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Proteomics‐based identification of proteins interacting with Smad3: SREBP‐2 forms a complex with Smad3 and inhibits its transcriptional activity
Author(s) -
Grimsby Susanne,
Jaensson Hanna,
Dubrovska Anna,
Lomnytska Marta,
Hellman Ulf,
Souchelnytskyi Serhiy
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2004.09.069
Subject(s) - immunoprecipitation , microbiology and biotechnology , proteomics , chemistry , dna binding protein , two hybrid screening , biology , transcription factor , biochemistry , gene
Smad3 is an important component of transforming growth factor‐β (TGFβ) 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 β 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.