Oligomerization of the human ARF tumor suppressor and its response to oxidative stress

The tumor suppressor ARF plays an important role as an inhibitor of the Mdm2-mediated degradation of p53. Here we demonstrate that human ARF (p14ARF) can form homo-oligomers. The stability of the oligomers is favored by oxidizing agents in a reversible fashion and involves all three cysteine residues in p14ARF. Furthermore, the effect of p14ARF in clonogenic assays is moderately but reproducibly increased by the mutation of its cysteine residues. We also observed that altering the amino terminus of p14ARF resulted in the appearance of remarkably stable oligomers. This indicates that the amino terminus of p14ARF interferes with the ability of the protein to form multimeric complexes. These observations suggest that p14ARF activity may be linked to its oligomerization status and sensitive to the redox status of the cell. Previous SectionNext SectionIn normal non-stressed cells p53 has a very short half-life (5–20 min) due to an autoregulatory feedback loop mechanism in which the Mdm2 protein plays a key role (reviewed in Refs. 1 and 2). It has been well established that wild type p53 acts as a transcriptional activator of the Mdm2 gene. In turn, Mdm2, which itself has a brief half-life due its auto-ubiquitination activity, has the ability to interact with p53 and to function as a ubiquitin E31 ligase that promotes the conjugation of p53 to ubiquitin (,3, 4, 5). This conjugation to ubiquitin serves as a tag that effectively targets p53 for degradation by the proteasome. In this way, in normal non-stressed cells, p53 is maintained at low levels, and cells are allowed to proliferate. The ARF tumor suppressor (p14ARF in human, p19ARF in mouse) is encoded by the INK4/ARF locus (6). This small protein has been shown to inhibit degradation of p53 mediated by Mdm2 (7, 8). Several models have been proposed to explain this effect of p14ARF. In vitro biochemical studies indicate that p14ARF inhibits the ubiquitin E3 ligase activity of Mdm2 (9), (10) and a decrease in the levels of polyubiquitinated p53 by p14ARF was also shown in vivo (11). In addition, p14ARF was reported to sequester the p53·Mdm2 complex in discrete subnuclear compartments and to inhibit the nuclear export of the complex, a step suggested to be essential for p53 degradation (12). According to Tao and Levine (13) this export step could occur via the nucleolus. Finally, in another study, overexpressed p14ARF was shown to induce the localization of Mdm2 in the nucleolus, which is where ARF is primarily detected (14, 15) and proposed to sequester Mdm2 in this compartment. This would prevent its effects on p53 (15). Whether this mechanism is entirely responsible for the activation of p53 by p14ARF has been questioned by others (16, 17, 18). p53-independent tumor suppressor effects of ARF have also been described (19, 20). ARF expression has been shown to be regulated at the transcriptional level (reviewed in Ref. 21). The expression of the ARF gene is induced by several oncogenic signals such as Myc, E1A, E2F, mutated ras, and v-Abl. ARF expression is inhibited by Twist and Tbx-2 and is actively repressed during development by the Bmi repressor. Additionally, the human ARF promoter can be silenced by methylation or expression of wild type p53 (22). However, to our knowledge, there are no reports on the regulation of p14ARF function at the protein level. Here we propose that p14ARF forms homo-oligomers and that these are stabilized by oxidative agents. Interestingly, the amino terminus of p14ARF, which is known to be critical for its function, can modulate the appearance of the oligomeric forms.