p53 in complex with DNA is resistant to ubiquitin-dependent proteolysis in the presence of HPV-16 E6.
The tumour suppressor p53 is a transcription factor with high affinity for specific DNA target sequences. Wild type p53 has a very short half life in normal cells but the protein shows transient accumulation in response to DNA damage, accompanied by up-regulation of target genes such as p21 and induction of growth arrest in G1 of the cell cycle. The rapid turnover of p53 may involve the ubiquitin-dependent proteolytic pathway. In order to investigate p53 turnover we have employed an in vitro system with rabbit reticulocyte lysate, in which ubiquitin-dependent degradation of p53 is mediated by the oncoprotein E6 of human papilloma virus type 16 (HPV-16). Using this system we have previously shown that E6-mediated degradation is preferential for p53 in the 1620+ conformation (reactive with the monoclonal antibody PAb1620). p53-1620+ is a pre-requisite for specific DNA binding and we have now asked if p53 in complex with DNA remains susceptible to ubiquitin-dependent proteolysis in the presence of E6. Our results indicate that p53-DNA complexes are resistant to degradation, whereas the 'free' protein is completely degraded within 20 min. Moreover, E6 did not complex with p53-DNA, possibly due to masking of sites recognised either by E6 or by the E6-associated protein (E6-AP) which facilitates E6-p53 interaction. Preincubation with E6 inhibited the DNA binding capacity of p53 and this effect could be explained, at least in part, by ubiquitination of the p53 protein.
Molinari
M
M
Department of Biology, University of York, UK.
Milner
J
J
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; D004247:DNA