Ubiquitin ligase E6AP mediates nonproteolytic polyubiquitylation of ß-catenin independent of the E6 oncoprotein.

Recently, we showed that the ubiquitin ligase E6AP stabilizes ß-catenin and activates its transcriptional activity. These activities were enhanced by the human papillomavirus (HPV) E6 protein. In the present study, we explored the function of E6AP, which increases ß-catenin stabilization and transcriptional activation. Here, we report that E6AP interacts with ß-catenin and mediates its nonproteolytic ubiquitylation, as evidenced in transiently transfected cell-based and in vitro reconstitution ubiquitylation assays. Overexpression of E6AP increased ß-catenin polyubiquitylation and, consistent with that, knockdown or knock-out of E6AP expression reduced ß-catenin polyubiquitylation. The ubiquitylation of ß-catenin by E6AP was dependent on its E3 ubiquitin ligase activity, but it was proteasome-independent and did not require HPV-E6, phosphorylation of ß-catenin by glycogen synthase kinase 3ß (GSK3ß) or activity of the ß-catenin 'destruction complex'. We also show that transcriptional activation of ß-catenin by E6AP is coupled with ß-catenin protein stabilization, but not its ubiquitylation. In contrast to ß-catenin ubiquitylation, ß-catenin protein stability and its transcriptional activity were absolutely dependent on the activity of the destruction complex and phosphorylation by GSK3ß. Collectively, our data uncover a dual role for E6AP in the regulation of ß-catenin ubiquitylation, stability and transcriptional activity, with HPV-E6 enhancing only part of E6AP activities.

HPV16 E6 and E6AP differentially cooperate to stimulate or augment Wnt signaling.

The present study investigated the roles of E6 and E6AP in the Wnt pathway. We showed that E6 levels are markedly reduced in cells in which Wnt signaling is activated. Coexpression of wild-type or mutant E6AP (C820A) in Wnt-activated cells stabilized E6 and enhanced Wnt/ß-catenin/TCF transcription. Expression of E6AP alone in nonstimulated cells elevated ß-catenin level, promoted its nuclear accumulation, and activated ß-catenin/TCF transcription. A knockdown of E6AP lowered ß-catenin levels. Coexpression with E6 intensified the activities of E6AP. Further experiments proved that E6AP/E6 stabilize ß-catenin by protecting it from proteasomal degradation. This function was dependent on the catalytic activity of E6AP, the kinase activity of GSK3ß and the susceptibility of ß-catenin to GSK3ß phosphorylation. Thus, this study identified E6AP as a novel regulator of the Wnt signaling pathway, capable of cooperating with E6 in stimulating or augmenting Wnt/ß-catenin signaling, thereby possibly contributing to HPV carcinogenesis.