Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins.
E6-AP is a 100-kDa cellular protein that mediates the interaction of the human papillomavirus type 16 and 18 E6 proteins with p53. The association of p53 with E6 and E6-AP promotes the specific ubiquitination and subsequent proteolytic degradation of p53 in vitro. We recently isolated a cDNA encoding E6-AP and have now mapped functional domains of E6-AP involved in binding E6, association with p53, and ubiquitination of p53. The E6 binding domain consists of an 18-amino-acid region within the central portion of the molecule. Deletion of these 18 amino acids from E6-AP results in loss of both E6 and p53 binding activities. The region that directs p53 binding spans the E6 binding domain and consists of approximately 500 amino acids. E6-AP sequences in addition to those required for formation of a stable ternary complex with E6 and p53 are necessary to stimulate the ubiquitination of p53. These sequences lie within the C-terminal 84 amino acids of E6-AP. The entire region required for E6-dependent ubiquitination of p53 is also required for the ubiquitination of an artificial E6 fusion protein.
Laboratory of Tumor Virus Biology, National Cancer Institute, Bethesda, Maryland 20892.
J M
JM
Huibregtse
M
M
Scheffner
P M
PM
Howley
D001665:Binding Sites; D015967:Gene Expression Regulation, Viral; D066298:In Vitro Techniques; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; D014764:Viral Proteins; D044767:Ubiquitin-Protein Ligases