The role of E6AP in the regulation of p53 protein levels in human papillomavirus (HPV)-positive and HPV-negative cells.
The E6 protein encoded by the oncogenic human papillomaviruses (HPVs) targets p53 for ubiquitin-dependent proteolysis. E6-mediated p53 degradation requires the 100-kDa cellular protein E6-associated protein (E6AP). E6AP and E6 together provide the E3-ubiquitin protein ligase activity in the transfer of ubiquitin to p53. In vitro studies have shown that E6AP can form a high energy thiolester bond with ubiquitin and, in the presence of E6, transfer ubiquitin to p53. In this study we have addressed the role of E6AP in vivo in the degradation of p53. Overexpression of wild-type E6AP in HeLa cells, which are HPV18-positive and express E6, resulted in a decreased steady state level of p53 and a decrease in the half-life of p53. Mutant forms of E6AP proteins were identified that were catalytically incapable of participating in E6-dependent ubiquitination of p53 and functioned in a dominant-negative manner in that they inhibited the E6-mediated ubiquitination of p53 by the wild-type E6AP in vitro. Transient transfection of one of these dominant negative (dn) mutants resulted in an increase in both the steady state level and half-life of p53 in vivo in HeLa cells. Consistent with this observation, overexpression of the dn E6AP resulted in a marked G1 shift in the cell cycle profile. In contrast, dn E6AP had no effect on p53 levels in U2OS cells, an HPV-negative cell line that contains wild-type p53. These studies provide evidence for the involvement of E6AP in E6-mediated p53 degradation in vivo and also indicate that E6AP may not be involved in the regulation of p53 ubiquitination in the absence of E6.
Talis
A L
AL
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Huibregtse
J M
JM
Howley
P M
PM
D002453:Cell Cycle; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D027383:Papillomaviridae; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases