Ayrault Olivier, Karayan Lucie, Riou Jean-François, Larsen Christian-Jacques, Séité Paule
Institut de Biologie Moléculaire et d'Ingénierie Génétique, Poitiers, France.
Oncogene. 2003 Apr 3;22(13):1945-54. doi: 10.1038/sj.onc.1206214.
We recently reported an interaction between the p14(ARF) 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 p14(ARF) 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 p14(ARF) to Topo I as well as for stimulatory activity of the enzyme. Exon 1 beta-encoded segment was not implicated in the interaction. Moreover, among p14(ARF) point mutants selected for their high conservation among different mammalian species, mutant p14(ARF) (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, p14(ARF) (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 p14(ARF) 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.
我们最近报道了p14(ARF)蛋白与人拓扑异构酶I（Topo I）之间的相互作用，该相互作用导致Topo I的松弛活性受到刺激。我们的数据表明，这两种蛋白之间的复合物位于核仁内。在本研究中，我们通过使用靶向点突变和缺失突变体，研究了参与这种相互作用的p14(ARF)区域。包含外显子2编码序列的区域对于p14(ARF)与Topo I的物理结合以及该酶的刺激活性是必需的。外显子1β编码片段与这种相互作用无关。此外，在因其在不同哺乳动物物种中具有高度保守性而选择的p14(ARF)点突变体中，突变体p14(ARF)（RR87, 88AA）尽管与该酶结合，但并未刺激Topo I，这表明其直接参与了ARF的功能活性。相反，一个突变体p14(ARF)（R71A）比野生型蛋白更有效地激活Topo I，这表明该残基是调节Topo I活性的关键元件。最后，仅发现含有p14(ARF)外显子2片段的ARF-Topo I复合物定位于核仁，这表明这种亚核定位与ARF-Topo I复合物的生物学功能相关。
