The DNA repair protein, O(6)-methylguanine-DNA methyltransferase is a proteolytic target for the E6 human papillomavirus oncoprotein.
We have previously shown that O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects tissues against toxic and carcinogenic effects of alkylating agents, is degraded through ubiquitination-dependent proteolysis. Here, we investigated the role of the human papillomavirus (HPV) E6 protein in MGMT degradation. In three pairs of isogenic human tumor cell lines in which a member of each pair expressed the E6 protein through stable transfection (HCT116/HCT116-E6, MCF7/MCF7-E6, and RKO/RKO-E6), we found a consistent 40-55% reduction in the MGMT protein level and its activity in all E6-expressing cells compared with the parent cells (P=<0.05). E6 expression did not, however, alter the levels of MGMT mRNA. Addition of the recombinant MGMT (rMGMT) protein to extracts of HCT116/E6 cells resulted in the binding of E6 to MGMT. Further, the purified E6 protein promoted the degradation of rMGMT in rabbit reticulocyte lysates. Immunoprecipitation assays showed the presence of a ternary protein complex between MGMT, E6, and the cellular ubiquitin-ligase E6-associated protein (E6-AP). Transient transfection of the p53-null H1299 lung tumor cells with an E6 construct also down-regulated the MGMT. The MGMT protein also showed structural features that are compatible for interaction with the E6, and E6-AP components. Collectively, these data suggest that the oncogenic E6 proteins enhance the ubiquitin-dependent proteolysis of MGMT.
Section of Molecular Therapeutics, Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston 77030-4009, USA. ksrivenu@mdanderson.org
Kalkunte S
KS
Srivenugopal
Francis
F
Ali-Osman
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004260:DNA Repair / Q000235:genetics; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D006801:Humans; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D012097:Repressor Proteins; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; C067713:lactacystin; D000255:Adenosine Triphosphate; D019853:O(6)-Methylguanine-DNA Methyltransferase; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D008274:Magnesium; D000111:Acetylcysteine