Identification of Three Functions of the Adenovirus E4orf6 Protein That Mediate p53 Degradation by the E4orf6-E1B55K Complex

Abstract

Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich alpha-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.

SUMO modification of E1B-55K oncoprotein regulates isoform-specific binding to the tumour suppressor protein PML

“…This possibility is nevertheless fascinating as it would offer a possible explanation for the different transformation capabilities of the large adenovirus E1Bs and earlier suggestions linking nuclear localization of HAdV5 E1B-55K (Endter et al, 2005) and presumably of subgroup HAdV12 E1B-54K (Kra¨tzer et al, 2000) to their oncogenic potential. However, it remains subject for further studies to evaluate how and whether E1B-55K facilitates PML and/or associated proteins to mediate its multiple functions during productive infection involving transcriptional repression of p53 (Yew et al, 1994;Berk, 1998, 1999), selective protein degradation and viral mRNA transport in combination with E4orf6 Querido et al, 2001;Stracker et al, 2002;Baker et al, 2007;Dallaire et al, 2009).…”

The Human Papillomavirus E6 protein and its contribution to malignant progression

“…To overcome this obstacle, several viruses encode proteins that functionally inactivate p53. SV40 LT prevents transactivation of p53 target genes through association with its DNA binding domain (Ruppert and Stillmann, 1993), Ad E1B-55K abolishes the same function by binding to the transactivating domain of p53 (Lin et al, 1994), yet in association with E4orf6 it can also lead to p53 degradation (Steegenga et al, 1998, Querido et al, 2001; TaÈ uber and Dobner, this issue), while the HBV X protein sequesters p53 in the cytoplasm (Elmore et al, 1997). A major strategy employed by the high-risk HPV E6 proteins to abrogate p53's oncosuppressive functions is to induce its degradation through the ubiquitin-proteasome pathway (Sche ner et al, 1990).…”

PCAF is a coactivator for p73-mediated transactivation

“…Moreover, unlike p53, which is phosphorylated by DNAPK and ATM (Ko and Prives, 1996;Prives and Hall, 1999), the p73 protein accumulates and undergoes c-Abl-dependent tyrosine phosphorylation in response to DNA damage (Agami et al, 1999;Gong et al, 1999;Yuan et al, 1999). Furthermore, p73 is not targeted for inactivation by viral oncoproteins such as simian virus 40 T antigen, adenovirus E1B 55-kDa and human papillomavirus E6 (Higashino et al, 1998;Marin et al, 1998;Prabhu et al, 1998;Wienzek et al, 2000;Gu et al, 2001;Querido et al, 2001), although cellular oncoprotein MDM2 was shown to inhibit p73-mediated transactivation (Kobet et al, 2000).…”