Significance of filamin A in mTORC2 function in glioblastoma.
Significance of filamin A in mTORC2 function in glioblastoma.
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DOI：
10.1186/s12943-015-0396-z
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发表时间：
2015-07-02
期刊：
影响因子：
37.3
通讯作者：
Tamanoi F
中科院分区：
文献类型：
--
作者：
Chantaravisoot N;Wongkongkathep P;Loo JA;Mischel PS;Tamanoi F
多形性胶质母细胞瘤（GBM）是最高转移性癌症之一。GBM与高水平的雷帕霉素复合物2（mTORC 2）活性的机制靶点相关。我们的目的是观察mTORC 2在GBM细胞中的作用，特别是在肌动蛋白细胞骨架重组，细胞迁移和侵袭中的作用，并进一步确定参与这些细胞过程调控的新的重要参与者。为了进一步研究mTORC 2在GBM中的意义，我们用PP 242（mTOR的ATP竞争性抑制剂）处理GBM细胞，并使用RICTOR siRNA敲低mTORC 2活性。对肌动蛋白细胞骨架，粘着斑，迁移和侵袭的GBM细胞的影响进行了检查。为了深入了解mTORC 2对细胞骨架排列和运动/侵袭作用的分子基础，我们从GBM细胞中亲和纯化mTORC 2，并通过质谱法鉴定感兴趣的蛋白质。对目的蛋白进行表征。除了mTORC 2活性的抑制，我们证明了肌动蛋白分布的显着改变，通过使用鬼笔环肽染色显示。此外，黏着斑蛋白染色发生改变，表明粘着斑发生变化。用PP 242观察到细胞迁移和侵袭的抑制。发现了与mTORC 2多蛋白复合物相关的两个主要蛋白，质谱鉴定了其中一个为细丝蛋白A（Filamin A，FLNA）。证实了FLNA与RICTOR而非mTOR相关。此外，在体外，纯化的mTORC 2可以磷酸化FLNA，类似于其已知的底物AKT。在GBM细胞中，观察到FLNA与RICTOR的共定位，并且FLNA蛋白以及磷酸化FLNA的总量高。在RICTOR siRNA或PP 242处理后，调节残基（Ser 2152）处的磷酸化FLNA水平降低。这种处理也破坏了肌动蛋白丝和FLNA的共定位。我们的研究结果支持FLNA作为mTORC 2控制GBM细胞运动的新下游效应子。这种新的mTORC 2-FLNA信号通路在胶质母细胞瘤细胞的运动和侵袭中起重要作用。本文的在线版本（doi：10.1186/s12943-015-0396-z）包含补充材料，可供授权用户使用。
Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. GBM has been associated with a high level of the mechanistic target of rapamycin complex 2 (mTORC2) activity. We aimed to observe roles of mTORC2 in GBM cells especially on actin cytoskeleton reorganization, cell migration and invasion, and further determine new important players involved in the regulation of these cellular processes. To further investigate the significance of mTORC2 in GBM, we treated GBM cells with PP242, an ATP-competitive inhibitor of mTOR, and used RICTOR siRNA to knock down mTORC2 activity. Effects on actin cytoskeleton, focal adhesion, migration, and invasion of GBM cells were examined. To gain insight into molecular basis of the mTORC2 effects on cellular cytoskeletal arrangement and motility/invasion, we affinity purified mTORC2 from GBM cells and identified proteins of interest by mass spectrometry. Characterization of the protein of interest was performed. In addition to the inhibition of mTORC2 activity, we demonstrated significant alteration of actin distribution as revealed by the use of phalloidin staining. Furthermore, vinculin staining was altered which suggests changes in focal adhesion. Inhibition of cell migration and invasion was observed with PP242. Two major proteins that are associated with this mTORC2 multiprotein complex were found. Mass spectrometry identified one of them as Filamin A (FLNA). Association of FLNA with RICTOR but not mTOR was demonstrated. Moreover, in vitro, purified mTORC2 can phosphorylate FLNA likewise its known substrate, AKT. In GBM cells, colocalization of FLNA with RICTOR was observed, and the overall amounts of FLNA protein as well as phosphorylated FLNA are high. Upon treatments of RICTOR siRNA or PP242, phosphorylated FLNA levels at the regulatory residue (Ser2152) decreased. This treatment also disrupted colocalization of Actin filaments and FLNA. Our results support FLNA as a new downstream effector of mTORC2 controlling GBM cell motility. This new mTORC2-FLNA signaling pathway plays important roles in motility and invasion of glioblastoma cells. The online version of this article (doi:10.1186/s12943-015-0396-z) contains supplementary material, which is available to authorized users.
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