Loss of the E6AP Ubiquitin Ligase Induces p53-Dependent Phosphorylation of Human Papillomavirus 18 E6 in Cells Derived from Cervical Cancer

Cancer-causing human papillomavirus (HPV) E6 oncoproteins contain a well-characterized phosphoacceptor site within the PDZ (PSD-95/Dlg/ZO-1) binding motif (PBM) at the C terminus of the protein. Previous studies have shown that the threonine or serine residue in the E6 PBM is subject to phosphorylation by several stress-responsive cellular kinases upon the induction of DNA damage in cervical cancer- derived cells. However, there is little information about the regulation of E6 phosphorylation in the absence of DNA damage and whether there may be other pathways by which E6 is phosphorylated. In this study, we demonstrate that loss of E6AP results in a dramatic increase in the levels of phosphorylated E6 (pE6) despite the expected overall reduction in total E6 protein levels. Furthermore, phosphorylation of E6 requires transcriptionally active p53 and occurs in a manner that is dependent upon DNA-dependent protein kinase (DNA PK). These results identify a novel feedback loop, where loss of E6AP results in upregulation of p53, leading to increased levels of E6 phosphorylation, which in turn correlates with increased association with 14-3-3 and inhibition of p53 transcriptional activity.