Liu X, Sun Y, Constantinescu S N, Karam E, Weinberg R A, Lodish H F
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA.
Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10669-74. doi: 10.1073/pnas.94.20.10669.
Drosophila Mad proteins are intracellular signal transducers of decapentaplegic (dpp), the Drosophila transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP) homolog. Studies in which the mammalian Smad homologs were transiently overexpressed in cultured cells have implicated Smad2 in TGF-beta signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-beta receptors has not been established. Here we stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-beta treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-beta-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-beta in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-beta signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-beta receptor complex is an essential step in signal transduction by TGF-beta for both inhibition of cell proliferation and activation of the PAI-1 promoter.
果蝇Mad蛋白是果蝇转化生长因子β（TGF-β）/骨形态发生蛋白（BMP）同源物——脱翅蛋白（dpp）的细胞内信号转导分子。在培养细胞中瞬时过表达哺乳动物Smad同源物的研究表明Smad2参与TGF-β信号传导，但Smad3蛋白在TGF-β受体信号传导中的生理相关性尚未确定。在此，我们使用一种结合了高效逆转录病毒基因转移和定量细胞分选的新方法，在上皮细胞中以可控水平稳定表达Smad蛋白。我们发现，在TGF-β处理后，Smad3在其C末端的SSVS基序处迅速磷酸化。无论是在C末端连接一个表位标签，还是用丙氨酸取代这三个丝氨酸残基，都能消除TGF-β诱导的Smad3磷酸化；这些蛋白以显性负性方式发挥作用，阻断TGF-β对貂肺上皮细胞的抗增殖作用。一种C末端三个丝氨酸被天冬氨酸取代的Smad3蛋白也是TGF-β信号传导的显性抑制剂，但当通过瞬时过表达迫使其核定位时，它能以不依赖配体的方式激活纤溶酶原激活物抑制剂1（PAI-1）转录。Smad3的C末端三个丝氨酸残基被激活的TGF-β受体复合物磷酸化，是TGF-β信号转导中抑制细胞增殖和激活PAI-1启动子的关键步骤。
