Cytoplasmic Death Signal Triggered by Src-Mediated Phosphorylation of the Adenovirus E4orf4 Protein

Abstract

In transformed cells, the adenovirus E4orf4 death factor works in part by inducing a Src-mediated cytoplasmic apoptotic signal leading to caspase-independent membrane blebbing and cell death. Here we show that Src-family kinases modulate E4orf4 phosphorylation on tyrosine residues. Mutation of tyrosines 26, 42, and 59 to phenylalanines inhibited Src-induced phosphorylation of E4orf4 in vivo and in vitro but had no effect on the molecular association of E4orf4 with Src. However, in contrast to wild-type E4orf4, the nonphosphorylatable E4orf4 mutant was unable to modulate Src-dependent phosphorylation and was deficient in recruiting a subset of tyrosine-phosphorylated proteins. Indeed, the Src substrates cortactin and p62dok were found to associate with wild-type E4orf4 but not with the nonphosphorylatable E4orf4. Importantly, the nonphosphorylatable mutant E4orf4 was preferentially distributed in the cell nucleus, was unable to induce membrane blebbing, and had a highly impaired killing activity. Conversely, an activated form of E4orf4 was obtained by mutation of tyrosine 42 to glutamic acid. This pseudophosphorylated mutant E4orf4 was enriched in the cytoplasm and plasma membrane, showed increased binding to phosphotyrosine-containing proteins, and induced a dramatic blebbing phenotype associated with increased cell death. Altogether, our findings strongly suggest that Srcmediated phosphorylation of adenovirus type 2 E4orf4 is critical to promoting its cytoplasmic and membrane localization and is required for the transduction of E4orf4-Src-dependent induction of membrane blebbing. We propose that E4orf4 acts in part by uncoupling Src-dependent signals to drive the formation of a signaling complex that triggers a cytoplasmic death signal.Apoptosis is a cell suicide program that plays a crucial role in the maintenance of cellular integrity (77). Two classical pathways for induction of apoptosis exist in mammalian cells, the intrinsic, or mitochondrial, and extrinsic, or death receptor, pathways, and both involve the activation of caspases, a family of cysteine proteases with aspartate specificity (3,4,28,39). This self-amplifying caspase cascade culminates in the proteolytic inactivation of critical components of survival pathways and activation of proapoptotic functions, which altogether lead to the disassembly of the cell. Despite the general role of caspases in apoptotic processes, several modes of caspaseindependent induction of cell death also exist, but the mechanisms involved are poorly understood. In general, the cytosolic hallmarks of apoptosis predominate (e.g., rounder and shrunken morphology, deformations of the plasma membrane, and membrane blebs) and are associated with DNA condensation but not with classical DNA degradation. This programmed cell death response has been termed type II apoptosis. Notably, such suicide programs can be driven by the growth suppressor PML (63), the c-myc-interacting protein Bin1 (17), the Fas-binding protein Daxx (7), and the adenoviral death factor E4orf4 (42). Caspase-...

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“…E4orf4 has also been linked to dysregulation of Src family kinases, which could lead to rearrangement of the actin cytoskeleton and loss of survival signals (Lavoie et al, 2000). E4orf4 becomes phosphorylated by Src kinase, which regulates its subcellular localization and aspects of the death signal (Gingras et al, 2002). The E4orf4 protein might therefore be exploited as a tumorspecific killing agent, and study of its mechanism of action might also identify cellular targets to aid in the development of novel small molecule anticancer drugs.…”

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“…Interestingly, cytoplasmic E4orf4 is also active in inducing cell death, but with a distinct mechanism, in which it interacts with the src family of kinases and thus deregulates the survival signals mediated by these kinases. 95,96 Unlike this, is the case with apoptin, where the nucleocytoplasmic shuttling of the protein is not affected by leptomycin B, an inhibitor of the CRM1 mediated nuclear export pathway. 97,98 E4orf4 gets phosphorylated at tyrosine residues Y26, Y42 and Y59 by src kinases, and this phosphorylation, and the interaction with the src kinases inhibits the nuclear import of the protein.…”

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“…E4orf4 Tyr phosphorylation is correlated with a shift of E4orf4 accumulation from the nucleus to the cytoskeleton and to cell membranes and enhances E4orf4-induced apoptosis (14,15).…”