Prokova Vassiliki, Mavridou Sofia, Papakosta Paraskevi, Petratos Kyriacos, Kardassis Dimitris
Laboratory of Biochemistry, Department of Basic Sciences, University of Crete Medical School, Heraklion 71110, Greece.
Biochemistry. 2007 Dec 4;46(48):13775-86. doi: 10.1021/bi701540u. Epub 2007 Nov 10.
Smad proteins are the key effectors of the transforming growth factor beta (TGFbeta) signaling pathway in mammalian cells. The importance of Smads for human physiology is documented by the identification and characterization of mutations that are frequently found in cancer patients. In the present study we have functionally characterized such a tumorigenic mutation in Smad4 (E330A) and shown that this mutant as well as a Smad3 mutant bearing the corresponding mutation (Smad3 E239A) failed to activate transcription in response to TGFbeta stimulation because of defects in homo-and hetero-oligomerization. In the case of Smad3, the E239A mutation also abolished the phosphorylation by the TGFbeta type I receptor (ALK5). Examination of the previously published crystal structure of a Smad3/Smad4 MH2 heterotrimer [Protein Data Bank accession code, 1U7F] showed that (a) residue E239 in Smad3 participates in a dense network of intermolecular hydrogen bond and ionic interactions with other conserved polar residues such as Y237 of beta1 strand, N276 and R279 of L2 loop, and R287 of helix H1; (b) residue R287 in Smad3 is also involved in intermolecular interactions by making hydrogen and ionic bonds with Y364 in Smad3 and D493 in Smad4, an amino acid residue that is also frequently mutated in cancer patients (mutation D493H). To investigate the contribution of these interactions to Smad function and TGFbeta signaling, we replaced two of these polar residues (R287 and Y237) with a nonpolar residue (alanine) and functionally characterized the resulting Smad3 mutants. Our analysis showed that Smad3 mutant R287A was phosphorylated by the ALK5 receptor but was unable to form homo-oligomers or hetero-oligomers with Smad4 and activate transcription whereas mutation Y237A had a wild type phenotype. In summary, our present work provides a molecular basis for the functional inactivation of the TGFbeta pathway in patients bearing previously uncharacterized mutations in Smad4 as well as new information regarding the importance of conserved polar amino acids for the structure and function of the MH2 domain of Smads.
Smad蛋白是哺乳动物细胞中转化生长因子β（TGFβ）信号通路的关键效应分子。癌症患者中常见突变的鉴定和特征描述证明了Smads对人体生理的重要性。在本研究中，我们对Smad4中的一种致瘤性突变（E330A）进行了功能表征，结果表明，由于同源和异源寡聚化缺陷，该突变体以及携带相应突变的Smad3突变体（Smad3 E239A）在TGFβ刺激下无法激活转录。就Smad3而言，E239A突变也消除了TGFβ I型受体（ALK5）的磷酸化作用。对先前发表的Smad3/Smad4 MH2异源三聚体的晶体结构[蛋白质数据库登录号，1U7F]进行分析表明：（a）Smad3中的E239残基与其他保守的极性残基，如β1链的Y237、L2环的N276和R279以及螺旋H1的R287，参与了密集的分子间氢键和离子相互作用网络；（b）Smad3中的R287残基也通过与Smad中的Y364和Smad4中的D493形成氢键和离子键参与分子间相互作用，D493也是癌症患者中经常发生突变的氨基酸残基（突变D493H）。为了研究这些相互作用对Smad功能和TGFβ信号传导的贡献，我们将其中两个极性残基（R287和Y237）替换为非极性残基（丙氨酸），并对所得的Smad3突变体进行了功能表征。我们的分析表明，Smad3突变体R287A被ALK5受体磷酸化，但无法与Smad4形成同源寡聚体或异源寡聚体并激活转录，而Y237A突变具有野生型表型。总之，我们目前的工作为携带先前未表征的Smad4突变的患者中TGFβ信号通路的功能失活提供了分子基础，同时也提供了关于保守极性氨基酸对Smads MH2结构域结构和功能重要性的新信息。
