The development of cancer is a multistep process involving mutations in proto-oncogenes, tumor suppressor genes, and other genes which control cell proliferation, telomere stability, angiogenesis, and other complex traits. Despite this complexity, the cellular pathways controlled by the p53 tumor suppressor protein are compromised in most, if not all, cancers. In normal cells, p53 controls cell proliferation, senescence, and/or mediates apoptosis in response to stress, cell damage, or ectopic oncogene expression, properties which make p53 the prototype tumor suppressor gene. Defining the mechanisms of regulation of p53 activity in normal and tumor cells has therefore been a major priority in cell biology and cancer research. The present study reveals a novel and potent mechanism of p53 regulation originating through alternative splicing of the human p53 gene resulting in the expression of a novel p53 mRNA. This novel p53 mRNA encodes an N-terminally deleted isoform of p53 termed p47. As demonstrated within, p47 was able to effectively suppress p53-mediated transcriptional activity and impair p53-mediated growth suppression. It was possible to select for p53-null cells expressing p47 alone or coexpressing p53 in the presence of p47 but not cells expressing p53 alone. This showed that p47 itself does not suppress cell viability but could control p53-mediated growth suppression. Interestingly, p47 was monoubiquitinated in an Mdm2-independent manner, and this was associated with its export out of the nucleus. In the presence of p47, there was a reduction in Mdm2-mediated polyubiquitination and degradation of p53, and this was also associated with increased monoubiquitination and nuclear export of p53. The expression of p47 through alternative splicing of the p53 gene thus has a major influence over p53 activity at least in part through controlling p53 ubiquitination and cell localization.

The E6 protein from high-risk human papillomaviruses (HPVs) targets the p53 tumor suppressor for degradation by the proteasome pathway. This ability contributes to the oncogenic potential of these viruses. However, several aspects concerning the mechanism of E6-mediated p53 degradation at the cellular level remain to be clarified. This study therefore examined the role of cell localization and ubiquitination in the E6-mediated degradation of p53. As demonstrated within, following coexpression both p53 and high-risk HPV type 18 (HPV-18) E6 (18E6) shuttle from the nucleus to the cytoplasm. Mutation of the C-terminal nuclear export signal (NES) of p53 or treatment with leptomycin B inhibited the 18E6-mediated nuclear export of p53. Impairment of nuclear export resulted in only a partial reduction in 18E6-mediated degradation, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. This was also consistent with the observation that 18E6 mediated the accumulation of polyubiquitinated p53 in the nucleus. In comparison, a p53 isoform that localizes predominantly to the cytoplasm was not targeted for degradation by 18E6 in vivo but could be degraded in vitro, arguing that nuclear p53 is the target for E6-mediated degradation. This study supports a model in which (i) E6 mediates the accumulation of polyubiquitinated p53 in the nucleus, (ii) E6 is coexported with p53 from the nucleus to the cytoplasm via a CRM1 nuclear export mechanism involving the C-terminal NES of p53, and (iii) E6-mediated p53 degradation can be mediated by both nuclear and cytoplasmic proteasomes.The p53 protein is a tumor suppressor that can cause cell cycle arrest or apoptosis in response to DNA damage and other forms of stress (19). The critical role played by p53 in regulating normal cell growth is highlighted by the observations that p53-deficient mice are rendered highly susceptible to sporadic cancers (6) and germ line mutations in p53 result in Li-Fraumeni syndrome, in which individuals are predisposed to a variety of cancer types (22). Furthermore, approximately 50% of tumors contain mutated p53 genes (15), while the remaining 50% frequently appear to have compromised p53 function. For example, wild-type p53-containing tumors may overexpress the mdm2 cellular oncogene or the E6 oncogene from high-risk human papillomavirus (HPV) types. Tight regulation of p53 activity involves a variety of posttranslational modifications including ubiquitination, proteasome-mediated degradation, and control of subcellular localization. Mdm2, a cellular ubiquitin ligase (16), is an important negative regulator of p53 activity and mediates the ubiquitin-dependent degradation of p53 by the proteasome (13, 17).High-risk HPV types, such as types 16 and 18, are the etiological agents for the development of cervical cancer (34). The oncogenic potential of these viruses correlates with the in vitro transforming activities of the viral oncogenes E6 and E7. The E7 protein promotes the upregulation of proliferation-related genes (25) thr...

Human papillomaviruses (HPVs) are aetiological agents for genital warts and cervical cancer, the different pathologies of which are dependent on the type of HPV infection. Oncogenic HPV types associated with cancer are carcinogens by virtue of their oncogene products, which target key regulators of cell proliferation and apoptosis. The viral E6 protein from oncogenic HPV types plays a central role in carcinogenesis by exploiting the cellular proteasome degradation pathway in order to mediate the degradation of cellular proteins, most notably the prototype tumour suppressor protein p53. Much less is known about the cellular targets of E6 from the non-oncogenic HPV types associated with genital warts. It is also unclear what factors influence the level and stability of the viral E6 proteins in cells. This report demonstrates that both oncogenic and non-oncogenic HPV E6 proteins (from types 18 and 11, respectively) are ubiquitinated and targeted for degradation by the 26S proteasome. E6 domains required for the induction of p53 or DLG degradation, or E6AP binding, are not involved in proteasome-mediated degradation of HPV-18 E6. These results provide insight into the cellular modulation of E6 protein levels from both high-risk and low-risk HPV types.About 30 HPV types can infect the genital area resulting in benign or malignant pathologies (zur Hausen 1991(zur Hausen , 1996Munoz et al., 2003). Low-risk HPV types (for example types 6 and 11) are non-oncogenic and mainly associated with benign genital warts, whereas the high-risk HPV types (i.e. types 16 and 18) are considered oncogenic and are frequently associated with cervical intraepithelial neoplasia (CIN), which can lead to invasive cancers. The oncogenic potential of high-risk HPV types has been correlated with the in vitro transforming activities of the viral oncogenes E6 and E7 (Storey et al., 1988). E6 and E7 proteins from oncogenic HPV types represent carcinogens by virtue of their ability to inactivate key regulatory cellular proteins involved in cell proliferation and apoptosis. Viral E6 proteins from oncogenic HPV types are able to bind p53 (Werness et al., 1990) and mediate its degradation (Scheffner et al., 1990) in order to abrogate p53-mediated apoptosis in infected cells. Oncogenic HPV E6 proteins target p53 for degradation through association with a cellular ubiquitin ligase termed E6AP (Huibregtse et al., 1991(Huibregtse et al., , 1993 resulting in the ubiquitination of p53 and its subsequent degradation by the 26S proteasome. In addition to p53, E6 from oncogenic HPVs has also been reported to mediate the degradation of a number of other cellular proteins (reviewed by Scheffner & Whitaker, 2003) including the ubiquitin ligase E6AP (Kao et al., 2000), c-Myc (Gross-Mesilaty et al., 1998), Bak (Thomas & Banks, 1998), hDLG (Kiyono et al., 1997Lee et al., 1997;Gardiol et al., 1999;Pim et al., 2000), MAGI-1 (Glausinger et al., 2000), hScrib (Nakagawa & Huibregtse, 2000) and MUPP-1 (Lee et al., 2000). E6 proteins from low-risk HPVs do not appear to t...

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