ATM-Mediated Phosphorylation of Cortactin Involved in Actin Polymerization Promotes Breast Cancer Cells Migration and Invasion.
ATM-Mediated Phosphorylation of Cortactin Involved in Actin Polymerization Promotes Breast Cancer Cells Migration and Invasion.
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ATM 介导的参与肌动蛋白聚合的 Cortactin 磷酸化可促进乳腺癌细胞迁移和侵袭。
DOI：
10.1159/000496048
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发表时间：
2018-01-01
期刊：
影响因子：
--
通讯作者：
Liu, Manran
中科院分区：
文献类型：
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作者：
Lang, Lei;Hou, Yixuan;Liu, Manran
背景/目的：共济失调-毛细血管扩张突变(ATM)蛋白激酶在维持基因组稳定性方面起着至关重要的作用，是肿瘤的抑制因子。虽然有证据表明DNA损伤非依赖的ATM(氧化ATM)可能参与了肿瘤的进展，但其潜在的机制仍不清楚。方法：应用免疫组织化学、免疫荧光和免疫印迹法检测氧化ATM的水平。用Transwell法检测不同处理组细胞的迁移和侵袭能力。利用缺氧的BT549细胞，在存在或不存在ATMK的特异性抑制剂Ku60019的情况下，进行定量的磷酸蛋白质组分析。免疫沉淀-Western印迹和体外蛋白激酶实验证实，氧化ATM的靶蛋白是磷酸化的皮质动蛋白。通过特异性短发夹状RNA、定点突变、Transwell实验和肌动蛋白聚合实验研究了磷酸化Cortactin的功能。结果：在没有DNA损伤的情况下，增强的氧化ATM蛋白不仅存在于晚期和浸润性乳腺肿瘤组织中，而且存在于恶性缺氧性乳腺癌细胞中。ATM表达的缺失或抑制氧化的ATM激酶活性减少了乳腺癌细胞的迁移和侵袭。应用定量磷蛋白质组学方法鉴定了333个氧化ATM靶蛋白，其中一些蛋白调控与缝隙连接、焦点黏附、肌动蛋白细胞骨架重排相关的关键信号转导。Cortactin是变化最大的磷酸化蛋白之一，是一种新的ATM氧化依赖的低氧反应靶标。我们从机制上揭示了低氧激活的ATM可以通过磷酸化Arp2/3复合体中的丝氨酸来增强Cortactin与Arp2/3复合体的亲和力，从而有利于乳腺癌细胞的迁移和侵袭。结论：氧化的ATM可以磷酸化丝氨酸113处的Cortactin，通过肌动蛋白聚合在促进乳腺肿瘤细胞的迁移和侵袭中发挥关键作用。
BACKGROUND/AIMS: The ataxia-telangiectasia mutated (ATM) protein kinase is critical for the maintenance of genomic stability and acts as tumor suppressor. Although evidence shows that a DNA damage-independent ATM (oxidized ATM) may be involved in cancer progression, the underlying mechanism is still unclear.METHODS: Immunohistochemistry, immunofluorescence and western blotting were applied to detect the levels of oxidized ATM. Transwell assay was used to detect the cell migration and invasion abilities in different treatments. Quantitative phosphoproteome analysis was performed using hypoxic BT549 cells, in the presence or absence of Ku60019, a specific inhibitor of ATM kinase. The phosphorylated cortactin, the target protein of oxidized ATM, was confirmed by immunoprecipitation-western blots and in vitro kinase assay. The functions of phosphorylated cortactin were studied by specific short hairpin RNA, site-directed mutation, transwell assay, and actin polymerization assay.RESULTS: Enhanced oxidized ATM proteins were present not only in the advanced and invasive breast tumor tissues but also malignant hypoxic breast cancer cells, in the absence of DNA damage. Loss of ATM expression or inhibiting oxidized ATM kinase activity reduced breast cancer cell migration and invasion. Using quantitative phosphoproteomics approach, 333 oxidized ATM target proteins were identified, some of these proteins govern key signaling associated with gap junction, focal adhesion, actin cytoskeleton rearrangement. Cortactin, one of the biggest changed phospho-protein, is a novel oxidized ATM-dependent target in response to hypoxia. Mechanically, we reveal that hypoxia-activated ATM can enhance the binding affinity of cortactin with Arp2/3 complex by phosphorylating cortactin at serine 113, and as a result, in favor of breast cancer cell migration and invasion.CONCLUSION: Oxidized ATM can phosphorylate cortactin at serine 113, playing a critical role in promoting breast tumor cell mobility and invasion via actin polymerization.