Mutations on the Smad3 MH2 interface that mediates binding to SARA [32] retain binding to Smad4 and Smad3 but have differential effects on interactions with other Smad-binding proteins including, in some cases, increased binding.

Binding between wild-type or mutant Renilla-Smad3 fusion proteins and eight different Flag epitope-tagged Smad-binding proteins was quantified by pull-down of protein complexes from cell lysates and detection of the Renilla luciferase activity per well. The Renilla luciferase counts were normalized to the number of counts recovered with phosphorylated wild-type Smad3 (100%). The proteins were co-expressed with the constitutively active Alk5. The average of the data from at least four independent transfections is shown. Total Renilla luciferase activity was measured in each transfected cell lysate as a surrogate measure for the level of Renilla-Smad fusion protein; data is only reported for experiments where the level of each Renilla-Smad3 mutant was within two-fold of the level of the Renilla-Smad3 wild type protein in the same experiment. FL indicates that the full-length protein was used. The amino acids comprising the SARA [32], Smurf2 [52], Ski [22], and Sip1 [94] Smad3-binding motifs expressed on the Flag-epitope tagged thioredoxin scaffold are indicated below each gene. The Smad3 Del-PPGY (182–185) mutation is included in the last row although it resides in the Smad3 linker region and not in the MH2 domain. Reduced binding interactions to the Smad interaction motif from Ski by W406A, Y297A and Y226A are consistent with previous studies on this protein interaction that used size exclusion chromatography with purified proteins [22], [64]. Standard deviations are indicated in parenthesis.