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

Grimsby, SusanneUppsala universitet,Ludwiginstitutet för cancerforskning (författare)

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.

Jaensson, HannaUppsala universitet,Ludwiginstitutet för cancerforskning (författare)
Dubrovska, AnnaUppsala universitet,Ludwiginstitutet för cancerforskning(Swepub:uu)andub498 (författare)
Lomnytska, MartaUppsala universitet,Ludwiginstitutet för cancerforskning(Swepub:uu)marlo829 (författare)
Hellman, UlfUppsala universitet,Ludwiginstitutet för cancerforskning(Swepub:uu)ulfhm (författare)
Souchelnytskyi, SerhiyKarolinska Institutet,Uppsala universitet,Ludwiginstitutet för cancerforskning(Swepub:uu)sergsouc (författare)
Uppsala universitetLudwiginstitutet för cancerforskning (creator_code:org_t)

Av författaren/redakt...: Grimsby, Susanne; Jaensson, Hanna; Dubrovska, Anna; Lomnytska, Marta; Hellman, Ulf; Souchelnytskyi, ...

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