The adenovirus early region 4 ORF4 protein (E4orf4) triggers a novel death program that bypasses classical apoptotic pathways in human cancer cells. Deregulation of the cell cytoskeleton is a hallmark of E4orf4 killing that relies on Src family kinases and E4orf4 phosphorylation. However, the cytoskeletal targets of E4orf4 and their role in the death process are unknown. Here, we show that E4orf4 translocates to cytoplasmic sites and triggers the assembly of a peculiar juxtanuclear actin-myosin network that drives polarized blebbing and nuclear shrinkage. We found that E4orf4 activates the myosin II motor and triggers de novo actin polymerization in the perinuclear region, promoting endosomes recruitment to the sites of actin assembly. E4orf4-induced actin dynamics requires interaction with Src family kinases and involves a spatial regulation of the Rho GTPases pathways Cdc42/N-Wasp, RhoA/Rho kinase, and Rac1, which make distinct contributions. Remarkably, activation of the Rho GTPases is required for induction of apoptotic-like cell death. Furthermore, inhibition of actin dynamics per se dramatically impairs E4orf4 killing. This work provides strong support for a causal role for endosome-associated actin dynamics in E4orf4 killing and in the regulation of cancer cell fate. INTRODUCTIONThe study of viral proteins and the cellular pathways they perturb is a powerful strategy for identifying critical functions that regulate cell growth and survival. The human adenovirus type 2 early region 4 ORF4 (Ad2 E4orf4) is a multifunctional early viral gene product that is not essential for adenovirus infection, but it seems to play several functions to facilitate viral replication (Branton and Roopchand, 2001;Ben-Israel and Kleinberger, 2002;O'Shea et al., 2005).However, when expressed alone in transformed and tumor cells, E4orf4 induces a p53-independent cell death program that shares several apoptotic hallmarks, including phagocytosis (Lavoie et al., 1998;Shtrichman and Kleinberger, 1998;Champagne et al., 2004). Nonetheless, the mechanisms involved clearly differ from those associated with apoptosis. E4orf4 killing proceeds in absence of classical caspase activation in most cancer cell types and resists to acute overexpression of Bcl-2 and caspase inhibitors (Lavoie et al., 1998;Livne et al., 2001;Robert et al., 2002). Furthermore, mitochondria depolarization is a late event and a consequence rather than a cause of cell death. Thus, E4orf4 activates a novel death pathway, which may involve calcium signaling and calpains (Robert et al., 2002). Evidence also suggests that E4orf4 killing is tumor selective, raising a great interest for the mechanisms involved (Kleinberger, 2004).Two major cellular targets are involved in the regulation of E4orf4 killing: the protein phosphatase 2A (PP2A) and the Src family of nonreceptor tyrosine kinases (Shtrichman et al., 1999;Lavoie et al., 2000;Marcellus et al., 2000). The Srcregulated death activity of E4orf4 that we call the cytoplasmic death pathway, requires a physical interaction wi...

The adenovirus type 2 Early Region 4 ORF4 (E4orf4) protein induces a caspase-independent death program in tumor cells involving changes in actin dynamics that are functionally linked to cell killing. Because an increase in myosin II-based contractility is needed for the death of E4orf4-expressing cells, we have proposed that alteration of cytoskeletal tension is part of the signals engaging the death pathway. Yet the mechanisms involved are poorly defined. Herein, we show that the Jun N-terminal kinase JNK is activated in part through a pathway involving Src, Rho, and ROCK (Rho kinase) and contributes to dysregulate adhesion dynamics and to kill cells in response to E4orf4. JNK supports the formation of atypically robust focal adhesions, which are bound to the assembly of the peculiar actomyosin network typifying E4orf4-induced cell death and which are required for driving nuclear condensation. Remarkably, the dramatic enlargement of focal adhesions, actin remodeling, and cell death all rely on paxillin phosphorylation at Ser-178, which is induced by E4orf4 in a JNK-dependent way. Furthermore, we found that Ser-178-paxillin phosphorylation is necessary to decrease adhesion turnover and to enhance the time residency of paxillin at focal adhesions, promoting its recruitment from an internal pool. Our results indicate that perturbation of tensional homeostasis by E4orf4 involves JNK-regulated changes in paxillin adhesion dynamics that are required to engage the death pathway. Moreover, our findings support a role for JNK-mediated paxillin phosphorylation in adhesion growth and stabilization during tension signaling.Cellular and viral gene products that display a so-called "tumor cell-selective killing activity" were recently discovered.