Activation of Adenovirus Type 2 Early Region 4 ORF4 Cytoplasmic Death Function by Direct Binding to Src Kinase Domain

Abstract

Adenovirus type 2 (Ad2) early region 4 ORF4 (E4orf4) triggers a major death pathway that requires its accumulation in cellular membranes and its tyrosine phosphorylation. This program is regulated by Src family kinases and triggers a potent ZVAD (benzyloxycarbonyl-VAD)-and Bcl2-resistant cell death response in humantransformed cells. How E4orf4 deregulates Src-dependent signaling is unknown. Here we provide strong evidence that a physical interaction requiring the kinase domain of Src and the arginine-rich motif of E4orf4 is involved. The Src binding domain of E4orf4 overlaps with, but is distinct from that of the B␣ subunit of protein phosphatase 2A (PP2A-B␣) and some E4orf4 complexes contain both PP2A and Src. Functional assays using mutant E4orf4 revealed that deregulation of Src signaling, activation of the Jun kinase pathway, and cell blebbing were all critically dependent on Src binding. In contrast, PP2A-B␣ binding per se was not required to engage the Src-dependent death pathway but was more critical for triggering a distinct death activity. Both E4orf4 death activities were manifested within a given cell population, were typified by distinct morphological features, and contributed to overall cell killing, although to different extents in various cell types. We conclude that E4orf4 binding to the Src kinase domain leads to deregulation of Src signaling and plays a crucial role in induction of the cytoplasmic death pathway. Nonetheless, both Src and PP2A enzymes are critical targets of E4orf4 that likely cooperate to trigger E4orf4-induced tumor cell killing and whose relative contributions may vary in function of the cellular background.

Adenovirus Overrides Cellular Checkpoints for Protein Translation

“…Of the known cellular targets of E4-ORF4, c-Src and PP2A seem the most likely to mediate E4-ORF4's effects on mTOR activation, given their well established roles in cellular signaling cascades. E4-ORF4 has been shown to bind to the c-Src kinase domain in vitro, 17 and to affect the tyrosine phosphorylation of c-Src substrates in transfected cells. 18 c-Src has previously been reported to activate mTOR, 19 although these effects are thought to be mediated by c-Src induced activation of the growth factor/PI3-kinase signaling pathway.…”

Adenovirus E4orf4 Protein-Induced Death of p53 −/− H1299 Human Cancer Cells Follows a G 1 Arrest of both Tetraploid and Diploid Cells due to a Failure To Initiate DNA Synthesis

“…E4orf4 is also toxic in yeast (Saccharomyces cerevisiae), and this effect is largely abrogated in yeast lacking CDC55, the B55 ortholog (22)(23)(24)(25)(26)(27)(28). E4orf4 also interacts with Src nonreceptor tyrosine kinases (29)(30)(31). This interaction results in actin remodeling and morphological changes and may contribute to cell death (32,33).…”

A RNA interference screen identifies the Protein Phosphatase 2A subunit PR55γ as a stress-sensitive inhibitor of c-SRC

“…Furthermore, polyoma MT was also found to interact with c-SRC [36] leading to its activation [37]. Moreover, it was reported that adenovirus E4orf4 can interact with both c-SRC and PR55a independently and that the interaction with c-SRC is required for E4orf4 to induce apoptosis [46,52]. Overexpression of E4orf4 phenocopies loss of PR55 in yeast [53], allowing the possibility that inhibition of PR55a is a prerequisite for E4orf4-induced apoptosis in mammalian cells.…”

“…For instance, both polyoma MT antigen and adenovirus E4orf4 were previously shown to interact with both c-SRC and PR55a independently, but the relevance of these interactions remained elusive [46][47][48]. To further address the functional relationship between PR55 and c-SRC, we asked whether PR55c could physically interact with c-SRC.…”