Structure- and Modeling-based Identification of the Adenovirus E4orf4 Binding Site in the Protein Phosphatase 2A B55α Subunit

The adenovirus E4orf4 protein selectively kills human cancer cells independently of p53 and thus represents a potentially promising tool for the development of novel antitumor therapies. Previous studies suggested that E4orf4 induces an arrest or a delay in mitosis and that both this effect and subsequent cell death rely largely on an interaction with the B55 regulatory subunit of protein phosphatase 2A. In the present report, we show that the death of human H1299 lung carcinoma cells induced by expression of E4orf4 is typified not by an accumulation of cells arrested in mitosis but rather by the presence of both tetraploid and diploid cells that are arrested in G 1 because they are unable to initiate DNA synthesis. We believe that these E4orf4-expressing cells eventually die by various processes, including those resulting from mitotic catastrophe.

Section: Discussionmentioning

confidence: 86%

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confidence: 86%

Adenovirus E4orf4 Protein-Induced Death of p53 ^−/− H1299 Human Cancer Cells Follows a G ₁ Arrest of both Tetraploid and Diploid Cells due to a Failure To Initiate DNA Synthesis

Cabon

Sriskandarajah

Mui

et al. 2013

“…PP2A functions vary extensively (40)(41)(42)(43)(44)(45), due in large part to the 15 or so mammalian B subunits, which have been divided into three structurally divergent classes, designated B/B55, B=/B56, and BЉ, as well as Bٞ striatin/SG2NA (42,46). Our group found that E4orf4 interacts solely with the B/B55 family (14), and more recent studies showed that E4orf4 associates with the B55␣ subunit in a region believed to be involved in substrate binding (1,35). Moreover, we have proposed that E4orf4 acts much like an inhibitory pseudosubstrate for PP2A B55␣ when expressed at high levels, preventing interactions with and dephosphorylation of substrates such as p107 (1) and inducing cell death by preventing dephosphorylation of key substrates required for cell viability (6,14).…”

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confidence: 94%

“…Moreover, we have proposed that E4orf4 acts much like an inhibitory pseudosubstrate for PP2A B55␣ when expressed at high levels, preventing interactions with and dephosphorylation of substrates such as p107 (1) and inducing cell death by preventing dephosphorylation of key substrates required for cell viability (6,14). Previous studies by our group and others indicated that such binding to B55 PP2A subunits is essential for E4orf4 toxicity (16,26,34,35), as E4orf4 mutants that are defective in B55␣ binding (we refer to them as class I mutants) also are defective in cell killing (10). However, members of another class of E4orf4 mutants exist that also are defective for cell killing, even though they bind high levels of B55␣ (10) (we refer to these as class II mutants), suggesting that B55␣ binding is necessary but not sufficient for E4orf4 toxicity and that other essential targets exist.…”

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confidence: 99%

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

Mui

Zhou

Blanchette

et al. 2015

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.

“…Nevertheless, E4orf4 expression also rapidly resulted in cell rounding, a hallmark of early mitosis, suggesting that E4orf4 might induce early mitotic events but create problems with normal passage through and exit from mitosis. In this context, it should be remembered that E4orf4 was shown to bind to the putative substrate binding groove of the B55 regulatory subunit of PP2A (7,21), thus inhibiting PP2A B55 activity by interfering with the access of substrates to the holoenzyme (7,9). Interestingly, such inhibition may mimic a natural process for entry into mitosis regulated by Greatwall (Gwl) and endosulfine alpha (ENSA), which are involved in the control and timing of M phase (22)(23)(24)(25)(26)(27)(28).…”

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confidence: 99%

Analysis by Live Imaging of Effects of the Adenovirus E4orf4 Protein on Passage through Mitosis of H1299 Tumor Cells

Sriskandarajah

Blanchette²,

Kucharski

et al. 2015

The adenovirus E4orf4 protein expressed at high levels kills cancer cells but not normal human primary cells. Previous studies suggested that disruption of processes that regulate mitosis may underlie E4orf4 toxicity. Here we have used live imaging to show that E4orf4 induces a slowed defective transit through mitosis, exhibiting a delay or often failure in cytokinesis that may account for an accumulation of G 1 tetraploids in the population of dying E4orf4-expressing cells. E4orf4 is a human adenovirus early protein that functions during infection to facilitate progression to the late phase (1-7); however, when overexpressed alone in cancer cells, it induces some accumulation of 4n (tetraploid) cells (8) and slow cell death, perhaps involving mitotic catastrophe (9-14). We showed previously that E4orf4-expressing tumor cells with both 2n and 4n DNA content are arrested in G 1 and die (8). This arrest in G 1 seems due to an inability to initiate new rounds of DNA synthesis (8). E4orf4's effects require an interaction with the B55 class of protein phosphatase 2A regulatory subunits (9, 14-17), and we have found that at high levels, E4orf4 inhibits PP2A B55 (18) to induce effects on anaphase-promoting complex (APC) and perhaps other processes that may disrupt normal passage through mitosis (18,19).In this report, we have specifically addressed the defect in mitosis by performing a series of live-cell imaging studies by timelapse microscopy on asynchronously growing as well as synchronized cells. H1299 cells expressing mCherry-tagged histone 2B (H2B) (H1299 cells that had previously been stably transfected with H2B-mCherry in pcDNA3 [catalog number 20972; Addgene, MA, USA]) (20) were infected at a multiplicity of infection (MOI) of 50 with adenoviral vectors expressing either E4orf4 or the reverse tetracycline-controlled transactivator (rtTA) protein. Construction of these viral vectors has been described previously (9,11). Following an 18-h expression period, the cells were plated on 6-well plates in medium containing 20% serum, and time-lapse microscopy was performed using an AxioVision 3 microscope equipped with AxioCam HR (Zeiss, Thornwood, NY) digital camera. Pictures were taken at 15-min intervals over 18 h at a ϫ10 magnification using both phase-contrast microscopy (to observe whole-cell morphology) and fluorescence microscopy (to observe histone 2B and hence the cell nuclei). For each condition, 50 cells were randomly chosen, monitored through the entire time course, and scored for their phenotypes. Cells were scored for the time required to round up (considered to be the start of mitosis), the time required from the first rounded phenotype to the appearance of two nuclei (telophase), the time required for the doubly nucleated cells to separate (cytokinesis), and the time required for the daughter cells to return to their flat morphology (considered returned to G 1 ).As summarized in Fig. 1A, asynchronously growing cells infected with the control rtTA viral vector mostly proceeded rapidly through M phase, a...