Identification of the Adenovirus E4orf4 Protein Binding Site on the B55α and Cdc55 Regulatory Subunits of PP2A: Implications for PP2A Function, Tumor Cell Killing and Viral Replication

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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...

supporting

confidence: 91%

mentioning

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

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“…Earlier work indicated that a major target responsible for E4orf4 toxicity in both human and yeast cells is the B55/Cdc55 regulatory subunit of PP2A (6,16,18,(20)(21)(22)(24)(25)(26)(27)(28). Our recent studies indicated that binding of E4orf4 to B55␣ blocks activity against at least some substrates, and we believe that the failure to dephosphorylate critical substrates involved in regulation of the cell cycle causes growth arrest and subsequent cell death (17,53). Previous results in yeast suggested that APC may be a target for E4orf4 (25), and we have shown, also in yeast, that expression of E4orf4 prematurely induces the early mitotic form of APC Cdc20 and inhibits activation of the late mitotic/early G 1 form APC Hct1/Cdh1 (27).…”

Section: Discussionmentioning

confidence: 92%

“…The E4orf4 polypeptide shares little homology with any known eukaryotic protein; however, two of its major cellular targets have been identified. Events in the nucleus appear to result largely from an interaction with B55 regulatory subunits of protein phosphatase 2A (PP2A) (6,(16)(17)(18)(19)(20)(21)(22) that we have shown in the case of B55␣ blocks the activity of PP2A against at least some substrates (17,53). E4orf4 is also toxic in yeast (Saccharomyces cerevisiae), and this effect is largely abrogated in yeast lacking CDC55, the B55 ortholog (22)(23)(24)(25)(26)(27)(28).…”

mentioning

confidence: 88%

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

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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.

“…Furthermore, overexpression of the PP2A-B55 subunit was reported to enhance E4orf4-induced cell death (18). In contrast, it was recently suggested that E4orf4 induces cell death by titrating out functional PP2A holoenzymes containing the B55 subunit, thus preventing dephosphorylation of substrates required for cell survival (22). It was also demonstrated that E4orf4 inhibited PP2A activity toward some substrates but not toward oth-ers (23).…”

mentioning

confidence: 99%

NTPDASE4 Gene Products Cooperate with the Adenovirus E4orf4 Protein through PP2A-Dependent and -Independent Mechanisms and Contribute to Induction of Cell Death

Avital-Shacham

Sharf

Kleinberger

2014

The adenovirus E4orf4 protein induces nonclassical apoptosis in mammalian cells through at least two complementing pathways regulated by the interactions of E4orf4 with protein phosphatase 2A (PP2A) and Src kinases. In Saccharomyces cerevisiae cells, which do not express Src, E4orf4 induces PP2A-dependent toxicity. The yeast Golgi apyrase Ynd1 was found to contribute to E4orf4-mediated toxicity and to interact with the PP2A-B55␣ regulatory subunit. In addition, a mammalian Ynd1 orthologue, the NTPDASE4 gene product Golgi UDPase, was shown to physically interact with E4orf4. Here we report that knockdown of NTPDASE4 suppressed E4orf4-induced cell death. Conversely, overexpression of the NTPDASE4 gene products Golgi UDPase and LALP70 enhanced E4orf4-induced cell killing. We found that similarly to results obtained in yeast, the apyrase activity of mammalian UDPase was not required for its contribution to E4orf4-induced toxicity. The interaction between E4orf4 and UDPase had two consequences: a PP2A-dependent one, resulting in increased UDPase levels, and a PP2A-independent outcome that led to dissociation of large UDPase-containing protein complexes. The present report extends our findings in yeast to E4orf4-mediated death of mammalian cells, and combined with previous results, it suggests that the E4orf4-NTPDase4 pathway, partly in association with PP2A, may provide an alternative mechanism for the E4orf4-Src pathway to contribute to the cytoplasmic death function of E4orf4. IMPORTANCEThe adenovirus E4orf4 protein contributes to regulation of the progression of virus infection from the early to the late phase, and when expressed alone, it induces a unique caspase-independent programmed cell death which is more efficient in cancer cells than in normal cells. The interactions of E4orf4 with cellular proteins that mediate its functions, such as PP2A and Src kinases, are highly conserved in evolution. The results presented here reveal that the NTPDASE4 gene product Golgi UDPase, first discovered to contribute to E4orf4 toxicity in Saccharomyces cerevisiae, associates with E4orf4 and plays a role in induction of cell death in mammalian cells. Details of the functional interaction between E4orf4, PP2A, and the UDPase are described. Identification of the evolutionarily conserved mechanisms underlying E4orf4 activity will increase our understanding of the interactions between the virus and the host cell and will contribute to our grasp of the unique mode of E4orf4-induced cell death.