Genetic Analysis of B55α/Cdc55 Protein Phosphatase 2A Subunits: Association with the Adenovirus E4orf4 Protein

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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.

Section: Discussionmentioning

confidence: 86%

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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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“…However, when expressed alone at high levels, E4orf4 induces the selective p53-independent death of a variety of human tumor cells (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) and is toxic in the yeast Saccharomyces cerevisiae (22)(23)(24)(25)(26)(27)(28)(29). Killing of cancer cells by E4orf4 is dose dependent and resembles apoptosis in some cell lines, but it seems to occur by mitotic catastrophe in others (8-13, 15, 30, 31).…”

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confidence: 99%

“…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

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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.

“…In adenovirus type 2 (Ad2) and Ad5, E4orf4 encodes a 114-amino-acid protein with no homology to eukaryotic proteins other than an arginine-rich nucleolar targeting sequence (6,7). E4orf4 has no intrinsic enzymatic activity and likely derives all of its reported functions through interaction with the cellular serine/threonine protein phosphatase 2A (PP2A) (6,(8)(9)(10)(11)(12)(13)(14)(15)(16) or other as-yet-unidentified polypeptides. E4orf4 interacts with the PP2A regulatory subunit, B␣, and recruits target phosphoproteins into complexes with PP2A, resulting in dephosphorylation of host and viral proteins.…”

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confidence: 99%

Interaction of Adenovirus Type 5 E4orf4 with the Nuclear Pore Subunit Nup205 Is Required for Proper Viral Gene Expression

Lü

Kucharski

Gamache

et al. 2014

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Adenovirus type 5 E4orf4 is a multifunctional protein that regulates viral gene expression. The activities of E4orf4 are mainly mediated through binding to protein phosphatase 2A (PP2A). E4orf4 recruits target phosphoproteins into complexes with PP2A, resulting in dephosphorylation of host factors, such as SR splicing factors. In the current study, we utilized immunoprecipitation followed by mass spectrometry to identify novel E4orf4-interacting proteins. In this manner we identified Nup205, a component of the nuclear pore complex (NPC) as an E4orf4 interacting partner. The arginine-rich motif (ARM) of E4orf4 was required for interaction with Nup205 and for nuclear localization of E4orf4. ARMs are commonly found on viral nuclear proteins, and we observed that Nup205 interacts with three different nuclear viral proteins containing ARMs. E4orf4 formed a trimolecular complex containing both Nup205 and PP2A. Furthermore, Nup205 complexed with E4orf4 was hypophosphorylated, suggesting that the protein is specifically targeted for dephosphorylation. An adenovirus mutant that does not express E4orf4 (Orf4 ؊ ) displayed elevated early and reduced late gene expression relative to that of the wild type. We observed that knockdown of Nup205 resulted in the same phenotype as that of the Orf4 ؊ virus, suggesting that the proteins function as a complex to regulate viral gene expression. Furthermore, knockdown of Nup205 resulted in a more than a 4-fold reduction in the replication of wild-type adenovirus. Our data show for first time that Ad5 E4orf4 interacts with and modifies the NPC and that Nup205-E4orf4 binding is required for normal regulation of viral gene expression and viral replication. IMPORTANCENuclear pore complexes (NPCs) are highly regulated conduits in the nuclear membrane that control transport of macromolecules between the nucleus and cytoplasm. Viruses that replicate in the nucleus must negotiate the NPC during nuclear entry, and viral DNA, mRNA, and proteins must then be exported from the nucleus. Several types of viruses restructure the NPC to facilitate replication, and the current study shows that adenovirus type 5 (Ad5) utilizes a novel mechanism to modify NPC function. We demonstrate that a subunit of the NPC, Nup205, is a phosphoprotein that is actively dephosphorylated by the Ad5-encoded protein E4orf4. Moreover, Nup205 is required by Ad5 to regulate viral gene expression and efficient viral replication. Nup205 is a nonstructural subunit that is responsible for the gating functions of the NPC, and this study suggests for the first time that the NPC is regulated by phosphorylation both during normal physiology and viral infection.