Rakefet Sharf

Adenovirus E4orf4 protein interacts with both Bα and B′ subunits of protein phosphatase 2A, but E4orf4-induced apoptosis is mediated only by the interaction with Bα

Adenovirus E4orf4 protein is a multifunctional viral regulator, which is involved in down regulation of virally-modulated signal transduction, in control of alternative splicing of viral mRNAs, and in induction of apoptosis in transformed cells. It has been previously shown that E4orf4 interacts with protein phosphatase 2A through the phosphatase Bα subunit. It was further shown that PP2A is required for performing the various E4orf4 functions. We report here that E4orf4 interacts with multiple isoforms of the PP2A-B′ subunit, as well as with Bα. We map the interaction sites of the B subunits on E4orf4 and show that they overlap but are not identical. We identify a dominant negative E4orf4 mutant, which disrupts the PP2A holoenzyme. We show that induction of apoptosis by E4orf4, which we previously reported to require the interaction with Bα, is not affected by the interaction with B′. Our results suggest that the interaction of E4orf4 with various PP2A subpopulations may mediate the different E4orf4 functions.

Selection of apoptosis-deficient adenovirus E4orf4 mutants in Saccharomyces cerevisiae.

ABSTRACT Adenovirus E4orf4 protein has been shown to induce p53-independent, protein phosphatase 2A (PP2A)-dependent apoptosis in transformed cells. Furthermore, E4orf4 also induces toxicity in Saccharomyces cerevisiae in a PP2A-dependent manner (D. Kornitzer and T. Kleinberger, submitted for publication). In this work, we utilized yeast cells to select for nonapoptotic E4orf4 mutants which, in turn, were shown to possess a diminished ability to bind PP2A. The success of this selection system will provide additional apoptosis-relevant mutants for E4orf4 research and strongly supports the relevance of E4orf4-induced toxicity in S. cerevisiae to E4orf4-induced apoptosis in mammalian cells.

The adenovirus E4orf4 protein targets PP2A to the ACF chromatin-remodeling factor and induces cell death through regulation of SNF2h-containing complexes

The adenovirus E4 open-reading-frame 4 (E4orf4) protein regulates the progression of viral infection and when expressed individually it induces non-classical apoptosis in transformed cells. Here we show that E4orf4 associates with the ATP-dependent chromatin-remodeling factor ACF that consists of a sucrose non fermenting-2h (SNF2h) ATPase and an Acf1 regulatory subunit. Furthermore, E4orf4 targets protein phosphatase 2A (PP2A) to this complex and to chromatin. Obstruction of SNF2h activity inhibits E4orf4-induced cell death, whereas knockdown of Acf1 results in enhanced E4orf4-induced toxicity in both mammalian and yeast cells, and Acf1 overexpression inhibits E4orf4′s ability to downregulate early adenovirus gene expression in the context of viral infection. Knockdown of the Acf1 homolog, WSTF, inhibits E4orf4-induced cell death. Based on these results we suggest that the E4orf4–PP2A complex inhibits ACF and facilitates enhanced chromatin-remodeling activities of other SNF2h-containing complexes, such as WSTF–SNF2h. The resulting switch in chromatin remodeling determines life versus death decisions and contributes to E4orf4 functions during adenovirus infection.

Adenovirus E4orf4 Protein Downregulates MYC Expression through Interaction with the PP2A-B55 Subunit

ABSTRACT The adenovirus E4 open reading frame 4 (E4orf4) protein is a multifunctional viral regulator that is involved in the temporal regulation of viral gene expression by modulating cellular and viral genes at the transcription and translation levels and by controlling alternative splicing of adenoviral late mRNAs. When expressed individually, E4orf4 induces apoptosis in transformed cells. Using oligonucleotide microarray analysis, validated by quantitative real time PCR, we found that MYC (also known as c-Myc) is downregulated early after the induction of E4orf4 expression. As a result, Myc protein levels are reduced in E4orf4-expressing cells. MYC downregulation is observed both when E4orf4 is expressed individually and within the context of viral infection. E4orf4 reduces MYC transcription but does not affect transcriptional elongation or RNA stability. An interaction with the PP2A-B55 subunit is required for the downregulation of MYC by E4orf4. Since Myc overexpression was previously shown to inhibit adenovirus replication, the downregulation of Myc by E4orf4 would contribute to efficient virus infection.

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

Background: The adenovirus E4orf4 protein must bind protein phosphatase 2A (PP2A) for its functions. Results: The E4orf4 binding site in PP2A was mapped to the α1,α2 helices of the B55α subunit. Conclusion: The E4orf4 binding site in PP2A-B55α lies above the substrate binding site and does not overlap it. Significance: A novel functional significance was assigned to the α1,α2 helices of the PP2A-B55α subunit. The adenovirus E4orf4 protein regulates the progression of viral infection and when expressed outside the context of the virus it induces nonclassical, cancer cell-specific apoptosis. All E4orf4 functions known to date require an interaction between E4orf4 and protein phosphatase 2A (PP2A), which is mediated through PP2A regulatory B subunits. Specifically, an interaction with the B55α subunit is required for induction of cell death by E4orf4. To gain a better insight into the E4orf4-PP2A interaction, mapping of the E4orf4 interaction site in PP2A-B55α has been undertaken. To this end we used a combination of bioinformatics analyses of PP2A-B55α and of E4orf4, which led to the prediction of E4orf4 binding sites on the surface of PP2A-B55α. Mutation analysis, immunoprecipitation, and GST pulldown assays based on the theoretical predictions revealed that the E4orf4 binding site included the α1 and α2 helices described in the B55α structure and involved at least three residues located in these helices facing each other. Loss of E4orf4 binding was accompanied by reduced contribution of the B55α mutants to E4orf4-induced cell death. The identified E4orf4 binding domain lies above the previously described substrate binding site and does not overlap it, although its location could be consistent with direct or indirect effects on substrate binding. This work assigns for the first time a functional significance to the α1,α2 helices of B55α, and we suggest that the binding site defined by these helices could also contribute to interactions between PP2A and some of its cellular regulators.

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

ABSTRACT 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-B55α 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. IMPORTANCE The 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.

The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition by E4orf4 contributes both to the efficiency of adenovirus replication and to the ability of E4orf4 to kill cancer cells.

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways(1,2). However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time(3-5). Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein(6). Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured(7-9). The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Using the Ras Recruitment System to identify PP2A-B55-interacting proteins.

The RRS system facilitated the discovery of hitherto unknown interactions with the PP2A-B55 subunit. The advantages of the system lie in its ability to identify interactions that may not be detected by traditional yeast two-hybrid systems. The RRS can thus provide a complementary genetic approach to the identification of protein-protein interactions.