The Human Adenovirus Type 5 E4orf4 Protein Targets Two Phosphatase Regulators of the Hippo Signaling Pathway

Abstract

When expressed alone at high levels, the human adenovirus E4orf4 protein exhibits tumor cell-specific p53-independent toxicity. A major E4orf4 target is the B55 class of PP2A regulatory subunits, and we have shown recently that binding of E4orf4 inhibits PP2A B55 phosphatase activity in a dose-dependent fashion by preventing access of substrates (M. Z. Mui et al., PLoS Pathog 9:e1003742, 2013, http://dx.doi.org/10.1371/journal.ppat.1003742). While interaction with B55 subunits is essential for toxicity, E4orf4 mutants exist that, despite binding B55 at high levels, are defective in cell killing, suggesting that other essential targets exist. In an attempt to identify additional targets, we undertook a proteomics approach to characterize E4orf4-interacting proteins. Our findings indicated that, in addition to PP2A B55 subunits, ASPP-PP1 complex subunits were found among the major E4orf4-binding species. Both the PP2A and ASPP-PP1 phosphatases are known to positively regulate effectors of the Hippo signaling pathway, which controls the expression of cell growth/survival genes by dephosphorylating the YAP transcriptional coactivator. We find here that expression of E4orf4 results in hyperphosphorylation of YAP, suggesting that Hippo signaling is affected by E4orf4 interactions with PP2A B55 and/or ASPP-PP1 phosphatases. Furthermore, knockdown of YAP1 expression was seen to enhance E4orf4 killing, again consistent with a link between E4orf4 toxicity and inhibition of the Hippo pathway. This effect may in fact contribute to the cancer cell specificity of E4orf4 toxicity, as many human cancer cells rely heavily on the Hippo pathway for their enhanced proliferation. IMPORTANCEThe human adenovirus E4orf4 protein has been known for some time to induce tumor cell-specific death when expressed at high levels; thus, knowledge of its mode of action could be of importance for development of new cancer therapies. Although the B55 form of the phosphatase PP2A has long been known as an essential E4orf4 target, genetic analyses indicated that others must exist. To identify additional E4orf4 targets, we performed, for the first time, a large-scale affinity purification/mass spectrometry analysis of E4orf4 binding partners. Several additional candidates were detected, including key regulators of the Hippo signaling pathway, which enhances cell viability in many cancers, and results of preliminary studies suggested a link between inhibition of Hippo signaling and E4orf4 toxicity.

Adenoviral protein E4orf4 interacts with the polarity protein Par3 to induce nuclear rupture and tumor cell death

“…We found that Par3 binding was mediated by a conserved motif across Ad serotypes, proximal to a K88 residue previously shown to impact the E4orf4-Par3 interaction ( Fig. 4 A; Mui et al, 2015). Substitution of a single negatively charged residue (D90A) reduced binding, while modification of three residues (DYV to GGG, to minimize structural alteration) blocked Par3 binding without impairing interaction with PP2A ( Fig.…”

“…3 A) for functional analyses, based on the role of Par polarity proteins in the spatial control of actin dynamics (Goldstein and Macara, 2007;Nance and Zallen, 2011). While Par3 was previously reported as an E4orf4 partner, the relevance of this interaction has not been addressed (Mui et al, 2015).…”

Biology of the adenovirus E4orf4 protein: from virus infection to cancer cell death

“…The Hippo pathway is often deregulated in cancer and further perturbations of the Hippo-regulated transcription program may contribute to E4orf4-induced cell death in cancer cells. It was indeed demonstrated that Yap knockdown aggravated E4orf4-induced reduction in cell viability [127].…”

“…E4orf4 also associates with protein phosphatase 1 (PP1) phosphatases that dimerize with apoptosisstimulating protein of p53 (ASPP) proteins, and with components of the Hippo signaling pathway [127] (Section 'The PP1 phosphatase and Hippo pathway components'). However, it is not clear in which cellular compartment these interactions take place.…”

“…It was further shown that E4orf4 reduced phosphorylation of the Hippo signaling component large tumor suppressor kinase (LATS) kinase but increased phosphorylation of its downstream effector, the Yes-associated protein (Yap) transcriptional activator, in a LATS-independent manner. The effect on Yap phosphorylation appeared to depend on the interaction of E4orf4 with PP1 complexes, and it was shown that E4orf4 reduced the interaction between Yap and one of the PP1 partners, ASPP2 [127]. Nuclear-cytoplasmic shuttling of Yap is regulated, at least in part, via Yap phosphorylation by kinases including LATS and Src [128], and Yap hyperphosphorylation may lead to increased cytosolic sequestration and degradation.…”