Proteomics-based identification of proteins interacting with Smad3 : SREBP-2 forms a complex with Smad3 and inhibits its transcriptional activity

• Smad3 is an important component of transforming growth factor-beta (TGFbeta) intracellular signalling. To identify novel interacting proteins of Smad3, we performed pull-down assays with Smad3 constructs fused to glutathione-S-transferase. Proteins which formed complexes with these constructs were analyzed by two-dimensional gel electrophoresis, and were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. We identified 14 proteins interacting with the Smad3 construct lacking the N-terminal Mad homology domain 1 (MH1), and 12 proteins interacting with the construct lacking the C-terminal MH2 domain. Proteins involved in signalling processes, in metabolism regulation, novel proteins, and components of cytoskeleton form four groups of interacting proteins. Interactions of AGP7, sex-determining region Y protein, actin beta and sterol regulatory element binding protein-2 (SREBP-2) proteins with Smad3 constructs were confirmed by immunoblotting with specific antibodies. Interaction of Smad3 with SREBP-2 was also confirmed by co-immunoprecipitation of myc-Smad3 and Flag-SREBP-2 upon expression in mammalian cells. We found that SREBP-2 inhibited the transcriptional activity of Smad3 in luciferase reporter assays.

Keyword

Animals

Cell Line

DNA-Binding Proteins/*metabolism

Electrophoresis; Gel; Two-Dimensional

Humans

Protein Binding

Proteome

Research Support; Non-U.S. Gov't

Spectrometry; Mass; Matrix-Assisted Laser Desorption-Ionization

Trans-Activators/*metabolism

Transcription Factors/*metabolism

Transcription; Genetic

Publication and Content Type

ref (subject category)

art (subject category)