LMP1) in NPC. Here, we further found that LMP1 promoted p53 accumulation via two distinct ubiquitin modifications. LMP1 promoted p53 stability and accumulation by suppressing K48-linked ubiquitination of p53 mediated by E3 ligase MDM2, which is associated with its phosphorylation at Ser20, while increasing the levels of total cellular ubiquitinated p53. LMP1 also induced K63-linked ubiquitination of p53 by interacting with tumor necrosis factor receptor-associated factor 2 (TRAF2), thus contributing to p53 accumulation. Furthermore, LMP1 rescued tumor cell apoptosis and cell cycle arrest mediated by K63-linked ubiquitination of p53. Collectively, these results demonstrate aberrant ubiquitin modifications of p53 and its biological functions by viral protein LMP1, which has broad implications to the pathogenesis of multiple EBVassociated tumors.
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blot and quantitated by densitometric measurement. Ubiquitination assays. H1299 cells were co-transfected with His-Ub, His-UbK48, His-UbK63 and other expression plasmids We thank Dr. George Tsao for some cell lines. This proj-as indicated. Forty hours after the transfection, the cells were ect was supported by National Basic Research Program (No. treated with 10 μM of MG132 (Sigma Aldrich) for 6 h prior to 2011CB504300), Key Program of National Natural Science harvest. Cells were split into two aliquots, one for immunoblot Foundation of China (No. 3090101), Joint Research Fund for and the other for ubiquitination assays. Cell lysates were affin-Hong Kong and Macao Young Scholars (No. 81028012) and ity purified with Ni-NTA-agarose beads (Qiagen) or incubated National Natural Science Foundation (No. 30801337). with specific antibodies targeting p53 or MDM2 and analyzed by immunoblot, as described previously. Supplemental Materials Flow cytometry for apoptosis and cell cycle analyses. Supplemental materials may be found here: Cells were co-transfected with GFP-p53, pcDNA3.1-TRAF2, www.landesbioscience.com/journals/cc/article/20771
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- Epstein-Barr virus