Role of PP2A in cell death induced by the adenoviral protein E4orf4

The E4orf4 protein of adenovirus induces cell death in human cancer cells and not normal primary cells when expressed alone. E4orf4 is also lethal to the yeast Saccharomyces cerevisiae, which we use as a model system. E4orf4-induced toxicity in both mammalian and yeast cells is largely dependent on its ability to associate with protein phosphatase 2A (PP2A), a highly conserved Ser/Thr phosphatase, more specifically, with the B class of regulatory subunits, of which Bα is best characterized in mammalian cells, as well as the yeast equivalent Cdc55. Binding to the B subunit appears to inhibit PP2A activity with some substrates. Consequently, E4orf4 is a useful agent in delineating specific functions of Bα/Cdc55-containing PP2A holoenzymes in vivo. E4orf4 induces mitotic arrest, suggesting that PP2ACdc55 plays a role in regulating mitotic exit. The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit complex involved in the timely degradation of cell cycle proteins to allow the mitotic exit of cells. Two forms of the APC, APCCdc20 and APCCdh1, control mitotic transitions and we demonstrate that E4orf4, through its interaction with Cdc55, induces unscheduled APCCdc20 activity in S-phase arrested cells, and results in the untimely degradation of both Pds1/securin and the cohesin Scc1. In contrast, E4orf4 prevents induction of APCCdh1. Thus, E4orf4 may uncouple PP2ACdc55 regulation of mitotic events. While the specific mechanism by which E4orf4 mediates cell death is unclear, its interaction with the B regulatory PP2A subunit plays a significant role. Cdc55 and Bα contain seven WD40 repeats organized into a seven-bladed propeller structure. In an attempt to functionally map the Cdc55 subunit, we identified regions in Cdc55 required for E4orf4 binding. A polypeptide composed only of blades 1 and 2 bound to E4orf4, and overexpression of this fragment blocked E4orf4 toxicity in yeast. Later on, with the knowledge of the Bα crystal structure, we delineated the precise regions of Bα/Cdc55 involved in E4orf4 binding. Mutational studies in both yeast Cdc55 and mammalian Bα suggest that E4orf4 binds across the substrate-binding groove of the B regulatory subunits, and binding interferes with the interaction of certain substrates, such as p107, with the PP2A holoenzyme. This deregulation of PP2A activity by E4orf4 may affect the functions of certain critical substrates, ultimately leading to cell death.

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