RAP80 responds to DNA damage induced by both ionizing radiation and UV irradiation and is phosphorylated at Ser 205.
RAP80 responds to DNA damage induced by both ionizing radiation and UV irradiation and is phosphorylated at Ser 205.
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DOI：
10.1158/0008-5472.can-07-5950
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发表时间：
2008-06
期刊：
影响因子：
11.2
通讯作者：
Jun Yan;Xiao‐ping Yang;Yong-Sik Kim;A. Jetten
中科院分区：
文献类型：
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作者：
Jun Yan;Xiao‐ping Yang;Yong-Sik Kim;A. Jetten
受体相关蛋白(RAP80)是一种含有两个泛素相互作用基序(UIM)的核蛋白，最近被发现与乳腺癌-1(BRCA1)相关并移位到电离辐射诱导灶(IRIF)。在这项研究中，我们发现BRCA1的BRCT突变体R1699W与乳腺癌风险增加相关，无法与RAP80相互作用。以前，我们已经证明了共济失调-毛细血管扩张症突变蛋白激酶(ATM)在体外可以在Ser(205)处磷酸化RAP80，但该位置是否为ATM在整个细胞中的靶标尚未确定。为了解决这个问题，我们产生了一种抗RAP80Ser205(P)的抗体，它能特异性地识别Ser(205)处磷酸化的RAP80。我们的数据显示，在暴露于电离辐射的细胞中，RAP80在Ser(205)处发生磷酸化，RAP80Ser205(P)易位到IRIF。我们证明这种磷酸化是由ATM介导的，不需要功能上的BRCA1。RAP80的磷酸化发生在照射后5分钟内，远在RAP80转位到IRIF之前。此外，我们还证明了紫外线照射可以诱导RAP80移位到与伽马-H_2AX共定位的DNA损伤部位。我们进一步证明这种易位也依赖于RAP80的UIMs，并且紫外线诱导的RAP80在Ser(205)的磷酸化是由ATM和Rad3相关的激酶介导的，而不是ATM。这些发现表明，RAP80在不同类型的DNA损伤反应中具有更普遍的作用。
Receptor-associated protein (RAP80), a nuclear protein containing two ubiquitin-interacting motifs (UIM), was recently found to be associated with breast cancer-1 (BRCA1) and to translocate to ionizing radiation-induced foci (IRIF). In this study, we show that the BRCT mutant of BRCA1, R1699W, which is associated with increased risk of breast cancer, is unable to interact with RAP80. Previously, we showed that ataxia-telangiectasia mutated protein kinase (ATM) can phosphorylate RAP80 in vitro at Ser(205), but whether this site is a target of ATM in whole cells was not established. To address this question, we generated an anti-RAP80Ser205(P) antibody that specifically recognizes RAP80 phosphorylated at Ser(205). Our data show that RAP80 becomes phosphorylated at Ser(205) in cells exposed to ionizing irradiation and that RAP80Ser205(P) translocates to IRIF. We show that this phosphorylation is mediated by ATM and does not require a functional BRCA1. The phosphorylation occurs within 5 minutes after irradiation, long before the translocation of RAP80 to IRIF. In addition, we show that UV irradiation induces translocation of RAP80 to DNA damage foci that colocalize with gamma-H2AX. We further show that this translocation is also dependent on the UIMs of RAP80 and that the UV-induced phosphorylation of RAP80 at Ser(205) is mediated by ATM- and RAD3-related kinase, not ATM. These findings suggest that RAP80 has a more general role in different types of DNA damage responses.