HPV E6 targeted degradation of the discs large protein: evidence for the involvement of a novel ubiquitin ligase.
The Discs Large (DLG) tumour suppressor protein is targeted for ubiquitin mediated degradation by the high risk human papillomavirus (HPV) E6 proteins. In this study we have used a mutational analysis of E6 in order to investigate the mechanism by which this occurs. We first show that the differences in the affinities of HPV-16 and of HPV-18 E6 proteins for binding DLG is reflected in their respective abilities to target DLG for degradation. A mutational analysis of HPV-18 E6 has enabled us to define regions within the carboxy terminal half of the protein which are essential for the ability of E6 to direct the degradation of DLG. Mutants within the amino terminal portion of E6 which have lost the ability to bind the E6-AP ubiquitin ligase, as measured by their ability to degrade p53, nonetheless retain the ability to degrade DLG. Significant levels of DLG degradation are also obtained using wheat germ extracts which lack E6-AP. Finally, we show that the transfer of the DLG binding domain onto the low risk HPV-6 E6 confers DLG binding activity to that protein and, most significantly, allows HPV-6 E6 to target DLG for degradation. These results indicate that E6 mediated degradation of DLG does not involve the E6-AP ubiquitin ligase and, in addition, shows that the high and low risk HPV E6 proteins most likely share a common cellular intermediary in the ubiquitin pathway.
Pim
D
D
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Thomas
M
M
Javier
R
R
Gardiol
D
D
Banks
L
L
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001859:Bone Neoplasms / Q000473:pathology; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D000074288:Discs Large Homolog 1 Protein; D005814:Genes, Viral; D006801:Humans; D008025:Ligases / Q000502:physiology*; D008565:Membrane Proteins; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000502:physiology*; D012516:Osteosarcoma / Q000473:pathology; D027383:Papillomaviridae / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D011506:Proteins / Q000502:physiology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012097:Repressor Proteins; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; D004268:DNA-Binding Proteins; D000074288:Discs Large Homolog 1 Protein; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D008565:Membrane Proteins; D009856:Oncogene Proteins, Viral; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014452:Ubiquitins; D015678:Viral Structural Proteins; D044767:Ubiquitin-Protein Ligases; D008025:Ligases