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Smad4 and FAST-1 in the assembly of activin-responsive factor

Nature volume 389, pages 85–89 (1997)Cite this article

Members of the TGF-β superfamily of signalling molecules work by activating transmembrane receptors with phosphorylating activity (serine–threonine kinase receptors)1; these in turn phosphorylate and activate2 SMADs3,4, a class of signal transducers. Activins are growth factors that act primarily through Smad25,6,7, possibly in partnership with Smad4, which forms heteromeric complexes with different ligand-specific SMADs after activation8,9. In frog embryos, Smad2 participates in an activin-responsive factor (ARF), which then binds to a promoter element of the Mix.2 gene10. The principal DNA-binding component of ARF is FAST-1 (ref. 11), 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.

Incorporation of Smad4 into ARF could reflect either the association of Smad2, Smad4 and FAST-1 into the same complex, or the formation of two distinct types of ARF: Smad2-containing ARF and Smad4-containing ARF. To distinguish between these possibilities, we overexpressed Smad2 bearing a Myc-epitope tag and Smad4 bearing a haemagglutinin (HA) epitope tag, and did an EMSA supershift with each anti-tag antibody alone or together. Addition of both anti-tag antibodies supershifted ARF-containing tagged Smad4 and Smad2 to a position higher than either antibody alone (Fig. 1b), indicating that Smad2 and Smad4 are present in the same complex, rather than in two discrete subsets of ARF complexes. Identical results were obtained when tagged Smad3 was expressed rather than Smad2, indicating that Smad3 can also participate in ARF in an activin-regulated manner (results not shown).

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Figure 1: Molecular composition of ARF.
Figure 2: Co-precipitation of SMADs with FAST-1.
Figure 3: Deletion analysis of FAST-1.
Figure 4: Blocking activin signalling by overexpressed SID.

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Acknowledgements

We thank M. Kawabata for permission to cite his correction of the FAST sequence, the laboratories of J. Massagué, J. Wrana, R. Derynck and J. Thomsen for cDNAs, M. Rubock for construction of GST-tagged FAST-1, and R. Miake-Lye for critically reading the manuscript. V. F. was supported by a postdoctoral fellowship from the American Heart Association. M.W. was supported by the Lucille P. Markey Charitable Trust and grants from the NIH.

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  1. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115-5730, Massachusetts, USA

    Xin Chen, Ellen Weisberg, Valerie Fridmacher, Minoru Watanabe, Grace Naco & Malcolm Whitman

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Chen, X., Weisberg, E., Fridmacher, V. et al. Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature 389, 85–89 (1997). https://doi.org/10.1038/38008

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