Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan.
PLoS Pathog. 2012;8(5):e1002690. doi: 10.1371/journal.ppat.1002690. Epub 2012 May 10.
Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), a human malignancy notorious for its highly metastatic nature. Among EBV-encoded genes, latent membrane protein 1 (LMP1) is expressed in most NPC tissues and exerts oncogenicity by engaging multiple signaling pathways in a ligand-independent manner. LMP1 expression also results in actin cytoskeleton reorganization, which modulates cell morphology and cell motility- cellular process regulated by RhoGTPases, such as Cdc42. Despite the prominent association of Cdc42 activation with tumorigenesis, the molecular basis of Cdc42 activation by LMP1 in NPC cells remains to be elucidated. Here using GST-CBD (active Cdc42-binding domain) as bait in GST pull-down assays to precipitate active Cdc42 from cell lysates, we demonstrated that LMP1 acts through its transmembrane domains to preferentially induce Cdc42 activation in various types of epithelial cells, including NPC cells. Using RNA interference combined with re-introduction experiments, we identified FGD4 (FYVE, RhoGEF and PH domain containing 4) as the GEF (guanine nucleotide exchange factor) responsible for the activation of Cdc42 by LMP1. Serial deletion experiments and co-immunoprecipitation assays further revealed that ectopically expressed FGD4 modulated LMP1-mediated Cdc42 activation by interacting with LMP1. Moreover, LMP1, through its transmembrane domains, directly bound FGD4 and enhanced FGD4 activity toward Cdc42, leading to actin cytoskeleton rearrangement and increased motility of NPC cells. Depletion of FGD4 or Cdc42 significantly reduced (∼50%) the LMP1-stimulated cell motility, an effect that was partially reversed by expression of a constitutively active mutant of Cdc42. Finally, quantitative RT-PCR and immunohistochemistry analyses showed that FGD4 and LMP1 were expressed in NPC tissues, supporting the potential physiologically relevance of this mechanism in NPC. Collectively, our results not only uncover a novel mechanism underlying LMP1-mediated Cdc42 activation, namely LMP1 interaction with FGD4, but also functionally link FGD4 to NPC tumorigenesis.
EB 病毒（EBV）与鼻咽癌（NPC）密切相关，NPC 是一种恶性肿瘤，以高度转移性而闻名。在 EBV 编码的基因中，潜伏膜蛋白 1（LMP1）在大多数 NPC 组织中表达，并通过非配体依赖性方式参与多种信号通路发挥致癌作用。LMP1 的表达还导致肌动蛋白细胞骨架重排，从而调节细胞形态和细胞运动——细胞过程受 RhoGTPases 调节，如 Cdc42。尽管 Cdc42 的激活与肿瘤发生密切相关，但 LMP1 在 NPC 细胞中激活 Cdc42 的分子基础仍有待阐明。在这里，我们使用 GST-CBD（活性 Cdc42 结合域）作为 GST 下拉测定中的诱饵，从细胞裂解物中沉淀活性 Cdc42，证明 LMP1 通过其跨膜结构域优先诱导各种类型的上皮细胞，包括 NPC 细胞中的 Cdc42 激活。通过 RNA 干扰结合再引入实验，我们确定 FGD4（FYVE、RhoGEF 和 PH 结构域包含 4）是负责 LMP1 激活 Cdc42 的 GEF（鸟嘌呤核苷酸交换因子）。连续缺失实验和共免疫沉淀实验进一步表明，过表达的 FGD4 通过与 LMP1 相互作用来调节 LMP1 介导的 Cdc42 激活。此外，LMP1 通过其跨膜结构域直接与 FGD4 结合，并增强 FGD4 对 Cdc42 的活性，导致肌动蛋白细胞骨架重排和 NPC 细胞迁移率增加。FGD4 或 Cdc42 的耗竭显著降低了（约 50%）LMP1 刺激的细胞迁移率，这一效应部分被 Cdc42 的组成性激活突变体的表达逆转。最后，定量 RT-PCR 和免疫组织化学分析显示 FGD4 和 LMP1 在 NPC 组织中表达，支持该机制在 NPC 中具有潜在的生理相关性。总之，我们的研究结果不仅揭示了 LMP1 介导的 Cdc42 激活的新机制，即 LMP1 与 FGD4 的相互作用，而且还将 FGD4 与 NPC 肿瘤发生功能联系起来。
