The Structural Basis for the Phosphorylation-Induced Activation of Smad Proteins: a Dissertation

The Smad proteins transduce the signal of transforming growth factor-p (TGF-P) and related factors from the cell surface to the nucleus. Following C-terminal phosphorylation by a corresponding receptor kinase, the R-Smad proteins form heteromeric complexes with Smad4. These complexes translocate into the nucleus, bind specific transcriptional activators and DNA, ultimately modulating gene expression. Though studied through a varety of means, the stoichiometr of the R-Smad/Smad4 complex is unclear. We investigated the stoichiometry of the phosphorylation-induced Smad/Smad4 complex by using acidic amino acid substitutions to simulate phosphorylatio . Size exclusion chromatography, analytical ultracentrfugation, and isothermal titration calorimetry analysis revealed that the R-Smad/Smad4 complex is a heterotrmer consisting of two R-Smad subunits and one Smad4 subunit. In addition , a specific mechanism for phosphorylation-induced R-Smad/Smad4 complex formation was studied. Although it had been previously established that par of the mechanism through which phosphorylation induces Smad oligomerization is through relieving MH1-domain mediated autoinhibition of the MH2 (oligomerization) domain, it is also evident that phosphorylatio serves to energetically drive Smad complex formation. Though mutational and size exclusion chromatography analysis, we established that phosphorylation induces oligomerization of the Smads by creating an electrostatic interaction between the phosphorylated C-terminal tail of one R-Smad subunit in a Smad trmer with a basic surface on an adjacent R-Smad or Smad4 subunit. The basic surface is defined largely by the L3 loop, a region that had previously been implicated in R-Smad