A Tumor-specific Kinase Activity Regulates the Viral Death Protein Apoptin

Abstract

Apoptin, a chicken anemia virus-encoded protein, is thought to be activated by a general tumor-specific pathway, because it induces apoptosis in a large number of human tumor or transformed cells but not in their normal, healthy counterparts. Here, we show that Apoptin is phosphorylated robustly both in vitro and in vivo in tumor cells but negligibly in normal cells, and we map the site to threonine 108. A gain-of-function point mutation (T108E) conferred upon Apoptin the ability to accumulate in the nucleus and kill normal cells, implying that phosphorylation is a key regulator of the tumorspecific properties of Apoptin. An activity that could phosphorylate Apoptin on threonine 108 was found specifically in tumor and transformed cells from a variety of tissue origins, suggesting that activation of this kinase is generally associated with the cancerous or precancerous state. Moreover, analyses of human tissue samples confirm that Apoptin kinase activity is detectable in primary malignancies but not in tissue derived from healthy individuals. Taken together, our results support a model whereby the dysregulation of the cellular pathway leading to the phosphorylation of Apoptin contributes to human tumorigenesis.

Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing

“…26 Similar to DEDD, Apoptin appears to be regulated by translocation to the nucleus, although nuclear localization of Apoptin is not sufficient to induce apoptosis and its phosphorylation appears to be required as well. 14 The finding that Apoptin binds to DEDAF is interesting, because it hints at a possible role of transcription repression in Apoptininduced apoptosis. Recently, we have shown that inhibitors of transcription or translation do not affect Apoptin's activity; however, this does not exclude a function in transcriptional repression.…”

“…12,13 These data suggest that the tumor-specific activity of Apoptin is not caused by nuclear localization per se. Rather, differential phosphorylation was found to be involved; 14 furthermore, binding to factors that are expressed at higher levels in transformed cells than in normal diploid cells may also play a role.…”

Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin

“…In cells resistant to apoptin-mediated cell death, the protein has a cytoplasmic localization, whereas in sensitive cells, apoptin is found in the nucleus (Danen-Van Oorschot et al, 2003). Apoptin was described to be specifically phosphorylated in transformed cells by an unknown kinase, which was proposed to be important for the nuclear localization and apoptotic activity of apoptin (Rohn et al, 2002). However, it was also shown that a C-terminally truncated apoptin mutant, in which the phosphorylation site was deleted, was still able to translocate to the nucleus and induce apoptosis (Guelen et al, 2004).…”

“…In agreement with the interaction data, only those mutants that had the intact interaction site for PI3-K were able to induce apoptosis both in PC-3 and MCF-7 cells (Figure 1c). Interestingly, the mutant Ala-108, which was reported to be noncytotoxic due the substitution of a Thr-phosphorylation site (Rohn et al, 2002), was still able to induce significant apoptosis, as long as its interaction site with the PI3-K remained intact.…”

“…Interaction of apoptin with PI3-kinase S Maddika et al death (Rohn et al, 2002). However, phosphorylation of apoptin was severely impaired in a mutant lacking amino acids 80-90, even though the residues within the phosphorylation site remained intact (Rohn et al, 2002).…”