Randall, Rebecca A; (2003) Smad-partner interactions and their roles in signalling by TGF-[beta] superfamily members. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Transforming growth factor-[beta] (TGF-[beta]) superfamily members signal to the nucleus via complexes of activated Smads, comprising phosphorylated R-Smads and the co-Smad, Smad4, which are recruited to DNA by specific transcription factors. During early Xenopus embryo development, Smad2, an R-Smad specific to the TGF-β /Activin/Nodal pathways, interacts with transcription factors such as those of the paired- like/homeodomain Mix family and of the forkhead/winged-helix Fast family. Integral to this interaction is a short, highly conserved motif, the SIM (Smad-interaction motif), present in the C-terminus of these transcription factors. I have fully characterised the SIM in the context of the Mix family member Mixer, and have defined the importance of individual residues for Smad2 interaction. Using this information I have identified which Mix family members contain a functional SIM, and determined which of these transcription factors constitute the endogenous Xenopus protein that binds to the Distal element of the goosecoid gene promoter in response to Activin/Nodal signals. Most importantly, I have identified significant sequence similarity between the SIM and the rigid coil region of the Smad-binding domain (SBD) of SARA, a cytoplasmic protein that recruits Smad2 to the receptors for phosphorylation. This region of the SBD has been demonstrated to interact with Smad2 through a shallow hydrophobic pocket and, using a combination of molecular modelling and site-directed mutagenesis, I have demonstrated that the SIM binds to this same pocket. This suggests that the SIM is a specialised case of a generic motif that is used by other components of the pathway to interact with Smad2. I have also identified a second Smad2-interaction motif present in all Fast family members, N-terminal to the SIM. This Fast motif (FM) shares little sequence similarity with the SIM and is distinct in that it discriminates between Smad2 and Smad3. In addition, the FM only interacts with Smad2 in the context of activated Smad complexes. I propose a binding pocket on the Smad2/Smad4 heterotrimer for FM binding.

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