c-Abl Phosphorylates E6AP and Regulates Its E3 Ubiquitin Ligase Activity

Abstract

In human papillomavirus (HPV)-infected cells, the p53 tumor suppressor is tightly regulated by the HPV-E6-E6AP complex, which promotes it for proteasomal degradation. We previously demonstrated that c-Abl tyrosine kinase protects p53 from HPV-E6-E6AP complex-mediated ubiquitination and degradation under stress conditions. However, the underlying mechanism was not defined. In this study, we explored the possibility that c-Abl targets E6AP and thereby protects p53. We demonstrated that c-Abl interacts with and phosphorylates E6AP. We determined that the E3 ligase activity of E6AP is impaired in response to phosphorylation by c-Abl. We mapped the phosphorylation site to tyrosine 636 within the HECT catalytic domain of E6AP, and using substitution mutants, we showed that this residue dictates the E3 ligase activity of E6AP, in a substrate-specific manner. On the basis of the crystal structure of the HECT domain of E6AP, we propose a model in which tyrosine 636 plays a regulatory role in the oligomerization of E6AP, which is a process implicated in its E3 ubiquitin ligase activity. Our results suggest that c-Abl protects p53 from HPV-E6-E6AP complex-mediated degradation by phosphorylating E6AP and impairing its E3 ligase activity, thus providing a molecular explanation for the stress-induced protection of p53 in HPV-infected cells.

The Active Form of E6-associated protein (E6AP)/UBE3A Ubiquitin Ligase Is an Oligomer

“…We were particularly interested in Tyr 533 , which is an Angelman syndrome mutation site and which participates in a pattern of side chain interactions between subunits (75). Tyrosine 533 also exists in the region of subunit interface recently suggested by Chan et al to be sensitive to cAbl-dependent regulation by phosphorylation of Tyr 636 (76). For two of the subunits in the trimer structure, the side chain phenolic group of Tyr 533 hydrogen bonds with the amide hydrogen of Asp 543 present on the same polypeptide chain (Fig.…”

“…In contrast, disrupting the hydrogen bond between Lys 688 in the small N-terminal subdomain and Glu 535 in the adjacent large N-terminal subdomain by mutation of the former has no consequence, indicating that the effects of the previous mutations are specific to those residues rather than a general feature of the interface. Interestingly, this subunit interface harbors Tyr 636 , which Chan et al (76) have identified as a substrate for c-Abl phosphorylation in the regulation of E6AP function. Chan et al (76) have speculated that the inhibition of E6AP activity observed on phosphorylation of Tyr 636 might result from blocking oligomerization of the enzyme.…”

Structural mechanisms of HECT-type ubiquitin ligases

“…This, in turn, may promote the formation of a RSP5 trimer, analogous to the crystallographic trimer formed by the truncated E6AP construct that is lacking the α1′-helix. It should be noted, however, that the trimerization of E6AP was suggested to be associated with hyper-activation (Chan et al, 2013;Ronchi et al, 2014), whereas the proposed RSP5 trimer model confers auto-inhibition. This model of RSP5 regulation has profound structural implications, in particular for the role of the α1′-helix, but also for the adjacent N-terminal regions and intramolecular domain interactions that await to be explored.…”

“…Moreover, various studies have referenced the crystallographic trimer to rationalize mutational results (Chan et al, 2013;Ronchi et al, 2014;Mortensen et al, 2015;Yi et al, 2015). For instance, several mutations at the crystallographic trimer interface, including F727D, R626A, D543A, Y533A (a mutation site in Angelman syndrome), and Y636D (a variant mimicking phosphorylation of Y636 by the tyrosine kinase c-ABL) were found to reduce the activity of E6AP, nourishing the idea that the E6AP trimer represents an activated state (Chan et al, 2013;Ronchi et al, 2014). …”

“…If applicable to E6AP, such a model could, perhaps, provide a way to integrate available experimental data. It will also be interesting to decipher at the structural level how the oligomeric state and activity of E6AP is regulated by posttranslational modifications other than phosphorylation as well as intra-and intermolecular interactions, indicated in several studies (Kühnle et al, 2011;Chan et al, 2013;Mortensen et al, 2015).…”