Adenovirus E4 open reading frame 4-induced apoptosis involves dysregulation of Src family kinases

The adenoviral early region 4 open reading frame 4 (E4orf4) death factor in duces p53-independent apoptosis in many cell types and appears to kill sele ctively transformed cells. Here we show that expression of E4orf4 in transf ormed epithelial cells results in early caspase-independent membrane blebbi ng, associated with changes in the organization of focal adhesions and acti n cytoskeleton. Evidence that E4orf4 can associate with and modulate Src fa mily kinase activity, inhibiting Src-dependent phosphorylation of focal adh esion kinase (FAK) and paxillin while increasing phosphorylation of cortact in and some other cellular proteins, is presented. Furthermore, E4orf4 dram atically inhibited the ability of FAK and c-src to cooperate in induction o f tyrosine phosphorylation of cellular substrates, suggesting that E4orf4 c an interfere with the formation of a signaling complex at focal adhesion si tes. Consistent with a functional role for E4orf4-Src interaction, over-exp ression of activated c-src dramatically potentiated E4orf4-induced membrane blebbing and apoptosis, whereas kinase dead c-src constructs inhibited E4o rf4 effects on cell morphology and death. Moreover treatment of E4orf4-expr essing cells with PP2, a selective Src kinase inhibitor, led to inhibition of E4orf4-dependent membrane blebbing and later to a marked decrease in E4o rf4-induced nuclear condensation. Taken together, these observations indica te that expression of adenovirus 2 E4orf4 can initiate caspase-independent extranuclear manifestations of apoptosis through a modulation of Src family kinases and that these are involved in signaling E4orf4-dependent apoptosi s. This study also suggests that Src family kinases are likely to play a ro le in the cytoplasmic execution of apoptotic programs.