Abstract
We recently reported an interaction between the p14ARF protein and human topoisomerase I (Topo I) resulting in the stimulation of the relaxation activity of Topo I. Our data showed that the complex between the two proteins was located within the nucleolus. In the present work, we have investigated the regions of p14ARF involved in this interaction by using targeted point mutagenesis and deletion mutants. A region encompassing exon 2-encoded sequence was required for physical binding of p14ARF to Topo I as well as for stimulatory activity of the enzyme. Exon 1β-encoded segment was not implicated in the interaction. Moreover, among p14ARF point mutants selected for their high conservation among different mammalian species, mutant p14ARF (RR87, 88AA) did not stimulate Topo I in spite of its association with the enzyme, suggesting its direct implication in the functional activity of ARF. In contrast, one mutant, p14ARF (R71A), was more efficient than wild-type protein to activate Topo I, suggesting that this residue is a key element to modulate Topo I activity. Finally, only ARF–Topo I complexes containing p14ARF exon 2 segment were found to be localized in the nucleolus, suggesting that this subnuclear location is linked to the biological function of the ARF–Topo I complex.
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Acknowledgements
This work was supported by a Grant (ARC 5915) from the Association pour la Recherche centre le Cancer (ARC) Villejuif, France. We are grateful to Dr Gazzeri for providing us with GST constructions. We also acknowledge the help of Dr A Cantereau for confocal microscopy studies.
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Olivier, A., Lucie, K., Jean-François, R. et al. Delineation of the domains required for physical and functional interaction of p14ARF with human topoisomerase I. Oncogene 22, 1945–1954 (2003). https://doi.org/10.1038/sj.onc.1206214
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DOI: https://doi.org/10.1038/sj.onc.1206214
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