Adenovirus E4orf4 hijacks rho GTPase-dependent actin dynamics to kill cells: a role for endosome-associated actin assembly. 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 contamination 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 malignancy 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 development. The results offered here reveal that this gene product Golgi UDPase, first discovered to contribute to E4orf4 toxicity in through to mammalian cells (9,C14), underscoring its importance to cell regulation. E4orf4 associates with several cellular proteins (10, ML348 11, 15,C19), and one of its major partners is protein phosphatase 2A (PP2A). The conversation with PP2A is required for all those E4orf4 functions known to date (1, 3, 7, 8, 16, 19). PP2A is composed of three subunits: the catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits encoded by at least four unrelated gene families, PR55/B55/B, PR61/B56/B, PR72/B, and PR93/PR110/B? (20), which dictate substrate specificity of the PP2A holoenzyme. The conversation of E4orf4 with the B55 subunit of PP2A, but not the B56 subunits, contributes to ML348 E4orf4-induced cell death and cell cycle arrest in both yeast and mammalian cells (10, 12, 18, 19). Earlier reports indicated that PP2A phosphatase activity was required for numerous E4orf4 functions within the context of virus contamination (4, 16, 21) and that E4orf4 recruited PP2A to novel substrates, such as the ACF chromatin remodeling complex, both in the context of virus contamination and when overexpressed alone (15). 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 made up of the B55 subunit, thus preventing dephosphorylation of substrates required for cell survival (22). It Mouse monoclonal to SMN1 was also exhibited that E4orf4 inhibited PP2A activity toward some substrates but not toward others (23). However, since physiological substrates of the E4orf4-PP2A complex have not been recognized to date, it is not clear yet how E4orf4 may impact PP2A activity toward them and whether it prevents PP2A from dephosphorylating them. In addition to its conversation with PP2A, E4orf4 associates with Src-family kinases, and this conversation produces a cytoplasmic death signal (24), which leads to remodeling of the actin cytoskeleton, alterations in recycling endosome trafficking, changes in Golgi membrane dynamics, and cell death (25, 26). Based on the findings that at least part of the E4orf4 effector network was conserved from yeast to mammalian cells, a genetic screen was utilized in to identify novel E4orf4 effectors. This screen revealed that yeast nucleoside diphosphatase (Ynd1) contributed to E4orf4-induced toxicity and actually interacted with the viral protein (11). Ynd1 is usually a Golgi apyrase whose enzymatic activity is required for regulation of nucleotide-sugar import into the Golgi lumen (27, 28). We reported previously that Ynd1 interacted both actually and functionally with Cdc55, the yeast orthologue of the PP2A-B55 regulatory subunit. Deletions of Cdc55 and Ynd1 were shown to confer additive resistance to E4orf4, suggesting that these proteins participated in more than one pathway involved in mediating E4orf4 toxicity. On the other hand, overexpression of Cdc55 was more toxic to the cells in the absence of Ynd1 than in the presence of Ynd1, ML348 indicating that there may be functional.