Randall Rebecca A, Howell Michael, Page Christopher S, Daly Amanda, Bates Paul A, Hill Caroline S
Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, London WC2A 3PX, United Kingdom.
Mol Cell Biol. 2004 Feb;24(3):1106-21. doi: 10.1128/MCB.24.3.1106-1121.2004.
Transforming growth factor beta (TGF-beta) superfamily members signal via complexes of activated Smads, comprising phosphorylated receptor-regulated Smads, such as Smad2 and Smad3, and Smad4. These complexes are recruited to DNA by specific transcription factors. The forkhead/winged-helix transcription factors, XFast-1/XFoxH1a and XFast-3/XFoxH1b, bind an activated Smad heterotrimer comprising two Smad2s and one Smad4. Here we identify a novel Smad2 interaction motif, the Fast/FoxH1 motif (FM), present in all known Fast/FoxH1 family members, N-terminal to the common Smad interaction motif (SIM). The FM is necessary and sufficient to bind active Smad2/Smad4 complexes. The FM differs from the SIM since it discriminates between Smad2 and Smad3, and moreover only binds phosphorylated Smad2 in the context of activated Smad complexes. It is the first Smad interaction motif with this property. Site-directed mutagenesis indicates that the binding site for the FM on a Smad2/Smad4 heterotrimer is a hydrophobic pocket that incorporates the Smad/Smad interface. We demonstrate that the presence of an FM and SIM in the Fast/FoxH1 proteins allows them to compete efficiently for activated Smad2/Smad4 complexes with transcription factors such as Mixer that only contain a SIM. This establishes a hierarchy of Smad-interacting transcription factors, determined by their affinity for active Smad complexes.
转化生长因子β（TGF-β）超家族成员通过活化的Smad复合物进行信号传导，该复合物由磷酸化的受体调节型Smad（如Smad2和Smad3）以及Smad4组成。这些复合物通过特定的转录因子被招募到DNA上。叉头/翼状螺旋转录因子XFast-1/XFoxH1a和XFast-3/XFoxH1b与包含两个Smad2和一个Smad4的活化Smad异源三聚体结合。在此，我们鉴定出一种新的Smad2相互作用基序，即Fast/FoxH1基序（FM），它存在于所有已知的Fast/FoxH1家族成员中，位于常见的Smad相互作用基序（SIM）的N端。FM对于结合活性Smad2/Smad4复合物是必需且充分的。FM与SIM不同，因为它能区分Smad2和Smad3，而且仅在活化的Smad复合物的背景下结合磷酸化的Smad2。它是首个具有此特性的Smad相互作用基序。定点诱变表明，FM在Smad2/Smad4异源三聚体上的结合位点是一个包含Smad/Smad界面的疏水口袋。我们证明，Fast/FoxH1蛋白中FM和SIM的存在使它们能够与仅含有SIM的转录因子（如Mixer）有效竞争活化的Smad2/Smad4复合物。这建立了由它们对活性Smad复合物的亲和力决定的Smad相互作用转录因子的层次结构。
