Involvement of a cellular ubiquitin-protein ligase E6AP in the ubiquitin-mediated degradation of extensive substrates of high-risk human papillomavirus E6.
Human scribble (hScrib), which was identified as substrate of human papillomavirus (HPV) E6 for ubiquitin-mediated degradation dependent on ubiquitin-protein ligase E6AP, is a human homolog of Drosophila neoplastic tumor suppressor scribble, in which mutation causes loss of polarity and overgrowth of epithelia. Drosophila discs large (Dlg) is one of neoplastic tumor suppressors, which genetically links to scribble. E6 also targets human Dlg (hDlg) for ubiquitin-mediated degradation. Ubiquitin-protein ligase involved in this process has not been identified thus far. Here we investigated mechanism underlying degradation of three target proteins of E6, hScrib, hDlg, and p53 by using eighteen HPV 16 E6 mutants with single amino acid substitution. In vitro degradation ability of each E6 mutant was equivalent for these tumor suppressors. We investigated whether E6AP is involved in ubiquitin-mediated degradation of hDlg. In vitro binding assay revealed that hDlg formed ternary complex with E6-E6AP complex. The ability of E6 mutants to degrade these tumor suppressors was correlated with their ability to interact with E6AP. Furthermore, hDlg was targeted for in vitro ubiquitination in the presence of both E6 and E6AP. These data revealed that E6AP is extensively involved in the ubiquitin-mediated degradation of E6-dependent substrates as a cellular E3 ubiquitin-protein ligase.
Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan.
Yoko
Y
Matsumoto
Shunsuke
S
Nakagawa
Tetsu
T
Yano
Shin
S
Takizawa
Kazunori
K
Nagasaka
Keiichi
K
Nakagawa
Takeo
T
Minaguchi
Osamu
O
Wada
Hajime
H
Ooishi
Koji
K
Matsumoto
Toshiharu
T
Yasugi
Tadahito
T
Kanda
Jon M
JM
Huibregtse
Yuji
Y
Taketani
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D045744:Cell Line, Tumor; D000074288:Discs Large Homolog 1 Protein; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; D000074288:Discs Large Homolog 1 Protein; C068651:E6 protein, Human papillomavirus type 16; D008565:Membrane Proteins; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; C480591:SCRIB protein, human; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases