Kuang Chenzhong, Chen Yan
Department of Medical and Molecular Genetics, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, IN 46202, USA.
Oncogene. 2004 Feb 5;23(5):1021-9. doi: 10.1038/sj.onc.1207219.
Smad4 is a critical component in transforming growth factor beta (TGF-beta) signaling and frequently mutated in pancreatic and colorectal cancers. Smad4 has two important functional domains, MH1 and MH2, that are involved in different biological processes. The MH1 domain comprises a DNA binding domain and the MH2 domain is mainly implicated in transcriptional activation and homo- and heteromeric complex formation among Smad proteins. In the present study, a total of nine Smad4 mutations at both MH1 and MH2 domains were analysed and all of them had a reduced activity to stimulate transcription of a TGF-beta-responsive reporter gene. All four MH1 mutations had a markedly reduced ability to bind a consensus Smad binding element by an in vitro assay using GST fusion proteins. Among the MH2 mutations, R497H, K507Q, and R515G mutations of Smad4 gave rise to a reduced DNA binding capacity. The R497H mutation had a slightly reduced interaction with Smad2 upon activation of TGF-beta receptor. However, the K507Q and R515G mutations greatly lost their ability to associate with Smad2. Using a GST pull-down assay, it was found that the Smad4 MH2 domain bearing R497H and R515G mutations had an enhanced interaction with the MH1 region of the Smad4 protein, indicating that an increased intramolecular interaction by these mutations may alleviate the DNA binding activity at the MH1 domain. Consistent with these observations, the MH2 domain with R497H mutation had an enhanced ability to inhibit TGF-beta receptor-mediated transcription. In addition, the full-length R497H mutation was able to antagonize TGF-beta signaling in a dominant-negative manner. Therefore, these studies revealed novel mechanisms by which the Smad4 mutations utilize to abrogate their functions in transducing the signaling of TGF-beta, which plays an important role in various stages of cancer formation.
Smad4是转化生长因子β（TGF-β）信号通路中的关键成分，在胰腺癌和结直肠癌中经常发生突变。Smad4有两个重要的功能结构域，即MH1和MH2，它们参与不同的生物学过程。MH1结构域包含一个DNA结合结构域，而MH2结构域主要参与转录激活以及Smad蛋白之间的同源和异源复合物形成。在本研究中，共分析了MH1和MH2结构域的9个Smad4突变，所有这些突变刺激TGF-β反应性报告基因转录的活性均降低。通过使用GST融合蛋白的体外试验，所有4个MH1突变结合共有Smad结合元件的能力均显著降低。在MH2突变中，Smad4的R497H、K507Q和R515G突变导致DNA结合能力降低。R497H突变在TGF-β受体激活后与Smad2的相互作用略有降低。然而，K507Q和R515G突变大大丧失了与Smad2结合的能力。使用GST下拉试验发现，携带R497H和R515G突变的Smad4 MH2结构域与Smad4蛋白的MH1区域的相互作用增强，这表明这些突变导致的分子内相互作用增加可能会减轻MH1结构域的DNA结合活性。与这些观察结果一致，具有R497H突变的MH2结构域抑制TGF-β受体介导的转录的能力增强。此外，全长R497H突变能够以显性负性方式拮抗TGF-β信号通路。因此，这些研究揭示了Smad4突变用于消除其在转导TGF-β信号中功能的新机制，TGF-β在癌症形成的各个阶段都起着重要作用。
