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Proteomics-based id...
Proteomics-based identification of proteins interacting with Smad3 : SREBP-2 forms a complex with Smad3 and inhibits its transcriptional activity
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- Grimsby, Susanne (author)
- Uppsala universitet,Ludwiginstitutet för cancerforskning
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- Jaensson, Hanna (author)
- Uppsala universitet,Ludwiginstitutet för cancerforskning
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- Dubrovska, Anna (author)
- Uppsala universitet,Ludwiginstitutet för cancerforskning
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- Lomnytska, Marta (author)
- Uppsala universitet,Ludwiginstitutet för cancerforskning
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- Hellman, Ulf (author)
- Uppsala universitet,Ludwiginstitutet för cancerforskning
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- Souchelnytskyi, Serhiy (author)
- Karolinska Institutet,Uppsala universitet,Ludwiginstitutet för cancerforskning
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(creator_code:org_t)
- 2004-10-07
- 2004
- English.
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In: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 577:1-2, s. 93-100
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Abstract
Subject headings
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- 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)
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