Adenovirus E4orf4 Hijacks Rho GTPase-dependent Actin Dynamics to Kill Cells: A Role for Endosome-associated Actin Assembly

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.

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confidence: 99%

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

“…Given the recent report that E4orf4 hijacks Rho GTPasedependent actin dynamics to kill cancer cells (39), it is of interest that Sit4p has been clearly implicated in the regulation of actin dynamics in yeast (3,23,52). SIT4 deletion increases the time necessary for repolarization of the actin cytoskeleton after cellular stress (3), and Sit4p interacts with Bem2p, a protein with GTPase-activating protein (GAP) homology that regulates actin dynamics and cellular morphogenesis.…”

Section: Discussionmentioning

confidence: 99%

Cdc55p-Mediated E4orf4 Growth Inhibition in Saccharomyces cerevisiae Is Mediated Only in Part via the Catalytic Subunit of Protein Phosphatase 2A

Wei

Hsieh

et al. 2008

The adenovirus early region 4 open reading frame 4 (E4orf4) protein specifically induces p53-independent cell death of transformed but not normal human cells, suggesting that elucidation of its mechanism may provide important new avenues for cancer therapy. Wild-type E4orf4 and mutants that retain cancer cell toxicity also induce growth inhibition in Saccharomyces cerevisiae, which provides a genetically tractable system for studying E4orf4 function. Interaction with the protein phosphatase 2A (PP2A) B regulatory subunit is required for E4orf4's effects, suggesting that E4orf4 may function by regulating B subunit-containing heterotrimeric PP2A holoenzymes (PP2A BAC ), which consist of a B subunit complexed with the PP2A structural (A) and catalytic (C) subunits. However, it is not known whether E4orf4-induced growth inhibition requires interaction with the PP2A C subunit or whether E4orf4 might have PP2A B subunit-dependent effects that are independent of PP2A BAC holoenzyme formation. To test these possibilities in S. cerevisiae, we disrupted the stable formation of PP2A BAC heterotrimers and thus E4orf4/C subunit association by PP2A C subunit point mutations or by deletion of the gene for the PP2A methyltransferase, Ppm1p, and assayed for effects on E4orf4-induced growth inhibition. Our results support a model in which E4orf4 mediates growth inhibition and cell killing both through PP2A BAC heterotrimers and through a B regulatory subunit-dependent pathway(s) that is independent of stable complex formation with the PP2A C subunit. They also indicate that Ppm1p has a function other than regulating the assembly of PP2A heterotrimers and suggest that selective PP2A trimer inhibitors and PP6 inhibitors may be useful as adjuvant anticancer therapies.The adenovirus protein E4orf4 is a multifunctional protein that plays an important role in controlling the alternative splicing pattern of adenovirus L1 mRNA by inhibiting the splicing repressor activity of certain host SR proteins (9, 22). E4orf4 also moderates transcriptional activation mediated by the adenovirus E1A protein, including AP-1 (c-fos/jun-B heterodimer) transcription factor activity and transactivation of the adenovirus E2 and E4 promoters (6,24,30,36). E4orf4 mediates these effects at least in part by inducing the dephosphorylation of viral and host proteins, including E1A, c-fos, and SR proteins, and by regulating the expression of jun-B (24, 36). Expression of E4orf4 alone in mammalian cells induces G 2 /M arrest (25) followed by p53-independent cell death in cancer cells, but not in normal human cells (32, 42), thus making E4orf4 a potentially useful tool for cancer therapy. Moreover, investigating the mechanisms underlying E4orf4 function may help identify important drug targets for cancer treatment.E4orf4 induction of cell death in transformed cells requires an interaction with the serine/threonine protein phosphatase PP2A (24, 43). PP2A is a highly conserved, ubiquitous eukaryotic protein phosphatase that has broad biological roles in important cel...

“…To date, several E4orf4 interacting partners besides PP2A have been reported and, in some cases, suggested to contribute to E4orf4-induced cell death, including c-Src (12,18,31,(70)(71)(72)(73)(74), the ATP-dependent chromatin-remodeling factor ACF (3), the NTPDASE4 gene product Golgi UDPase (20,29), and Nup 205 (2). In an attempt to discover more E4orf4 binding partners, we performed affinity purification/mass spectrometry (AP-MS) using both wild-type E4orf4 and class I and class II E4orf4 mutants.…”

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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.