Abstract
Deregulation of the HECT ubiquitin ligase UBE3A/E6AP has been implicated in Angelman syndrome as well as autism spectrum disorders. We and others have previously identified the 26S proteasome as one of the major UBE3A-interacting protein complexes. Here, we characterize the interaction of UBE3A and the proteasomal subunit PSMD4 (Rpn10/S5a). We map the interaction to the highly conserved Zn2+-binding N-terminal (AZUL) domain of UBE3A, the integrity of which is crucial for binding to PSMD4. Interestingly, two Angelman syndrome point mutations that affect the AZUL domain show an impaired ability to bind PSMD4. Although not affecting the ubiquitin ligase or the estrogen receptor α-mediated transcriptional regulation activities, these AZUL domain mutations prevent UBE3A from stimulating the Wnt/β-catenin signaling pathway. Taken together, our data indicate that impaired binding to the 26S proteasome and consequential deregulation of Wnt/β-catenin signaling might contribute to the functional defect of these mutants in Angelman syndrome.
Highlights
Deregulation of the HECT ubiquitin ligase UBE3A/E6-associated protein (E6AP) has been implicated in Angelman syndrome as well as autism spectrum disorders
UBE3A was originally named E6-associated protein (E6AP) after its initial identification as an interaction partner of the E6 protein expressed by a subset of HPVs that are associated with cervical cancer, other anogenital cancers, and oropharyngeal cancer [2,3,4]
The E6 proteins encoded by this group of cancer-associated HPVs, referred to as high-risk HPVs, hijack UBE3A to form a ternary complex with the tumor suppressor p53 protein, resulting in p53 ubiquitylation and proteasomemediated degradation [5]
Summary
The 26S proteasome is one of the major cellular complexes interacting with UBE3A [21,22,23,24,25,26,27,28,29]. UBE3A-CA did not reduce endogenous p53 or mRuby-p53(R273C) levels, a UBE3A mutant lacking the AZUL domain (UBE3A-⌬NT) was able to induce degradation of both endogenous and mRubyp53(R273C) (Fig. 4C), confirming previously published results that the AZUL domain is not necessary for UBE3A ubiquitin ligase activity [53]. Both UBE3A(G20V) and UBE3A(C21Y) were able to induce p53 degradation when compared with mock-infected cells (Fig. 4C). Deletion of the AZUL domain (UBE3A-⌬NT) abolished the Wnt/-catenin stimulating effect of UBE3A, indicating that both the ubiquitin ligase activity and proteasome binding are necessary for this effect (Fig. 6A). Knockout of either function impairs UBE3A’s ability to activate Wnt/-catenin signaling, consistent with a potential role in the development of Angelman syndrome
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