Nuclear localization of the adenovirus E4orf4 protein is mediated through an arginine-rich motif and correlates with cell death

Abstract

The adenovirus E4orf4 protein induces p53-independent death of human cancer cells by a mechanism requiring interactions with the Balpha subunit of protein phosphatase 2A. When expressed alone E4orf4 localizes predominantly in the nucleus, although significant levels are also present in the cytoplasm. While tyrosine phosphorylation of E4orf4 and recruitment of Src have been linked with E4orf4 cytoplasmic cell death functions, little is known about the functions of E4orf4 in the nucleus. In this study, we identified an arginine-rich motif (E4ARM; residues 66-75) that is necessary and sufficient for nuclear and nucleolar localization. This motif, which is highly homologous to the arginine-rich nuclear and nucleolar localization motif of some lentiviral proteins, was shown to target heterologous proteins to the nucleus and to nucleoli, functions found to be dependent on the overall charge of the motif rather than on specific residues. Furthermore, mutation of arginine residues to alanines but not to lysines in E4ARM was shown to block such targeting activity and, when introduced into full-length E4orf4, to decrease induction of cell death. Finally, coexpression of the ARM motifs of E4orf4, HIV-1 Tat or Rev along with full-length E4orf4 was seen to decrease E4orf4-dependent cell killing. Thus it appears that targeting of E4orf4 to the nucleus and cell nucleoli by E4ARM is an important component of E4orf4-induced cell death.

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

“…The E4orf4 polypeptide shares little homology with any known eukaryotic protein; however, two of its major cellular targets have been identified. Events in the nucleus appear to result largely from an interaction with B55 regulatory subunits of protein phosphatase 2A (PP2A) (6,(16)(17)(18)(19)(20)(21)(22) that we have shown in the case of B55␣ blocks the activity of PP2A against at least some substrates (17,53). 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).…”

Cancer-selective therapy of the future: Apoptin and its mechanism of action

“…The ARM directs E4orf4 to the nucleus and further promotes the nucleolar accumulation of the protein. 74,77 Like apoptin, E4orf4, is selectively toxic towards cancer cells. E4orf4 specifically induces apoptosis in a wide range of cancer cells www.landesbioscience.com…”

Adenovirus Protein E4orf4 Induces Premature APC Cdc20 Activation in Saccharomyces cerevisiae by a Protein Phosphatase 2A-Dependent Mechanism

“…Nevertheless, it is difficult to determine what particular form of PP2A actually functions in specific PP2A-regulated processes in all eukaryotic cells. PP2A has previously been implicated in the control of mitotic events in yeast and higher eukaryotes that are essential for cell survival (12, 15); however, the precise role of PP2A regarding mitotic progression and cell cycle regulation has yet to be fully defined, especially with regard to the specific form of PP2A that is involved.The E4orf4 (early region 4 open reading frame 4) protein of human adenoviruses is a 114-residue polypeptide containing an arginine-rich nuclear/nucleolar targeting sequence (27) shown previously to induce the p53-independent death of human cancer cells when expressed alone in the absence of other viral proteins (1,(19)(20)(21)(22)(23)(36)(37)(38). More importantly, E4orf4 protein was shown to bind to the B55␣ regulatory subunit of PP2A (1,22,37,38), and analysis of a series of E4orf4 mutants revealed that functional interaction between E4orf4 and B55␣ correlates with tumor cell killing (22, 37).…”