title: Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif.
abstract: 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.
authors.affiliation: Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, London WC2A 3PX, United Kingdom.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Randall
authors.forename: Michael
authors.initials: M
authors.lastname: Howell
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Page
authors.forename: Amanda
authors.initials: A
authors.lastname: Daly
authors.forename: Paul A
authors.initials: PA
authors.lastname: Bates
authors.forename: Caroline S
authors.initials: CS
authors.lastname: Hill
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D011485:Protein Binding; D051899:Smad2 Protein; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C497782:SMAD2 protein, human; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta