Smad4 and FAST-1 in the assembly of activin-responsive factor.
Members of the TGF-beta superfamily of signalling molecules work by activating transmembrane receptors with phosphorylating activity (serine-threonine kinase receptors); these in turn phosphorylate and activate SMADs, a class of signal transducers. Activins are growth factors that act primarily through Smad2, possibly in partnership with Smad4, which forms heteromeric complexes with different ligand-specific SMADs after activation. In frog embryos, Smad2 participates in an activin-responsive factor (ARF), which then binds to a promoter element of the Mix.2 gene. The principal DNA-binding component of ARF is FAST-1, a transcription factor with a novel winged-helix structure. We now report that Smad4 is present in ARF, and that FAST-1, Smad4 and Smad2 co-immunoprecipitate in a ligand-regulated fashion. We have mapped the site of interaction between FAST-1 and Smad2/Smad4 to a novel carboxy-terminal domain of FAST-1, and find that overexpression of this domain specifically inhibits activin signalling. In a yeast two-hybrid assay, the FAST-1 carboxy terminus interacts with Smad2 but not Smad4. Deletion mutants of the FAST-1 carboxy terminus that still participate in ligand-regulated Smad2 binding no longer associated with Smad4 or ARF. These results indicate that Smad4 stabilizes a ligand-stimulated Smad2-FAST-1 complex as an active DNA-binding factor.
Chen
X
X
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115-5730, USA.
Weisberg
E
E
Fridmacher
V
V
Watanabe
M
M
Naco
G
G
Whitman
M
M
D028341:Activins; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004586:Electrophoresis; D004625:Embryo, Nonmammalian / Q000378:metabolism; D000939:Epitopes; D051858:Forkhead Transcription Factors; D006801:Humans; D007265:Inhibins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D011233:Precipitin Tests; D015398:Signal Transduction; D051785:Smad Proteins; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; D000939:Epitopes; C102067:FOXH1 protein, Xenopus; C510350:FOXH1 protein, human; D051858:Forkhead Transcription Factors; C491117:Foxh1 protein, mouse; D046911:Macromolecular Substances; D009414:Nerve Growth Factors; C497782:SMAD2 protein, human; C497796:SMAD4 protein, human; D051785:Smad Proteins; D051899:Smad2 Protein; C497785:Smad2 protein, Xenopus; C497783:Smad2 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D029867:Xenopus Proteins; C490808:smad4.1 protein, Xenopus; C117263:smad4.2 protein, Xenopus; D028341:Activins; D007265:Inhibins; D004247:DNA