Human papillomavirus E6-induced degradation of E6TP1 is mediated by E6AP ubiquitin ligase.
High-risk human papilloma viruses are known to be associated with cervical cancers. We have reported previously that the high-risk human papillomavirus (HPV) E6 oncoprotein interacts with E6TP1, a novel Rap GTPase-activating protein (RapGAP). Similar to p53 tumor suppressor protein, the high-risk HPV E6 oncoproteins target E6TP1 for degradation. The HPV16 E6-induced degradation of E6TP1 strongly correlates with its ability to immortalize human mammary epithelial cells. In this study, we used treatment with a proteasome inhibitor MG132, analysis in CHO-ts20 cells with a thermolabile ubiquitin-activating enzyme, and direct detection of ubiquitin-modified E6TP1 to demonstrate that E6TP1 is targeted for degradation by the ubiquitin-proteasome pathway both in the presence and in the absence of E6. Using deletion mutants of E6TP1, we mapped the region required and sufficient for E6 binding to COOH-terminal 40 amino acid residues and showed this region to be necessary for E6-dependent degradation of E6TP1. Furthermore, the E6-binding region of E6TP1 complexes with E6AP via E6. Importantly, the purified E6AP enhanced the ubiquitination and degradation of E6TP1 in the presence of E6 in vitro. Additionally, the expression of a dominant-negative E6AP mutant (C833A) in cells inhibited the E6-induced degradation of E6TP1. These findings demonstrate that the E6-induced decrease in the levels of E6TP1 protein involves the E6AP-mediated ubiquitination followed by proteasome-dependent degradation.
Gao
Qingshen
Q
Division of Radiation and Cancer Biology, Department of Radiation Oncology, New England Medical Center, Tufts University School of Medicine, 750 Washington Street, Boston, MA 02111, USA.
Kumar
Ajay
A
Singh
Latika
L
Huibregtse
Jon M
JM
Beaudenon
Sylvie
S
Srinivasan
Seetha
S
Wazer
David E
DE
Band
Hamid
H
Band
Vimla
V
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008025:Ligases / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins; D013696:Temperature; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D015853:Cysteine Proteinase Inhibitors; C068651:E6 protein, Human papillomavirus type 16; C116934:E6-target protein 1; D020690:GTPase-Activating Proteins; D007976:Leupeptins; D009097:Multienzyme Complexes; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde