Structural Analysis Of RB-Smad3Interaction

The transforming growth factor-β (TGF-β) signaling plays an important role in cellular differentiation, apoptosis and proliferation. Smads are key signal transducers for TGF-P signaling. Upon activation by the ligand-bound receptor complex, Smads cooperate with many other transcriptional factors and co-factors to regulate transcription.Transforming growth factor-β (TGF-β) induces up-regulation of cyclin dependent kinase inhibitors (CKIs), including members of the INK4family p15Ink4B and Cip/Kip family p21Cip1, and as a result, blocks the activity of G1cyclin-dependent kinases (CDKs) to hyperphosphorylate RB, thereby ensuring inhibition of growth-promoting activities of E2F proteins. In an attempt to address the physiological function of RB, we are surprised to find that RB can interact with Smad3directly.RB could interact with Smad3, but not Smad1, Smad2, Smad4in co-immunoprecipitation assay. Recombinant RB directly bound to Smad3but not Smad2in the cell-free binding assay.To further characterize the RB-Smad3interaction, we examined a series of RB deletion mutants. We found that the pocket B (RB-B) as well as the C terminal region (RB-C) of RB interacted with Smad3. At the same time, we detected the interaction. The MH2domain, but not the MH1and/or the linker region of Smad3with RB in co-immunoprecipitation. These data demonstrate that RB-Smad3interaction requires the pocket domain B and C terminal region of RB and the MH2domain of Smad3.The MH2domain in Smad3(aa232-425) shares～97%identity with that of Smad2(aa274-467). The differences between the two Smads lie only in six amino acids. We evaluated5amino acids in Smad3by substituting them to the corresponding ones in Smad2. Among them, Smad3-S236A, Smad3-L285M and Smad3-I419V/V424M double mutant all retained the ability to interact with RB. We found that A282T was sufficient to abolish the RB-Smad3interaction.To further prove the involvement of Ala282in the RB-Smad3interaction, we synthesized a cell permeable peptide containing Ala282and tested whether it could compete with Smad3for RB interaction. And the result indicated that the peptide could completely abolish the RB-Smad3interaction.We tried to analyze the RB-Smad3interaction based on the crystal structure. In order to get crystals, we carried out molecular biology, protein expression, purification and forming a complex in vitro. But unfortunately, we did not get the complex.Implication:in this paper, we first reported the interaction between the RB and Smad3. RB may play a role in TGF-β signaling pathway through its interaction with Smad3. Further study will be helpful to understand the specific regulatory functions of RB in TGF-β signaling pathway.