DNA Damage Disrupts the p14ARF-B23(Nucleophosmin) Interaction and Triggers a Transient Subnuclear Redistribution of p14ARF

Abstract

The p14 alternate reading frame (ARF) tumor suppressor plays a central role in cancer by binding to mdm2 (Hdm2 in humans) and enhancing p53-mediated apoptosis following DNA damage and oncogene activation. It is unclear, however, how ARF initiates its involvement in the p53/mdm2 pathway, as p53 and mdm2 are located in the nucleoplasm, whereas ARF is largely nucleolar in tumor cells. We have used immunofluorescence and coimmunoprecipitation to examine how the subnuclear distribution and protein-protein interactions of ARF change immediately after DNA damage and over the time course of the DNA damage response in human tumor cells. We find that DNA damage disrupts the interaction of ARF with the nucleolar protein B23(nucleophosmin) and promotes a transient p53-independent translocation of ARF to the nucleoplasm, resulting in a masking of the ARF NH 2 terminus that correlates with the appearance of ARF-Hdm2 complexes. The translocation also results in an unmasking of the ARF COOH terminus, suggesting that redistribution disrupts a nucleolar interaction of ARF involving this region. By 24 hours after irradiation, DNA repair has ceased and the pretreatment immunofluorescence patterns and complexes of ARF have been restored. Although the redistribution of ARF is independent of p53 and likely to be regulated by interactions other than Hdm2, ARF does not promote UV sensitization unless p53 is expressed. The results implicate the nucleolus and nucleolar interactions of the ARF, including potentially novel interactions involving its COOH terminus as sites for early DNA damage and stress-mediated cellular events. (Cancer Res 2005; 65(21): 9834-42)

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

“…For a broader list of responses to different stress stimuli, please refer to Boulon et al (20). Along with the inhibition of ribosomal biogenesis, massive reorganization occurs, with a rapid outflow of ''nucleolar effectors'', such as p14 Arf , NCL, and NPM1, which slow down or arrest the cell-cycle in both p53-dependent and independent manners (20,31,34,49,86). Concurrently, also DNA repair factors stored within nucleoli and frequently bound to NCL and NPM1 are released into the nucleoplasm; the transient arrest of the cell-cycle progression possibly facilitates the DNA repair process.…”

NSC348884, a nucleophosmin inhibitor disrupts oligomer formation and induces apoptosis in human cancer cells

“…DNA damaging treatments disrupt the p14 ARF -NPM interaction and trigger a transient subnuclear redistribution of p14 ARF to the nucleoplasm, where it interacts with HDM2 and prevents p53 degradation (Bertwistle et al, 2004;Korgaonkar et al, 2005;Lee et al, 2005;Gjerset, 2006). In addition, NPM has been shown to interact with the p53 N-terminal domain directly and inhibits p53 phosphorylation on Ser15 in response to low doses of UV radiation (Colombo et al, 2002;Maiguel et al, 2004).…”

“…In addition, NPM is frequently found in chromosomal translocations associated with hematological malignancies (Naoe et al, 2006). The role of NPM as an oncogene is further enhanced as it binds several tumor suppressor genes, including pRb (Takemura et al, 1999), p14 ARF (Brady et al, 2004;Zhang, 2004;Lee et al, 2005;Gjerset, 2006) and p53 (Colombo et al, 2002;Li et al, 2004;Maiguel et al, 2004). The p53/p14 ARF /Mdm2 (Hdm2) stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability and therapy-induced DNA damage.…”

Beyond Repair Foci: Subnuclear Domains and the Cellular Response to DNA Damage

“…This inhibits the nucleo-cytoplasmic shuttling of Mdm2 and disrupts its interaction with p53, thereby stabilizing p53. 61,68 Although human p14ARF appears to recruit Hdm2 to the nucleolus in response to ionizing radiation, 69 the nucleolar accumulation of murine p19ARF-Mdm2 complexes following UV-damage has not been observed. 68 The difference between the human and murine effect may reflect a dependence on the mode of DNA damage.…”

“…61,68 Although human p14ARF appears to recruit Hdm2 to the nucleolus in response to ionizing radiation, 69 the nucleolar accumulation of murine p19ARF-Mdm2 complexes following UV-damage has not been observed. 68 The difference between the human and murine effect may reflect a dependence on the mode of DNA damage. A precedent for this is observed with WRN, where ionizing radiation causes a translocation from nucleolus to nucleoplasm, whereas UV irradiation does not cause this translocation.…”