Identification and characterization of constitutively active Smad2 mutants: Evaluation of formation of Smad complex and subcellular distribution
Identification and characterization of constitutively active Smad2 mutants: Evaluation of formation of Smad complex and subcellular distribution
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DOI：
10.1210/me.14.10.1583
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发表时间：
2000-10-01
影响因子：
--
通讯作者：
Mathews, LS
中科院分区：
文献类型：
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作者：
Funaba, M;Mathews, LS
Smads介导激活素、转化生长因子β和骨形态发生蛋白从受体到细胞核的信号转导。根据目前的模型，激活的激活素/转化生长因子β受体使Smad2和Smads的羧基末端丝氨酸磷酸化(SSMS-COOH)；磷酸化的Smad2/3与Smad4寡聚，转位到细胞核，调节特定基因的转录。为了详细测试这一模型的关键特征，我们探索了结构性活性Smad2突变体的构建。为了模拟磷酸化的Smad2，我们用羧基末端丝氨酸的酸性氨基酸取代了两个Smad2突变体：Smad2-2E(Ser465,467Glu)和Smad2-3E(Ser464,465,467Glu)。突变体增强了水貂肺上皮细胞系L17的基础转录活性。在Smad4缺失的细胞系SW480.7中，Smad2-2e不影响基本信号转导；然而，与全长Smad4共转染，而不是单独转染Smad4，导致基本转录活性增强，这表明结构性活性的Smad2突变体也需要Smad4来发挥作用。体外蛋白质相互作用分析表明，Smad2-2E与Smad4结合更为紧密，野生型Smad2：Smad4复合体的解离常数为270+/-66 nM，Smad2-2E：Smad4复合体的解离常数为79+/-18 nM。对Smad2亚细胞定位的测定表明，Smad2-2E定位于细胞核的比例高于野生型Smad2。这些结果表明，Smad2的磷酸化导致与Smad4更紧密的结合和核浓度的增加；这些变化可能是Smad2转录激活的原因。
Smads mediate activin, transforming growth factor beta (TGF beta), and bone morphogenetic protein signaling from receptors to nuclei. According to the current model, activated activin/TGF beta receptors phosphorylate the carboxyl-terminal serines of Smad2 and Smads (SSMS-COOH); phosphorylated Smad2/3 oligomerizes with Smad4, translocates to the nucleus, and modulates transcription of defined genes. To test key features of this model in detail, we explored the construction of constitutively active Smad2 mutants. To mimic phosphorylated Smad2, we made two Smad2 mutants with acidic amino acid substitutions of carboxyl-terminal serines: Smad2-2E (Ser465, 467Glu) and Smad2-3E (Ser464, 465, 467Glu). The mutants enhanced basal transcriptional activity in a mink lung epithelial cell line, L17. In a Smad4-deficient cell line, SW480.7, Smad2-2E did not affect basal signaling; however, cotransfection with full-length Smad4, but not transfection of Smad4 alone, resulted in enhanced basal transcriptional activity, suggesting that the constitutively active Smad2 mutant also requires Smad4 for function. In vitro protein interaction analysis revealed that Smad2-2E bound more tightly to Smad4 than did wild-type Smad2; dissociation constants were 270 +/- 66 nM for wild-type Smad2:Smad4 complexes and 79 +/- 18 nM for Smad2-2E:Smad4 complexes. Determination of the subcellular localization of Smad2 revealed that a greater percentage of Smad2-2E was localized in the nucleus than wild-type Smad2. These results suggest that Smad2 phosphorylation results in both tighter binding to Smad4 and increased nuclear concentration; those changes may be responsible for transcriptional activation by Smad2.