Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates
Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates
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DOI：
10.1158/0008-5472.can-10-0382
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发表时间：
2010-09-01
期刊：
影响因子：
11.2
通讯作者：
Ohta, Tomohiko
中科院分区：
文献类型：
--
作者：
Koike, Ayaka;Nishikawa, Hiroyuki;Ohta, Tomohiko
DNA双链断裂（DSB）时的蛋白质积累对基因组稳定性至关重要;然而，这些事件的机制尚未完全了解。在这里，我们报告了一个新的作用，核磷蛋白NPM 1在这些机制。Thr 199-磷酸化NPM 1（pT 199-NPM 1）被募集到电离辐射（IR）诱导的核DNA损伤灶中。E3泛素连接酶RNF 8和RNF 168或E2 Ubc 13的耗竭损害了病灶形成，并且pT 199-NPM 1在体外结合Lys 63连接的泛素聚合物。因此，磷酸化的NPM 1可能与RNF 8依赖的泛素缀合物在DNA损伤位点相互作用。发现这种相互作用依赖于T199磷酸化、酸性通道和邻近的泛素相互作用基序样结构域。乳腺癌抑制因子BRCA 1或其伴侣RAP 80的耗竭增强了IR诱导的NPM 1病灶，并延长了病灶的持续时间，这可能与BRCA 1参与pT 199-NPM 1的作用和动力学有关。用其非磷酸化的T199 A突变体替换内源性NPM 1延长了IR诱导的RAD 51灶的持久性，伴随着未修复的DNA损伤。总的来说，我们的研究结果表明，磷酸化NPM 1是一种新的组成部分，在DSB修复，是由泛素缀合物下游的RNF 8和RNF 168。Cancer Res; 70（17）; 6746-56.（C）2010年AACR。
Protein accumulation at DNA double-strand breaks (DSB) is essential for genome stability; however, the mechanisms governing these events are not fully understood. Here, we report a new role for the nucleophosmin protein NPM1 in these mechanisms. Thr199-phosphorylated NPM1 (pT199-NPM1) is recruited to nuclear DNA damage foci induced by ionizing radiation (IR). Foci formation is impaired by depletion of the E3 ubiquitin ligases RNF8 and RNF168 or the E2 Ubc13, and pT199-NPM1 binds to Lys63-linked ubiquitin polymers in vitro. Thus, phosphorylated NPM1 may interact with RNF8-dependent ubiquitin conjugates at sites of DNA damage. The interaction was found to rely on T199 phosphorylation, an acidic tract, and an adjacent ubiquitin-interacting motif-like domain. Depletion of the breast cancer suppressor BRCA1 or its partner, RAP80, enhanced IR-induced NPM1 foci and prolonged persistence of the foci, possibly implicating BRCA1 in pT199-NPM1 action and dynamics. Replacement of endogenous NPM1 with its nonphosphorylable T199A mutant prolonged persistence of IR-induced RAD51 foci accompanied by unrepaired DNA damage. Collectively, our findings suggest that phosphorylated NPM1 is a novel component in DSB repair that is recruited by ubiquitin conjugates downstream of RNF8 and RNF168. Cancer Res; 70(17); 6746-56. (C)2010 AACR.