Proteomics-based identification of proteins interacting with Smad3: SREBP-2 forms a complex with Smad3 and inhibits its transcriptional activity
2004 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 577, no 1-2, 93-100 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2004. Vol. 577, no 1-2, 93-100 p.
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
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-72965DOI: 10.1016/j.febslet.2004.09.069PubMedID: 15527767OAI: oai:DiVA.org:uu-72965DiVA: diva2:100876