Proteomics-based identification of proteins interacting with Smad3: SREBP-2 forms a complex with Smad3 and inhibits its transcriptional activity
详细信息 查看全文
- 作者：Grimsby ; Susanne ; Jaensson ; Hanna ; Dubrovska ; Anna ; Lomnytska ; Marta ; Hellman ; Ulf ; Souchelnytskyi ; Serhiy
- 关键词：TGFβ ; transforming growth factor-β ; 2D-GE ; two-dimensional gel electrophoresis ; MALDI TOF MS ; matrix-assisted laser desorption-ionization time-of-flight mass spectrometry ; SREBP ; sterol regulatory element binding protein ; GST ; glutathione-S-tran
- 刊名：FEBS Letters
- 出版年：2004
- 出版时间：November 5, 2004
- 年：2004
- 卷：577
- 期：1-2
- 页码：93-100
- 全文大小：417 K
文摘
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.