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 Src-mediated 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.

We thank Aroussen Laflamme for contributing to the construction of E4orf4(3Y-F)-GFP and E4orf4(Y42E)-GFP expression vectors. We are most grateful to André Lévesque for his major contribution in establishing the microinjection technique as well as for his assistance in all microscopic and imaging analyses. We thank Stephen M. Dilworth, Thomas J. Parsons, and Joan S. Brugge for providing us with expression vectors for middle-T antigen, FAK, and chicken c-src, respectively, and Jacques Landry for helpful discussions and critical reading of the manuscript.

This work was supported by a Terry Fox Research Grant (no. 009058) from the National Cancer Institute of Canada (NCIC) and partly by a research grant from the Canadian Institutes of Health Research (CIHR) (MOP-49450). J. N. Lavoie is a Scholar of the Canadian Institutes of Health Research (CIHR).