Regulation and function of the ubiquitin ligase E6AP

Abstract

Post-translational modification of proteins by ubiquitin (''ubiquitination'') determines proteolytic as well as non-proteolytic fates of the target proteins. It is catalyzed by the ubiquitination cascade, which is the sequential action of E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases. The substrate specificity relies on the E3 ligases, which covalently link ubiquitin to substrate proteins under formation of an isopeptide bond. A prominent member of the E3 ligase family is E6AP, which plays a key role in the development of distinct human diseases.In cervical cancer, high-risk human papillomavirus (HPV) E6 proteins hijack E6AP to target the tumor suppressor p53 for ubiquitination and subsequent proteasomal degradation. In the absence of E6, p53 is not a substrate of E6AP. Besides, genetic evidence indicates that E6AP plays a crucial role in neurodevelopment. In particular, loss of E6AP expression is causally associated with the development of Angelman syndrome, while amplification of the E6AP gene causes autistic traits. Thus, level and/or activity of E6AP have to be tightly regulated; however, only little is known how this is achieved. Similarly, the physiological role of E6AP in HPV-negative cells is still largely unknown. To gain insights into the regulation of E6AP's E3 ligase activity and the function of E6AP within cells, in vitro and in cellulo studies were employed.The first part of this thesis addressed the role of ubiquitin and HPV E6 proteins in E6AP-mediated catalysis in vitro. Previous studies revealed ubiquitin's ''canonical'' hydrophobic patch to play a crucial role in E6AP-mediated ubiquitination. However, once E6AP is bound to E6, this patch is not required for an efficient ubiquitination reaction anymore, suggesting that E6 does not only alter E6AP's substrate spectrum, but also acts as an allosteric activator of E6AP. To further clarify the role of ubiquitin in E6AP-mediated ubiquitination, studies were extended towards the ''non-canonical'' hydrophobic patch of ubiquitin. The obtained results clearly show that both patches of ubiquitin make a substantial contribution to E6AP-catalyzed isopeptide bond formation, but by different mechanisms. Eventually, experimental evidence indicates that E6 stimulates E6AP activity by enhancing its ability to catalyze isopeptide bond formation.In the second part of this thesis, studies were focused on the role of E6AP in cell signaling pathways. Previously, we and others have reported that E6AP modulates estrogen receptor (ER) signaling – an effect that may not require E6AP's E3 ligase activity. Therefore, E6AP affects protein homeostasis not only at the post-translational, but also at the transcriptional level. However, in contrast to other reports, we could not observe a direct interaction between E6AP and ER. Hence, it was hypothesized that the effect of E6AP on ER might occur indirectly rather than directly. Indeed, experimental evidence suggests that E6AP affects the transactivation of ER by modulating protein kinase cascades, in particular the PI3K/Akt/GSK3 pathway, which in turn regulate ER activity by phosphorylation. Notably, the PI3K/Akt pathway regulates a variety of different signaling events and plays a crucial role in neurodevelopment. Thus, the pathology of the neurological disorders caused by altered E6AP expression may at least partially depend on E6AP's action on kinase-mediated signaling pathways.